主题：DCs

1-7. 特异抗原递呈细胞的活化是诱导适应性免疫必须的首要步骤Activation of specialized antigen-presenting cells is a necessary first step for induction of adaptive immunity

当在感染组织，病原体被未成熟的DCs细胞摄入时，适应性免疫反应就已启动。这些特异的吞噬细胞大部分居住在组织，具有相对长的生命周期。象巨噬细胞一样，它们来自相同的骨髓前体细胞，从骨髓迁徙到周围组织，它们的作用就是监视病原体可能侵入的局部环境。最终，所有的居住在组织的DCs细胞通过淋巴液迁徙到区域淋巴结，在区域淋巴结，它们与再循环的天然淋巴细胞相互作用（图A-1.14）。一旦DCs细胞没有被激活，则会诱导一个对它们负荷的自身抗原的耐授。

未成熟的DCs细胞表面的受体可识别大多数病原体的公共特性，如细菌细胞壁的蛋白聚糖。与巨噬细胞和中性粒细胞类似，当细菌与DCs细胞的受体结合时就会刺激DCs细胞吞食病原体并在胞内降解它。未成熟的DCs细胞通过受体非依赖的机制（巨噬胞饮作用）也可持续不断地吸收胞外物质，包括病毒颗粒与细菌。然而，DCs细胞的功能主要地不是消灭病原体，而是携带病原体抗原到外周淋巴器官，并在外周淋巴器官将抗原递呈给淋巴细胞。当DCs细胞吸收感染组织的病原体，DCs细胞被活化，并转移到附近的淋巴结。一旦DCs细胞被活化，它就成熟为一种高效的抗原递呈细胞（antigen-presenting cell，APC），APC细胞，去激活病原体特异的淋巴细胞（Figure A-1.14）。活化的DCs细胞分泌细胞因子影响固有免疫反应和适应性免疫反应。这些细胞是基本的看门人，由它们来决定对存在的病原体在何处并如何启动免疫系统的反应。DCs细胞的成熟及其作用见免疫学（IIIV）部分。

IL-12基因转染的人树突状细胞加强细胞免疫反应的体外研究

中国免疫学杂志 2000年第12期第16卷 分子与细胞免疫学

作者：曲春枫　顾培娣　鞠吉雨　孙宗棠

单位：曲春枫 鞠吉雨（山东潍坊医学院免疫学教研室， 潍坊261042）；顾培娣 孙宗棠（中国医科院肿瘤研究所分子肿瘤学国家重点实验室分子免疫室，北京100021）

　　关键词: IL-12；基因转染；树突状细胞；肿瘤疫苗；Th1/Th2分化

　　摘　要　目的：探讨IL-12基因转染的树突状细胞（DCs）能否体外加强细胞免疫反应。方法：脂质体转染（经过Flt-3L、SCF、GM-CSF刺激）增殖周期中的DCs前体细胞。加入GM-CSF、IL-4和TNF-α扩增转染细胞。在流式细胞仪上分析细胞表型及IL-12的表达。成熟DCs装载上分别能与HLA-I 类 和HLA-II类分子结合的多肽，观察IL-12基因转染DCs对特异性Th细胞发育及特异性CTL诱导和功能的影响。结果：IL-12基因转染细胞在上述细胞因子作用下发育成为能有效表达IL-12并具有典型形态学与表型特征的DCs。在装载上CD4⁺T细胞表位HBcAg_50-69后，能诱导这些细胞发育成为IFN-γ产生型的Th1细胞。HLA-A201亚型DCs在其同时装载CD4⁺T和CD8⁺T细胞相应的表位后，IL-12 基因转染DCs所诱导产生的自身淋巴细胞的CTL效应增强。结论：IL-12基因转染的DCs能增强对Th1细胞的刺激和CTL效应的诱导能力，提示它在肿瘤疫苗发展中的潜力。

　　中国图书分类号　R398

IL-12 engineered human dendritic cells in enhancing antigen presenting functions to augment cell mediated immune response in vitro

QU Chun-Feng　GU Pei-Di　JU Ji-Yu

　　（National Laboratory of Molecular Oncology, Cancer Institute, Chinese Academy Medical Sciences,Beijing 100021）

　　Abstract　Objective:To explore whether IL-12 engineered human dendritic cells (DCs) might enhance its antigen presenting functions to augment cell mediated immune responses. Methods: The early replicating progenitors of DCs (stimulated by Flt-3L, SCF and GM-CSF) were transfected with IL-12 recombinant retroviral vector by lipofection. The transfected cells were driven through committed expansion into DCs lineage by adding the above cytokines plus IL-4 and TNF-α. IL-12 transfected and untransfected DCs pulsed with HBcAg_50-69 and p53_264-272 were used to induce specific Th and CTL immune responses in vitro. Results: After culturing in the cocktail of the above mentioned cytokines, IL-12 transfected DCs developed and maintained the characteristic morphological and phenotypic features of DCs. When the DCs were pulsed with a broad spectrum class II epitope HBcAg_50-69, the IL-12 transfected DCs stimulated the autologous lymphocytes to proliferate to a higher level, and the primed Th cells released more IFN-γ when compared with the effects shown in untransfected DCs. Meanwhile, the production of IL-4 was decreased. Preliminary experiment in HLA-A201 subtype system showed that the autologous lymphocytes, primed by IL-12 transfected DCs loaded with class I and class II restricted epitopes, secreted more IFN-γ when they were subsequently co-cultured with A201 target cells loaded only with class I restricted peptide. The latter fact indicated that the specific CTL response was enhanced. Conclusion: IL-12 engineered human dendritic cells enhanced its functions to stimulate Th1 cells as well as to induce specific CTL effect, showing its promise for developing cancer vaccine.

　　Key words　IL-12　Dendritic cells　Gene transfection　Cancer vaccine　Th1/Th2 differentiation

　　IL-12通过增强特异性与非特异性细胞免疫应答，而成为一种有效的抗肿瘤、抗感染免疫的生物佐剂^［1］，但其全身应用毒性严重。因此，寻找合适的应用途径具有重要的临床意义。我们以前对p53蛋白的一些多肽抗原表位研究显示：p53_264-272多肽是HLA-A201个体CD8⁺T细胞识别表位^［2］。HBcAg_50-69多肽是一段能够较广泛地被CD4⁺T淋巴细胞所识别的HBV核心抗原表位^［3］。本文报道采用这两种多肽抗原表位观察了IL-12基因转染的DCs在呈递抗原多肽后对特异性Th细胞的增殖与分化、以及对特异性CTL的诱导是否有增强的效应，籍以探讨其在肿瘤疫苗发展中的应用潜力。

　　1　材料与方法

　　1.1　 载体与细胞　人IL-2逆转录病毒双亚基共表达载体pL35P40SN是在pLXPXSN载体（由NIH的Dr. R. Morgen 赠送）基础上构建^［4］。174CEM T2细胞株由美国NCI的Dr. E. Appella赠送。

　　1.2　抗原性多肽　p53_264-272为HLA-201 分子限制的CD8⁺T细胞识别表位。氨基酸序列为LLGRNSFEV, 由 Dr. E. Appella 赠送，HBcAg_50-69为HLA-II类分子限制的CD4⁺T细胞识别的较广谱的抗原表位，氨基酸序列为PHHTALRQAILCWGELMTLA，由青岛大学医学院分子病毒学实验室合成。

　　1.3　主要试剂　细胞因子SCF、GM-CSF、Flt-3L、IL-4，IL-12定量ELISA检测试剂均为R&D产品，TNF-α为北方同正公司产品，IL-4和IFN-γ检测试剂为晶美公司进口分装试剂, DOTAP 为B.M. 产品。

　　1.4　 PCR-SSP进行HLA-A2型及A201亚型分析　方法参见文献［2，5］，所用引物购自英国Bristol大学移植科学部。

　　1.5　人脐带血DCs的培养及IL-12基因转染　 25 ng/ml SCF溶于0.1 mol/L pH8.5 Na₂HPO₄中，每孔0.8 ml加入到6孔细胞培养板中，4℃过夜。经淋巴细胞分离液分离的新生儿脐带血（产后12 h内）单个核细胞加入到上述细胞培养板中，37℃孵育1～2 h，用预温到37℃的培养液进行洗涤。粘壁细胞加入Flt-3L、GM-CSF、SCF刺激48～50 h，进行DOTAP脂质体介导的基因转染，DOTAP与DNA之比为4：1，转染过程中继续加入上述细胞因子，转染共进行8～8.5 h。此后于培养的第5、第8天再转染两次，每次2～3 h。转染终止后加入含细胞因子的培养液继续培养。第8天后加入IL-4和TNF-α，12～14 d收集细胞进行其表型鉴定及细胞因子的表达检测及功能分析。

　　1.6　培养细胞表型分析　收集培养12～14 d的细胞，进行CD3、CD14、CD19、CD80、CD86、MHC-I、MHC-II的间接免疫荧光染色（所用试剂均为DAKO产品），在Coulter流式细胞仪上进行检测。

　　1.7　肽特异性淋巴细胞增殖实验　12～14 d常规培养成熟的DCs、重组人IL-12基因载体及空载体pLXPXSN转染的DCs，经洗涤后悬浮于AIM-V培养液中（1×10⁶ ml^-1），每孔0.2 ml加到24孔板中。然后加入HBcAg_50-69多肽使其终浓度达到10 μg/ml，混匀后放置于37℃ 2 h。加入液氮冻存复苏后的自身淋巴细胞0.5 ml，使刺激细胞与应答细胞数之比为1：10。放置于37℃培养，24 h后加入IL-2（10 U/ml）。第7天按上述方法重复刺激1次。收集培养细胞上清进行IFN-γ、IL-4的检测（按厂家说明进行）。将上述增殖细胞分3份均匀加入到96孔培养板中，随后加入³H-TdR继续培养16～18 h，收获细胞进行β计数。采用刺激指数（Stimulating Index, SI）进行分析：

　　1.8　多肽特异性CTL的体外诱导及其活性的检测　参照文献［2，5］对所收集到的脐带血通过PCR-SSP法筛选HLA-A201亚型个体，其DCs前体细胞按照前述方法进行IL-12基因转染及DCs的扩增培养。部分培养DCs于第7天加入TNF-α诱导24 h促进其成熟。将细胞洗涤后悬浮于AIM-V中，置于24孔板中加入HBcAg_50-69及p53_264-272 两种多肽，终浓度均分别为10 μg/ml。余同上。通过此方法诱导的细胞作为效应细胞。174CEM T2细胞按前述方法进行p53_264-272多肽的结合，多肽浓度为50 μg/ml，洗涤后作为CTL的靶细胞。两者共同培养，收集培养48 h的上清检测其IFN-γ（按厂家说明进行）的分泌能力。

　　2　结果

　　2.1　人脐带血造血干细胞的富集　在CD34阳性的造血干细胞的表面通常也存在着一种酪氨酸激酶受体——c-kit， 其配体为干细胞因子（Stem Cells Factor，SCF）^［6］。本实验采用预包被于细胞培养板上的SCF来富集人脐带血中的c-kit阳性造血干细胞，在光镜下观察发现，富集得到的造血干细胞较牢固地粘附于细胞培养板上。采用FITC标记的CD34抗体进行直接免疫荧光染色，流式细胞仪测定分析显示，通过上述方法所富集到的细胞，其CD34阳性率约80%。

　　2.2　IL-12基因表达载体转染DCs前体细胞后DC的发育和IL-12的表达　通过形态学观察和流式细胞仪分析，发现IL-12基因转染的DCs在培养12～14 d后，发育为具有典型形态学及表型特征的DCs细胞，细胞表面有大量的CD80、CD86、HLA-I、HLA-II分子表达。通过细胞内细胞因子染色法检查发现，IL-12在转染细胞中的表达可达到50%，每10⁶细胞中IL-12的产量为1.2～1.8 ng/24 h。

　　2.3　IL-12基因转染的DCs对Th细胞增殖和分化的影响　IL-12基因转染DCs对CD4⁺T淋巴细胞增殖水平的影响结果如图1所示。为确定上述对HBcAg_50-69多肽产生应答反应的Th细胞的性质，实验中收集在Th应答反应过程中的细胞上清，分别测定了IFN-γ和IL-4的产量，结果如图2所示。以DCs作为抗原提呈细胞， IL-12基因转染的DCs在诱导特异性Th细胞分化过程中，IFN-γ的产量明显高于空载体转染的DCs，而IL-4的产量则降低。这种细胞因子产量的显著差异在第2次刺激后更为明显（见图2）。表明IL-12基因转染的DCs在负载上HBcAg_50-69多肽后能够诱导IFN-γ产生型的Th1细胞的分化。

图1　IL-12基因转染的DCs对Th细胞增殖的影响

　　Fig.1　IL-12 engineered DCs enhanced the proliferation of Th cells

图2　IL-12基因转染的DCs对IFN-γ和IL-4产量的影响

　　Fig.1　The production of IFN-γ and IL-4 by IL-12 transfected and nontransfected DCs

　　2.4　IL-12基因转染的DCs对CTL的诱导及其活性的影响　IL-12基因转染的HLA-A201亚型个体脐带血DCs，在负载上HBcAg_50-69和p53_264-272时与自身淋巴细胞作用7天后，光镜下可见到有较多的淋巴细胞克隆在DCs周围形成。收集这些增殖的细胞作为效应细胞。采用负载了p53_264-272多肽的T2细胞作为靶细胞，按效：靶=10：1的比例混合，进行特异性CTL活性测定，通过检测其与靶细胞作用后IFN-γ的释放来反映其CTL效应。结果发现IL-12基因转染的DCs所诱导的CTL在与靶细胞作用后，IFN-γ的产生明显升高。每10⁵细胞可产生550 pg/ml，而未转染的DCs所诱导的同样数量的CTL在与靶细胞作用后，IFN-γ的产量为150 pg/ml。

　　3　讨论

　　在机体生理条件下，抗原递呈细胞（APC）通过局部分泌产生IL-12诱导Th0→Th1的分化，有效产生细胞介导的免疫应答反应^［1］。然而，在感染及肿瘤发生过程中，APC自然分泌的IL-12不能有效拮抗其微环境中IL-4和IL-10的作用，致使Th1的发育受到一定的阻碍^［7］。采用预包被于细胞培养板上的SCF富集脐带血中的造血干细胞，加入Flt-3L、SCF、GM-CSF刺激后，进行IL-12基因的转染。随后，在GM-CSF、IL-4和TNF-α作用下，细胞发育成为能够表达外来IL-12基因并具有典型DCs形态学和表型特征的细胞，为促进T淋巴细胞识别抗原后发育成Th1提供了可能。

　　实验结果显示，IL-12基因转染的DCs在负载CD4⁺T细胞的抗原表位HBcAg_50-69多肽后， T淋巴细胞产生明显的增殖反应，并且IFN-γ的产量也明显高于空载体转染的DCs，而IL-4的产生量则显著降低，这种细胞因子产量的差异在第2次刺激后更为明显。提示IL-12基因转染DCs在CD4⁺T细胞抗原表位存在下，抑制了IL-4产生型Th2细胞的分化，促进IFN-γ产生型Th1细胞的产生。

　　在DCs同时负载上能够与HLA-I、HLA-II类分子相结合的多肽后，初步实验结果显示，IL-12基因转染DCs所诱导产生的效应细胞，在与相应靶细胞作用后，特异性CTL所释放的IFN-γ产量是空载体转染DCs诱导细胞释放IFN-α产量的3倍。提示在CD4⁺和CD8⁺T细胞抗原表位的共同存在下，IL-12在抗原提呈局部通过诱导Th1细胞的发育，而有可能促进CTL的应答反应，并能加强CTL对其特异性靶细胞的杀伤活性。

　　采用p53_264-272和HBcAg_50-69作为肿瘤抗原多肽研究模型，在体外实验中发现：与基因未转染的DCs相比，IL-12基因转染DCs在装载上CD4⁺T细胞表位后，能增强这类淋巴细胞的增殖水平，并促进IFN-γ的分泌，向着有利于Th1细胞分化的方向发育；同时装载上CD4⁺和CD8⁺ T细胞表位后，显著增强特异性CTL的应答反应。表明IL-12基因转染DCs的生物佐剂效应较单纯DCs更强，在其装载肿瘤相关的抗原后，可能成为一种值得探索的抗肿瘤的治疗性疫苗。

　　作者简介：曲春枫，女，免疫学博士；孙宗棠，男，教授，博士生导师，主要从事肝癌的发生及预防研究

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收稿2000-01-28

　　修回2000-04-20

    Department of Respiratory Diseases, Ghent University Hospital, Ghent, Belgium

    ABSTRACT

    Dendritic cells (DCs) are leukocytes that are emerging as chief orchestrators of immune responses. The crucial task of DCs is the continuous surveillance of antigen-exposed sites throughout the body, and their unique responsibility is to decide whether to present sampled antigen in an immunogenic or tolerogenic way. Any misstep can either lead to a flawed immune defense or to allergy, even autoimmunity. It comes as no surprise that the lungs become increasingly the subject of DC-related investigations, as they represent a vast interface between the body and the outer world. This constitutes an enormous challenge for the immune system: "firing up" immune responses inappropriately could have devastating results for the fragile gas exchange structures. Evidence accumulates that DCs play a pivotal role in maintaining the delicate balance between tolerance and active immune response in our respiratory system. The exponentially growing body of DC-related publications is a big challenge. This article aims to provide researchers and clinicians with an up-to-date view on DC biology and its relevance to pulmonary medicine. A developing trend in the field of DCs is the shift from fundamental immunologic research toward exciting clinical insights and applications. For the pulmonary clinician, this heralds the dawn of promising therapies in various domains such as infections, allergy, and cancer.

    Key Words: airway; chemokine; chronic obstructive pulmonary disease; dendritic cell; lymph node

    INTRODUCTION

    The "Sentinel" Paradigm of Dendritic Cell Biology

    The first dendritic cells (DCs) were described by Paul Langerhans in 1868 in the basal layers of the epidermis (eer die Nerven der menschlichen Haut. Virchows Archiv fe pathologische Anatomie und Physiologie, und fe klinische Medicin 1868;44:325eC337). These "Langerhans' cells" displayed a typical dendritic morphology, with long, branched arms interdigitating between surrounding epithelial cells. This peculiar shape prompted Dr. Langerhans to consider these cells as a type of neuron. It took more than a century to generate insights through which DCs were correctly identified as white blood cells related to macrophages and monocytes. Subsequently, DCs were described in all lymphoid organs (1), in the blood (2), in the bone marrow (3), and in several other organs, including the lung (4), gut (5), liver (6), heart (7), kidney (8), and urogenital tract (9). Meanwhile, the crucial role of DCs in the control of immunity started to emerge slowly, thanks in part to a series of seminal articles by Steinman and coworkers in the 1970s (1, 10eC13). It was known that T lymphocytes, the effector "muscles" of the immune response, cannot recognize antigens (e.g., microbes, tumor fragments, or allergens) in their native form. So-called antigen-presenting cells (APCs) are required to first sample and then process antigens into short fragments, which are presented to T cells on major histocompatibility complex (MHC) molecules. In addition, APCs provide "costimulatory" molecular signals that are required to raise T-cell activation above a threshold allowing an active response. Before the era of DCs, the only known professional APCs were the macrophages, monocytes, and B lymphocytes. With time, however, it became clear that the antigen-presenting power of DCs greatly exceeded what was documented so far (14, 15). This appeared to rely on some unique features in the biology of these cells (for a comprehensive review, see Reference 16).

    First, DCs are strategically mobilized to anatomic sites with high antigen exposure (e.g., skin, mucosal surfaces, spleen), thanks in part to their sensitivity to a whole array of chemoattracting inflammatory signals. At this stage, DCs are termed "immature," which implies a high capacity to sense, sample, and process incoming antigen, but a poor ability to stimulate T cells. When antigen exposure is accompanied by so-called danger signals (typically, molecular signatures from pathogens or tissue destruction), DCs undergo a series of dramatic changes, which are defined as "maturation" or "activation" (reviewed in Reference 17). From this point on, the antigen-uptake and antigen-processing function is shut down while large amounts of processed antigen are displayed on cell surface MHC molecules, together with a whole battery of T-cell costimulatory factors. At the same time, DCs become specifically attracted to chemokines emanating from the T-celleCrich areas of local lymphoid organs (this is in sharp contrast to more sedentary sentinel cells, such as macrophages). After migration into the lymph node (LN), DCs induce the proliferation of antigen-specific T lymphocytes, which differentiate into cytokine-producing effector T cells capable of recirculating to the endangered tissues. A summary of this "sentinel" paradigm of DC biology and the molecules generally involved is shown in Figure 1.

    Although it constitutes a very efficient defense system, the sentinel function of DCs carries an inherent danger. Invasion by pathogens is usually accompanied by tissue destruction, and there is a substantial risk that activated DCs might take up and present released self-antigens along the way, leading to the induction of autoimmunity. This is especially worrisome because experimental evidence points to the necessity for maintaining self-tolerance throughout life. Fortunately, it appears that DCs play an active role here as well. Indeed, apoptotic cell fragments originating from normal tissue turnover are continuously taken up and processed by DCs (18eC20). Those "resting" DCs that present self-material and home to the T-cell areas in the steady state lead to the priming of immunosuppressive regulatory T cells or even T-cell deletion (21eC24). Understandably, the role of DCs in the protection against, or the induction of, autoimmunity is currently drawing more and more interest.

    In summary, the DC system of leukocytes has evolved into a fine-tuned, highly sensitive sentinel network capable of both igniting as well as shutting down immune responses as appropriate. This article first provides general notions on DC subsets, where these subsets are found in the lung, and how DCs control the pulmonary immune response. In a second part, we summarize current insights on the possible role of DCs in several major pulmonary pathologies.

    Some of the results of the studies described have been previously reported in the form of abstracts (25, 26).

    Defining Features of the Human DC Family

    Despite the arsenal of techniques available to modern-day researchers, DCs are still difficult to study due to a number of issues, as follows, all of which also apply to the lung:

    DCs are rare cells in situ: they represent at most a few percent of the total cell population in a given organ.

    Isolating DCs from tissue samples easily induces activation artifacts: DCs are exquisitely sensitive to stress signals arising from the environment. Classical methods for extraction of these cells involve a whole sequence of procedures, including enzymatic organ digestion, gradient centrifugations, and/or overnight incubation steps. All these interventions can cloud the original state of the DC in situ.

    In contrast to other leukocytes, DCs cannot be immunophenotypically identified using one single antigenic marker. Human DCs are commonly identified by the abundant expression of MHC class II (HLA-DR is commonly used as a marker) and the absence of T, B, and natural killer cell, monocytic, and granulocytic lineage markers. Mature or activated human DCs are characterized by a further increase of HLA-DR and costimulatory molecules (e.g., CD40, B7-1, B7-2) on their surface. Additional specific markers of mature human DCs are the immunoglobulin superfamily member CD83 (27), the 55-kD actin-bundling protein Fascin (28, 29), and the DC-specific lysosome-associated membrane protein DC-LAMP (30).

    An overview of the human DC family tree, together with typical identifying markers, is shown in Figure 2. Currently, an important distinction is made between two main DC subtypes: myeloid DCs (mDCs) and plasmacytoid DCs (pDCs). Human mDCs develop from bone marroweCderived monocytic precursors, whereas pDCs are developmentally related to the lymphoid lineage (31). In addition to differences in identifying markers (32, 33), the most relevant distinction between mDCs and pDCs is functional because these cells are activated by a different set of pathogenic stimuli. This reflects a differential expression of specific Toll-like receptors (TLRs) on both subsets (34).

    TLRs are primitive sensors for "danger signals," which are, for the most part, microbial molecular signatures but also include products released by cell necrosis and extracellular matrix breakdown (reviewed in Reference 35). Human mDCs primarily express TLR2 and TLR4 and become mature in response to LPS and mycobacterial cell wall components. In contrast, human pDCs do not possess LPS-sensing receptors but typically express TLR7 (a target for the immunomodulatory molecule imiquimod) and TLR9 (the receptor for unmethylated microbial DNA sequences). Typically, immature pDCs exposed to viral products secrete massive amounts of type I interferon, indicating that these cells are in fact identical to the long-known natural interferon-producing cell (36). Thus, pDCs first deliver a powerful innate response designed to limit viral replication, after which these cells differentiate into mature DCs capable of triggering an adaptive immune response. In addition to mDCs and pDCs, Langerhans' celleCtype (i.e., intraepithelial) DCs are often considered as a separate DC subset in humans. Their origin and developmental relationship to other DCs is subject to differing views (37eC40). Langerhans' cells (LCs) are typically located above the basement membrane in epithelial layers of antigen-exposed organs and extend long, branched pseudopods between surrounding epithelial cells (41). LCs have specific requirements in terms of hematopoietic growth factors (42), and can be identified by the presence of typical Birbeck granules (tennis racketeCshaped intracellular organelles with unknown function) and a set of markers such as E-cadherin (which anchors LCs to neighboring epithelial cells), CD1a, and Langerin (43). Another subset of DCs are the so-called interstitial-type DCs, first described in the dermis. These share some markers with resident macrophages (e.g., CD68), but in contrast to macrophages, exhibit a potent T-cell stimulatory activity (42). In addition, interstitial DCs appear to be the only DC subset capable of inducing B-cell differentiation and drive humoral immune responses (44).

    DCs OF THE LUNG: LOCALIZATION AND PHENOTYPE

    Sertl and colleagues (4) was one of the first to describe the presence of DCs in the airway epithelium, lung parenchyma, and visceral pleura of human and mouse specimens. These cells displayed a typical dendritic morphology, expressed copious amounts of MHCII on their surface, and acted as potent T-cell stimulators in vitro. Later studies have provided more detail into the features and localization of DCs in the different pulmonary compartments. From a methodologic point of view, immunofluorescent techniques allow identification of pulmonary DCs among low-autofluorescent leukocytes (next to lymphocytes and granulocytes), in contrast to the highly autofluorescent pulmonary macrophages (45eC47). Using this approach, a small number of DCs can be detected in the bronchoalveolar lavage fluid (BALF). A fraction of these cells express LC markers (CD1a, S100) and exhibit potent T-cell stimulatory capabilities in an allogeneic mixed leukocyte reaction (45, 48). A recent study succeeded in distinguishing different DC subsets in human BALF (i.e., an mDC subset; 0.06% of BALF cells) and a pDC subset (0.02%) (49). Within the bronchial epithelium, DCs are found with typical features of LCs, such as the presence of Birbeck granules, the expression of CD1a, and Langerin (Figure 3) (43, 50, 51). Specific tissue preparation techniques applied to rodent specimens have succeeded in visualizing this superficial airway DC network (Figure 4) (52eC54). In the absence of inflammation, DCs are distributed at an average density of several hundred cells per millimeter squared in the large airways, decreasing to less than a hundred DCs per millimeter squared within smaller intrapulmonary airways (52). DCs in the lung parenchyma are present in larger numbers. They are mostly found within interalveolar septa (55). Cochand and coworkers (56) have clearly described the immature character of human lung parenchymal DCs, in terms of low T-cell costimulary molecule expression, the presence of receptors for inflammatory chemokines, and a strong antigen-uptake activity. Recently, our group succeeded in identifying three previously undescribed types of DCs in normal human lung parenchyma: two subsets of mDCs (one, CD11c+/BDCA[blood DC antigen]-1+; the other, CD11c+/BDCA-3+) and one pDC subset (CD11ceC/BDCA-2+) (51). We are currently trying to characterize the functional differences between these subsets, and the impact of lung disease on these cells.

    LUNG DCs AND THE SHAPING OF THE PULMONARY IMMUNE RESPONSE

    A growing body of experimental evidence indicates that the "sentinel" paradigm of DC function applies extremely well to the respiratory system. This implies (1) a constant recruitment of DCs into the lung, (2) a capacity for these cells to sense and capture inhaled antigen, and (3) an efficient transport of processed antigen to the draining pulmonary LNs, where DCs decide which type of immune response will ensue.

    DC Recruitment to the Lung Is Driven by Environmental Stimuli

    Even in the absence of overt inflammation, DCs or their precursors are constantly recruited from the blood into the lung. Holt and coworkers (57) were the first to demonstrate that respiratory tract DCs are continuously replenished by a steady-state bone marrow output. Further studies in animals have shown that the steady-state deployment of the respiratory tract DC network is clearly correlated with age (58): although MHCII+ DCs already colonize the fetal lung mesenchyme, airway mucosal DCs only appear a few days after birth, reaching adultlike density and distribution after weaning. Interestingly, DCs spread in a "wave," which starts at the nasal mucosa and progressively spreads deeper into the trachea, all the way down to the alveolar walls. Also, the degree of maturation or activation seems to correlate with the proximity to the outside environment.

    Not surprisingly, inflammatory stimuli in the inhaled air have a profound impact on these steady-state dynamics. In a series of hallmark experiments, McWilliam and colleagues (59) described how the inhalation of pathogenic material, such as bacteria or viral particles, induced a very rapid influx of DCs into the airways of rats, in some cases peaking already 2 hours after challenge. Remarkably, this DC recruitment was as fast or sometimes ahead of the prototypic neutrophil influx. It was an isolated phenomenon among mononuclear cells because macrophage numbers were not increased and lymphocytes were not present at that time point. This surprising discovery overthrows the classical view of host defense in which a first phase of neutrophilic influx is gradually taken over by a mononuclear infiltrate. It indicates that DC recruitment is an integral part of the early phase of the innate immune response, with the potential to progress toward a powerful adaptive defense.

    Resolution of the inflammation usually restores baseline numbers of pulmonary DCs. However, in some cases, this normalization can be considerably delayed, as observed after Sendai virus infection in rat airways (60), and in a mouse model of respiratory syncytial virus infection in which pulmonary DC numbers were still increased weeks after remission of the acute inflammatory phase (61).

    On a molecular level, isolated respiratory tract DCs were shown to be attracted by a whole array of stimuli, including chemokines, complement cleavage products, and bacterial peptides (62). By far the most studied among these mediators are the chemokines (reviewed in Reference 63). Chemokine concentration gradients elicit a directed cellular movement, a phenomenon known as chemotaxis. In vitro studies show that many cells of the lung produce a wide array of chemokines with a known effect on DCs in general (see Table 1 and References 64eC81), although only a few have been studied in detail with respect to pulmonary DC mobilization. In vivo, different chemokines orchestrate the recruitment of DCs into the lung depending on the inflammatory stimulus present. In a rat model of inhaled heat-killed Moraxella catarrhalis, the phenomenon seemed dependent on the expression of CCR1 and CCR5, which are receptors for the chemokines CCL5 (regulated on activation, normal T-cell expressed and secreted [RANTES]) and/or CCL3 (macrophage inflammatory protein [MIP]-1) produced at the airway level (82). Interestingly, freshly isolated human pulmonary DCs were found to specifically express both CCR1 and CCR5. Meanwhile, chemokines of the monocyte chemotactic protein (MCP) family appear to be important for the recruitment of DCs in a murine model of pulmonary Mycobacterium tuberculosis infection: mice genetically deficient for the corresponding chemokine receptor, CCR2, have a defect in the progressive accumulation of pulmonary DCs after inoculation with M. tuberculosis. This results in a secondary shortage in pulmonary T-cell recruitment, correlating with premature death, compared with wild-type animals (83, 84). In addition to chemokines, the airway epithelium could attract DCs by means of defensins. These are small cationic peptides with bactericidal activity, produced by epithelial cells after contact with pathogenic stimuli (85). Remarkably, human -defensin 2 (which is produced by airway epithelial cells [86]) was shown to engage CCR6, a chemokine receptor typically expressed by immature DCs homing to epithelia (87).

    Finally, nonmicrobial stimuli can also affect the steady-state recruitment of pulmonary DCs. Animals that are housed in specific pathogen-free, yet dusty conditions show an increased influx of DCs into the lung (58). Interestingly, ultrafine particulate matter triggers airway epithelial cells to produce the chemokine CCL20 (MIP-3), a major chemoattractant for CCR6-bearing immature DCs (88). In a model of allergic airway inflammation, CCR6 appears to mediate the recruitment of DCs into the inflamed lung, correlating with a burst of pulmonary CCL20 production a few hours after allergen challenge (89).

    It is not clear yet whether lung DCs are recruited from the blood in a differentiated form or as early precursors. Suda and coworkers (90) have shown that the pulmonary vasculature is enriched with direct DC precursors compared with the peripheral blood circulation. This pool of cells combines a close relationship to the lung tissue environment as well as the potential to differentiate rapidly in strongly immunogenic DCs. Also, the possibility of lung DC population renewal through local proliferation of intrapulmonary progenitors cannot be excluded. It is already known that steady-state pulmonary alveolar macrophage populations are predominantly maintained by in situ cell proliferation, and to a lesser extent by recruitment of monocytic precursors from the circulation (91). Moreover, in the skin, local cell division is the sole mechanism by which LCs appear to regenerate under noninflammatory conditions (92). It is important to note that granulocyte-monocyte colonyeCstimulating factor (GM-CSF), a key growth factor for "DC-poiesis," is expressed in significant amounts in the lung. GM-CSF production in the airways is increased in an inflammatory context (e.g., respiratory viral infection or exposure to environmental pollutants). Wang and colleagues (93) demonstrated how local transgenic overexpression of GM-CSF in the airways can result in a dramatic increase of pulmonary DC numbers. Another important hematopoietic growth factor for DCs is Fms-like tyrosine kinase 3 ligand (Flt-3L) (94). In contrast to GM-CSF, the potential source of Flt-3L within pulmonary tissues has not been mapped. Nevertheless, repeated injection of this cytokine greatly expands DC numbers in the lung parenchyma (95), and conversely, Flt-3LeCdeficient animals have a severe reduction in the number of lung interalveolar DCs, whereas airway mucosal DCs remain unaffected (96). These observations suggest that, similar to DCs in lymphoid organs, lung DCs do not constitute a homogenous population. Rather, lung DCs could fall into separate subsets with possibly different hematopoietic origins.

    In summary, DCs can populate the lung through different mechanisms (Figure 5). DCs present in the blood circulation could be directly recruited by means of appropriate pulmonary chemokine signals. Alternatively, monocytic DC precursors could first be chemoattracted from the blood into the lung and subsequently differentiate into DCs under the influence of cytokines secreted by resident pulmonary cells (e.g., GM-CSF produced by respiratory epithelium). In addition, a hypothetical mechanism for maintaining lung DC homeostasis might be proliferation of putative intrapulmonary DC progenitors, induced by growth factors secreted by resident pulmonary cells (GM-CSF or Flt-3L). Finally, indirect evidence points to the possibility of transdifferentation of pulmonary macrophages into DCs under the influence of GM-CSF (93).

    Lung DCs Continuously Report Antigenic Information from the Airways to Pulmonary LNs

    One of the most crucial specialized functions of DCs is the capture and delivery of antigen to local lymphoid tissues. These are the privileged sites for potential encounters between APCs and antigen-specific lymphocytes (i.e., the basis for mounting an adaptive immune response). The dense superficial network of airway mucosal DCs is ideally positioned for the interception of any potential antigen in the inhaled air. In addition, pulmonary DCs possess the necessary molecular equipment for efficient sensing and sampling of a wide variety of airborne antigens. Receptors of the C-type lectin family are an important family of molecules in this respect (97). These transmembrane sugar-binding proteins act as "pathogen recognition receptors," recognizing carbohydrate motifs present on the surface of several microbial organisms. In addition, they deliver captured antigens to endocytic vesicles for further processing and routing to the MHC class II presentation pathway. Several C-type lectins have been described on pulmonary DCs. Cochand and colleagues (56) revealed a high expression of mannose receptor on lung parenchymal DCs, endowing these cells with a robust endocytic capacity for mannosylated antigen. It is known that several important lung pathogens, such as M. tuberculosis, Pneumocystis carinii, as well as many fungi, display mannose sugars on their surface. DC-SIGN (DC-specific intercellular adhesion molecule [ICAM]-3eCgrabbing nonintegrin), a newly described C-type lectin, appears to be the principal molecule through which M. tuberculosis enters pulmonary DCs (98). DC-SIGN also has affinity for galactomannans present on the cell wall of Aspergillus fumigatus (99). Consequently, binding and internalization of A. fumigatus conidia by DCs correlate with DC-SIGN expression on the surface of these cells. Other lectin-type receptors that have been documented on subsets of lung DCs are Langerin (43, 51), BDCA-2 (51), and, at least in the mouse, DEC-205 (100). However, their function with respect to specific antigen recognition and endocytosis is currently poorly defined. Additional receptors with a role in antigen uptake have been detected on pulmonary DCs: these include IgG-Fc receptors, for the capture of antibodyeCantigen complexes, and C3bi-R (or Mac-1), which binds opsonizing complement fragments (4).

    Using a mouse model, we have shown that antigen uptake by airway DCs can proceed without any breach to the mucosal barrier, and in the absence of any inflammatory stimulus (101). This is surprising given that DCs of mouse intrapulmonary airways are predominantly located underneath the airway epithelial basement membrane, and that these experiments involved administration of large (i.e., nonpermeant) macromolecules in the airways using a rigorously nontraumatic and sterile instillation technique. This could point to an antigen capture mechanism as described in a recent study on the gut mucosa: indeed, despite their subepithelial location, DCs in the intestinal lamina propria are perfectly capable of capturing noninvasive antigens by extending interepithelial pseudopods toward the lumen without disrupting the epithelial barrier's integrity (102).

    The instillation of inert fluorescent macromolecules into the airways enabled us to track DCs migrating and transporting antigen from the airway mucosa toward pulmonary LNs (Figure 6) (101). This transport was rapid, occurred constitutively (i.e., in the absence of inflammation), and the migratory DCs were seen to specifically penetrate the T-cell zones of the mediastinal lymph nodes (MLNs). These observations confirm earlier studies suggesting that the steady-state recruitment of DCs into the lung is balanced by a continuous emigration of these cells to the MLNs (57). Also, intratracheal administration of soluble protein antigen results in the appearance of strong antigen-presenting activity in the draining MLNs, with similar kinetics (103). Analogous conclusions were reached using intratracheal transfer of in vitroeCderived DCs (104, 105), although these cultured DCs might not reflect the exact nature of the endogenous pulmonary DC populations in terms of phenotype, activation level, and antigen-uptake capacity.

    Much research has been devoted to the molecules that help DCs "navigate" toward the T-cell areas of local LNs. Insights into the mechanisms involved might lead to therapeutic strategies in which elicitation of inappropriate immune responses could be averted by blocking antigen transport to LNs. Alternatively, this knowledge could lead to improved vaccine design by optimizing DC homing to LNs. In general, activated DCs use the chemokine receptor CCR7 to guide themselves along chemokine gradients within afferent lymphatic vessels, all the way down to the LN's T-cell areas (106). The CCR7-triggering chemokines expressed in these structures are CCL21 (= SLC [secondary lymphoid organ chemokine]) and CCL19 (= MIP-3) (107, 108). Surprisingly little is known regarding the involvement of this molecular network in the trafficking of lung DCs. In one report, pulmonary CCL21 expression was detected in a peribronchial and perivascular pattern, with virtual absence in the alveolar zones (109). This pattern probably corresponds to the distribution of the deep pulmonary lymphatic vessel plexus and might delineate the migratory route of LN-homing lung DCs. Accordingly, in vivo neutralization of CCL21 could prevent DC homing to pulmonary LNs and the subsequent triggering of a T-celleCdriven immune response (110). In addition to chemokines, lipid metabolites such as leukotrienes and prostaglandins are emerging as important upstream controllers of DC migration toward LNs. It was shown that DCs require the presence of the Cys-leukotriene LTD4 in the immediate extracellular space to be responsive to chemoattraction by CCL19 (111). Prostaglandin E2 (PGE2) produced by epithelial cells after antigen exposure can also stimulate DC emigration toward draining LNs (112, 113). In contrast, PGD2 exerts an opposite effect (114): a recent study showed that PGD2 could inhibit the emigration of airway DCs toward MLNs and consequently prevent the induction of a primary immune response. The same effect was obtained with pharmacologic agonists of the peroxisome proliferator-activated receptor  (PPAR-), an important intracellular mediator of prostaglandin signaling (115, 116).

    As mentioned in the INTRODUCTION, the presence of an inflammatory process at antigen-exposed surfaces is a strong stimulus for the migration of antigen-transporting DCs toward LNs. Transposed to the lung, this would result in an increased flow of antigenic information from the airways toward the MLNs. We have studied this phenomenon in a model of allergic airway disease, and found that ongoing airway inflammation causes a massive and accelerated flux of allergen-transporting DCs from the airway mucosa to the MLNs (117). This is probably due to the intense release of DC-activating inflammatory molecules, such as tumor necrosis factor  (TNF-) and prostaglandins, during allergic airway inflammation. Whether this phenomenon contributes to the amplification of the allergen-driven immune response remains to be investigated.

    DCs Translate Signals from the Pulmonary Environment into a Specific Immune Response

    After arriving into the LNs, DCs face their last and most important task: that is, instruct T cells to respond to presented antigen in the most adequate way. The type and activation state of the DC, the dose of antigen, as well as the nature of concomitant environmental factors present at the time of antigen encounter determine the nature of the resulting T-cell response. Currently, three different possible outcomes for effector T cells are distinguished. A T-helper 1 (Th1) response is characterized by the production of IFN- and TNF by T cells. It is the normal outcome after DC exposure to viruses or bacteria and is crucial for the control of intracellular pathogens such as Mycobacterium spp. It is also the basis of the delayed type hypersensitivity reaction. Th2 differentiation usually occurs after contact with extracellular parasites (e.g., helminths) and involves production of cytokines such as interleukin 4 (IL-4), IL-5, IL-9, and IL-13, resulting in IgE production as well as accumulation of eosinophils and mast cells. Allergens are nonpathogenic environmental antigens that elicit an inappropriate Th2 response. A third main outcome is the induction of tolerogenic or regulatory T cells producing immunosuppressive cytokines, such as IL-10 or transforming growth factor  (TGF-). This is probably the most prevalent response in steady-state conditions. It forms a constant safeguard against the emergence of inappropriate inflammatory reactions to harmless antigen. The way this paradigm can be transposed to the lung's immune homeostasis is summarized in Figure 7.

    It has long been proposed that the airway mucosa is inherently Th2-biased, providing a counterbalance to potentially tissue-damaging Th1 reactions to harmless inhaled antigen. A few studies were aimed at demonstrating the involvement of pulmonary DCs in this phenomenon. Lung DCs were found to produce cytokines such as IL-10 and IL-6, which have often been considered as Th2-skewing (although this remains controversial). In addition, pulmonary DCs showed an impaired ability to produce the Th1-inducing cytokine IL-12 (118, 119). However, a DC-promoted Th2 climate does not necessarily imply protection from inflammation. Lambrecht and colleagues (104) showed that antigen-loaded DCs transferred into the airways of healthy animals migrate to the MLNs and evoke a highly inflammatory Th2-type sensitization. This experiment left open the question concerning the role of the lung's own DCs in the control of the default pulmonary immune response. The latter is more and more regarded as strongly tolerogenic or regulatory by nature, thereby ensuring the protection of the delicate gas exchange structures from excessive inflammation, be it Th1- or Th2-driven. This raises two important questions: (1) Can the induction of tolerance or immunity to inhaled antigen be assigned to specific DC subtypes and (2) What are the molecular mechanisms involved in this process Recently, pDCs in the lung have been described as important actors in the maintenance of tolerance to inhaled antigen (120). This property was attributed to these cells transporting antigen to the thoracic LNs while maintaining low levels of T-cell costimulatory molecules, and high levels of the inhibitory molecule PD-L1. As a result, pulmonary pDCs failed to trigger antigen-specific T-cell proliferation and production of effector cytokines, but induced immunosuppressive T cells instead. Meanwhile, several studies support a concept of duality in pDC function: in baseline conditions, pDCs would exist in a quiescent state with weak immunostimulatory capacity and the ability to induce tolerance, whereas after appropriate stimuli (most notably viral exposure), pDCs would switch to an immunogenic state characterized by an impressive production of type I interferon (121). This paradigm sheds a new light on the recent observation that a totally inert and tolerogenic protein can become immunogenic when coinhaled with a respiratory virus (122).

    Nevertheless, induction of tolerance in the lung is unlikely to be the exclusive function of pDCs. Mice deficient for Flt-3L, a key growth factor for pDCs, show no impairment in the development of tolerance to harmless inhaled antigen. In addition, the mDCs that transport protein antigen to the MLNs produce IL-10 and express high amounts of inducible costimulator ligand (ICOS-L), both of which lead to the induction of antigen-specific regulatory T cells with the power to inhibit airway inflammation (123, 124).

    As expected, the presence of microbial organisms in the lung modifies the outcome of antigen presentation by DCs in the pulmonary LNs. As an example, a single pulmonary delivery of influenza virus leads to a transient amplified flux of DCs toward the MLNs, generating influenza-specific CD8+ T cells that produce large amounts of IFN- (125). Heat-killed Listeria monocytogenes (126) or Aspergillus fumigatus (127) delivered into the airways are also rapidly transported to the MLNs by DCs, leading to the priming of CD4+ T cells. Remarkably, the pulmonary DC is able to discriminate among different fungal components: phagocytosis and processing of Aspergillus conidia induces a Th1 response, whereas uptake of hyphae results in Th2 polarization.

    The immune response initiated by pulmonary DCs is not only preconditioned by stimuli from the inhaled air. The lung's own cells provide numerous molecular signals that are known to affect DC function in some way. Significant insight has been gained regarding the coexistence of pulmonary DCs and macrophages. In the lung parenchyma, macrophages in the alveolar lumen and DCs within interalveolar septa are separated by a fraction of a micrometer. It has repeatedly been reported that alveolar macrophages inhibit DC functions through the production of soluble mediators (128). Alveolar macrophageeCderived nitric oxide was shown to inhibit MHCII expression on lung DCs and suppress their T-cell stimulatory activity (129). Other DC-inhibiting factors secreted by alveolar macrophages include prostaglandins, H2O2, TGF- (130), and IL-10 (131), as well as decoy receptors for IL-1 and TNF (132). Together, these data suggest that pulmonary macrophages exert their well-known immunomodulatory function through an important restraining influence on DCs. In this view, noninvasive aeroantigens could be taken care of by scavenging macrophages crawling over the airway surface, and these cells would produce mediators that prevent the triggering of DC activation. However, damage to the macrophageeCsurfactanteCepithelium barrier would allow antigen to reach deeper sentinel DCs and would shift the local cytokine environment in favor of DC activation and the initiation of an adaptive immune response. Intriguingly, interstitial macrophages can exert a supportive influence on pulmonary DC immune function by preprocessing particulate antigen into smaller peptides that are then loaded on the surface of neighboring DCs (133, 134).

    In contrast to DCeCmacrophage dialogs, the interaction of pulmonary DCs with other prominent cells of the lung has been less studied so far. Nevertheless, airway and alveolar epithelial cells, fibroblasts, mast cells, nerve endings, and lymphocytes all express mediators that are generally known to affect the immunomodulatory function of DCs. The putative interactions of all these pulmonary cell types with DCs (summarized in Figure 8) are a vast and largely unexplored research terrain with important therapeutic implications.

    DCs IN HUMAN LUNG DISEASE: FRIEND OR FOE

    Given the DC's pivotal role in controlling pulmonary immunity, it follows that any aberration in DC function can have a considerable clinical impact. This section reviews the emerging knowledge on the role of DCs in several major lung pathologies.

    Allergic Asthma

    Asthma is a chronic inflammatory disorder of the airways featuring bronchial smooth muscle hyperreactivity, congestion of the airway mucosa, and excessive mucus secretion. This results in characteristic symptoms of wheezing, coughing, and dyspnea. These manifestations are typically variable and the airflow limitation is often reversible. Importantly, there is a relentless increase in asthma incidence throughout the world (www.ginasthma.org).

    The last decades have seen a growing insight in the cellular and molecular mechanisms underlying allergic asthma. Cutting-edge experimental research has progressively shifted the focus from downstream effectors, such as mast cells, IgE, and eosinophils, up to the Th2 lymphocytes and their proallergic cytokine products (e.g., IL-4, IL-5, IL-13) (135). More recent research by our group and others has implicated the DC as an even more upstream instigator in the allergic inflammatory cascade. Introduction of antigen-loaded DCs into the airways of healthy animals is sufficient to induce allergic sensitization to that antigen (104). Conversely, selective elimination of DCs during ongoing allergic airway inflammation results in the virtual disappearance of the inflammatory symptoms (54). Importantly, other APCs in the lung, such as macrophages or B cells, are unable to take over the fundamental role of DCs in the initiation or maintenance of the eosinophilic inflammation (136, 137).

    Experimental studies point to several properties of lung DCs that could account for their crucial role in the maintenance of Th2-based inflammation to inhaled antigen. First, DCs are recruited in massive amounts to the lung during allergic airway inflammation. At the plateau of eosinophilic inflammation, recovery of DCs from BALF is increased 30- to 100-fold (117, 138eC140). This increase is supported by a proliferation of early DC precursors in the bone marrow (140), suggesting possible bone marroweCstimulating signals emanating from the inflamed airways, as was already reported for eosinophils (141). Second, DCs within inflamed airways are unusually activated compared with DCs from healthy lungs: cell surface expression of MHCII and T-cell costimulatory molecules is markedly enhanced, whereas levels of ICOS-L, a molecule involved in the induction of inhalation tolerance (142), are decreased (117). In addition, DCs are the chief producers of the chemokine CCL17 in the lung (139). CCL17 ("TARC" [thymus- and activation-regulated chemokine] in the old nomenclature) is a molecule that specifically attracts Th2 lymphocytes toward APCs (143). The interaction of airway DCs with these allergen-specific T cells leads to reciprocal activation and precedes the appearance of the late-phase asthmatic response (144). Seen as a whole, the local DC activation within the airways, together with the immediate exposure to aeroallergen and the concomitant recruitment of airway T and B cells, could provide an adequate microenvironment for an in situ perpetuation of the chronic airway inflammation. In one report, it was even suggested that the chronicity of allergic airway disease could be attributed to the existence of an exceptionally long-lived subset of allergen-presenting DCs in the BALF (145).

    As would be expected from these data, molecular defects that impair the mobilization of DCs into the lung during allergen exposure should lead to diminished airway inflammation. We and others have identified matrix metalloproteinase 9 (MMP-9) as a critical molecule enabling the entry of DCs into the airways (139, 146). MMP-9 is an MMP that breaks down basement membranes and allows migratory cells to pass from one tissue compartment to another. MMP-9 deficiency impairs DC accumulation after allergen exposure, resulting in a local shortage of CCL17 and ultimately a collapse of the allergic airway inflammation. A summary of experimental insights concerning the role of DCs in the sensitization and maintenance of allergic airway inflammation is provided in Figure 9.

    The relative contribution of mDCs versus pDCs in the pathogenesis of allergic airway disease is a relatively recent subject of debate. Originally, pDCs were termed "DC2," based on a Th2-polarizing character. However, this Th2 effect was probably an artifact of specific in vitro culture conditions (147). It is now clear that, depending on the stimulus, pDCs can initiate Th1- and Th2-biased responses alike (148). It was recently shown that pulmonary pDCs possess some "antiallergic" properties: selective in vivo elimination of pDCs from healthy lungs unleashes severe allergic responses to otherwise tolerogenic inhaled antigen (120). There is additional evidence supporting a potential antiallergic effect of pDC function. It has been shown that administration of CpG oligonucleotides can provide protection from allergic airway inflammation, in a way that is mostly independent of Th1 cytokine production (149). CpG oligodeoxynucleotides are typically known as powerful activators of pDCs, because these cells express the corresponding TLR (TLR9). In vitro studies show that CpG-stimulated pDCs can inhibit Th2 cell proliferation via IFN-/ production (150). These experimental data also suggest that immunomodulatory molecules emerging as antiasthmatic therapies primarily achieve their effect by targeting lung DCs.

    Research on human subjects has provided some important insights on the function of DCs in asthma as well. In patients with asthma, allergen challenge causes a sharp decline in the number of circulating blood mDCs within a few hours (151). During the same timeframe, a rapid accumulation of mDCs occurs within the bronchial mucosa (152). This suggests that, in subjects with asthma, an alteration occurs in the lung's chemokine networks, allowing a rapid entry of blood-borne mDCs into the allergen-exposed lung (pDCs do not seem to be affected in the same way in these studies). Recently, the acute drop in circulating mDCs after allergen challenge was shown to be inhibited by treatment with the cysteinyl leukotriene 1 (CysLT1) receptor antagonist pranlukast (153). This was associated with a decreased production of the DC-attracting chemokine CCL20 (measured in induced sputum), and consistent with the expression of the CysLT1 receptor on circulating mDCs. Earlier studies using bronchial biopsies had already documented an increase in the number of intraepithelial airway DCs in subjects with asthma compared with healthy individuals, and this number returned to control levels after inhaled corticosteroid treatment (it is known from animal studies that glucocorticosteroids induce apoptotic depletion of the airway DC network [53]) (154). A functional comparison between bronchial epithelial DCs isolated from healthy atopic versus atopic donors with asthma yielded interesting results: the respiratory epithelium of subjects with asthma was strongly enriched with CD1a+ DCs, and these cells preferentially induced the production of IL-4 and IL-5 from sensitized autologous Th cells (50). Also, CD1a+ airway mucosal DCs from patients with asthma displayed increased surface levels of FcRI, the high-affinity receptor for IgE, which could help DCs collect IgE-coated allergen on their surface (155).

    Additional studies using human blood as a starting material further confirm that DCs from patients with asthma or allergy are "altered" in a way that secures a perpetuation of the inappropriate Th2 response. The possible role of environmental agents in this phenomenon is the subject of intense research. Diesel exhaust particles have often been proposed as Th2-inducing "adjuvants," and it was recently shown that these pollutants can establish a Th2 milieu by influencing both the DC as well as the responder T cell (156). In addition, diesel exhaust particles cause inflammation at the level of the airways by means of reactive oxygen species (157). This would result in DC activation and thus amplify DC-mediated antigen transport toward MLNs, where antigen would be presented in the context of the Th2-biasing signals. More recently, pollen-derived isoprostanes were shown to activate DCs and turn them into Th2 inducers (158). Additional indoor sources of proallergic factors have also been identifed, such as house dust mites thriving in overinsulated houses or cockroach allergens in inner city areas. Der p-1, a protein found in fecal pellets of the house dust mite Dermatophagoides pteronyssinus, has interesting properties with respect to DC biology. This molecule exhibits proteolytic activity and was shown to cleave epithelial tight junction proteins (159), thereby facilitating the delivery of antigen to intra- or subepithelial DCs. Meanwhile, the disturbance in the epithelial integrity would constitute a danger signal and lead to DC activation. Also, bronchial epithelial cells produce several DC-attracting chemokines after exposure to Der p-1 (160). In several studies, human bloodeCderived DCs were exposed to house dust miteeCderived proteins in vitro. From these investigations, it appears that only DCs obtained from atopic subjects exhibit a proallergic function on exposure to house dust mite protein, whereas DCs from healthy subjects induce no T-cell polarization or even maintain tolerogenic properties (110, 161, 162). Intriguingly, only DCs from atopic individuals secrete high levels of the Th2-attracting chemokines CCL17 and CCL22 after Der p-1 exposure (163). The reason why DCs from atopic individuals are "hard-wired" to preferentially induce and attract proallergic T cells is still obscure. To date there is virtually no evidence for genetic polymorphisms determining the T-celleCpolarizing function of DCs in atopy. Much more research is addressing the question of how environmental influences can help establish an inappropriately Th2-oriented immune climate, sometimes for life. Further exploration of this issue from a DC standpoint might provide a mechanistic basis to the so-called hygiene hypothesis for the increasing prevalence of allergy. This theory proposes that the immune system of infants needs Th1-inducing stimuli (typically provided by microbial exposure) to evolve from an immature, Th2-biased immune response. The more "aseptic" Westernized urban lifestyle, where use of antibiotics and vaccines is widespread and orofecal pathogen burden is low, implies a shortage of such Th1-promoting triggers, thus creating an allergy-prone situation. Alternatively, inappropriate Th2 responses might arise from a failure of tolerogenic mechanisms, rather than from a lack of Th1 stimuli: Wills-Karp and coworkers (164) recently proposed a refinement to the hygiene hypothesis, in which a continuous low level of microbial stimulation (presumably from gut commensals) would ensure a tolerogenic climate dominated by the production of the immunosuppressive cytokine IL-10. Absence of such "counterregulatory" stimulus would thus remove an important inhibition on inappropriate Th2 (allergic) or even Th1 (autoimmune) immune responses.

    There are some interesting elements pointing to the immediate relevance of DCs to the hygiene hypothesis, although many aspects of this complex relationship still need to be resolved. The "immature" character of mucosal immunity in early life might be attributed to the state of the DC system during that period: compared with the adult situation, DCs within neonatal airways are not only less numerous and less mature in terms of MHC expression (58) but also hyporesponsive to inflammatory stimuli (165). In addition, DCs from neonates show an impaired production of IL-12, leading to a defective capacity to induce Th1 responses (166). Holt (167) suggested that a delay in the postnatal acquisition of Th1 competence could explain the difference between atopic and nonatopic individuals. However, in line with the ideas put forth by Wills-Karp and colleagues, we argue that chronic low-level exposure to microbial stimuli would help mucosal DCs evolve toward a tolerogenic rather than a Th1-inducing function in the first place. From that point on, tolerogenic DC networks at mucosal surfaces would constitute the prime sensors for any additional Th1- or Th2-inducing environmental signals, acting alone or in combination. As an experimental illustration of this concept, Eisenbarth and coworkers (168) showed how inert protein antigen coinhaled with low levels of LPS endotoxin triggers airway DCs to induce a Th2 rather than a tolerogenic antigen-specific response. In contrast, coinhalation of high doses of LPS led to a shift from a Th2- to a Th1-biased reaction (168). The modulation of DC-induced airway allergic responses by endotoxin was also confirmed in a report by Kuipers and colleagues (169). These experimental findings are in line with epidemiologic data in which exposure to house dust endotoxin was found to decrease allergic sensitization in infants at high risk of developing asthma (170). In another study, instillation of Dermatophagoides farinae proteineCexposed DCs into the airways of healthy mice led to allergic sensitization to that antigen. However, prior coexposure of the DCs to D. farinae extract together with respiratory syncytial virus shifted the pulmonary immune response in vivo from Th2 toward Th1 (171). It should be noted that this experiment involved exposure of mature DCs to respiratory syncytial virus. Primo-infection with respiratory syncytial virus during early infancy, a time when airway DCs are defective in Th1 polarization, is known to establish a Th2-biased climate in the airways, increasing the risk for the development of asthma in later life.

    Next to environmental stimuli, a number of studies have stressed the importance of tissue-derived factors in the conditioning of DCs toward a proallergic function. PGE2, which can be produced by airway epithelial cells, suppresses IL-12 production by DCs, leading to the development of IL-4eC and IL-5eCproducing T cells (172). Histamine, the prototypic mediator released by mast cells, triggers human DCs to induce Th2 immune responses as well (173), thus creating the possibility of a positive feedback loop involving IgE and activated mast cells (174). This finding sheds new light on the results of a large prospective study, in which intake of an antihistamine during infancy significantly decreased the risk of developing asthma in a subset of allergen-sensitized children (ETAC [Early Treatment of the Atopic Child] study [175]).

    GM-CSF, another product of the airway epithelium, is not only a DC differentiation factor but also a strong Th2-polarizing cytokine in vivo (176). This might explain why in vitroeCgenerated DCs (using an abundance of GM-CSF in the culture medium) induce allergic sensitization after intratracheal transfer, whereas endogenous airway DCs are tolerogenic by default. Thymic stromal lymphopoietin (TSLP), an IL-7eCrelated cytokine produced by epithelial cells and mast cells, is one of the most powerful triggers to date for DC-dependent allergic immune responses in humans (177). TSLP-exposed DCs secrete high amounts of the Th2-attracting chemokines CCL17 (TARC) and prime naive Th cells to produce proallergic cytokines (IL-4, IL-5, IL-13). Although TSLP is expressed in the normal lung and overexpressed in the skin of patients with atopic dermatitis, TSLP levels in asthmatic lungs have not been investigated yet, something that could have important therapeutic implications. Equally important is knowing which environmental factors might trigger an overproduction of TLSP by pulmonary cells.

    In summary, there is compelling evidence to conclude that DCs play a pivotal role in the pathogenesis of allergic asthma. Experimental data indicate that DCs are obligatory APCs for the initiation and maintenance of allergic airway inflammation. In humans, the presence of asthma is associated with an increase in DC numbers in the bronchial mucosa. This increase can be caused by a strikingly rapid movement of circulating DCs into the lung tissue after allergen exposure. Meanwhile, DCs from allergic individuals seem to lose their tolerogenic character and instead perpetuate the allergic state through the expression of costimulatory molecules, cytokines, and chemokines that favor Th2 responses.

    Chronic Obstructive Pulmonary Disease

    In contrast to allergic asthma, the role of DCs in chronic obstructive pulmonary disease (COPD) has been much less investigated. COPD encompasses two clinical entities, each of which correlates with specific histopathologic features. On one hand, there is chronic obstructive bronchiolitis characterized by inflammatory infiltrates surrounding peripheral airways, leading to poorly reversible airflow obstruction (www.goldcopd.com). On the other hand, there is emphysema, which involves a destruction of interalveolar septa with a progressive decrease of the gas exchange surface. The inflammatory infiltrate in COPD contains mainly neutrophils, CD8+ (cytotoxic) T cells, and monocytes/macrophages (reviewed in Reference 178), a combination of cells that has not been fitted into a clear pathogenetic mechanism yet (in contrast to the Th2 lymphocyte/eosinophil presence in asthma).

    Recently, we documented a relentless increase in lung DCs in a murine model of tobacco smokeeCinduced pulmonary emphysema. After 6 months of smoke inhalation, DC numbers in the BALF were 10-fold greater than those observed in control animals (25). An earlier study by Zeid and Muller (179) documented a similar increase in pulmonary Langerhans-like cells on tissue sections of tobacco smokeeCexposed mice. In contrast to these studies, Robbins and coworkers (180) reported a decrease in pulmonary DC numbers after smoke inhalation. The reason for these divergent observations can be attributed in part to the methodology used for the detection of DCs and also to large differences in the smoke exposure regimen (a relatively low dose was used in the latter study).

    In humans, increased numbers of CD1a+ DCs were detected in the airway mucosa of patients with COPD and these numbers declined after fluticasone proprionate inhalation (181). It should be noted that smoking by itself induces an increase in DC numbers in the airways, regardless of the presence of COPD (182, 183). The association of COPD with smoking suggests an innate inflammatory response to inhaled toxic components. It is very likely that the airway DC network is acutely sensitive to smoke inhalation: tobacco smoke contains many potential DC-activating components such as reactive oxygen species and endotoxin. With respect to the latter, we have recently observed a TLR4-dependent activation of pulmonary DCs after smoke inhalation (TLR4 is the signaling receptor for LPS; Maes and colleagues, unpublished manuscript, and Reference 26). In addition, tobacco smoke could trigger airway epithelial cells to release DC-activating factors, such as TNF-, GM-CSF, or heat-shock proteins. It is tempting to speculate that the smoke-activated DCs would lose their default tolerogenic character and induce a sustained immune response to otherwise harmless antigens present in the tobacco smoke (e.g., tobacco glycoprotein). Alternatively, intracellular components released through toxic damage could be chemically altered by smoke and become immunogenic. DCs would then take up, process, and present these modified intracellular antigens to CD8+ cytotoxic T cells. The dangerous combination of activated DCs and an abundance of these "new" antigens could set the stage for a self-perpetuating, tissue-directed immune response with devastating consequences in the long term. This acquired immune response could form the basis for the relentless progression of inflammation observed in patients with severe COPD even after smoking cessation. This disturbing phenomenon was revealed in a study by Retamales and coworkers (184), but was rather attributed to latent adenoviral infection of lung epithelial cells, the degree of which was correlated with emphysema severity and associated with persistent inflammatory cell recruitment despite cessation of smoking. Chronic viral infection could indeed provide a plausible explanation for the relative increase in CD8+ T cells in the airway infiltrates of patients with COPD. Interestingly, smokers with airway obstruction have elevated levels of a specific latent adenoviral DNA sequence compared with smokers without obstructive disease (185). In addition, CD8+ T cells were shown to mediate cytotoxic damage to airway epithelial cells when these were expressing a viral antigen (186). Here again, DCs are known to be required for the initial sensitization of T cells to the viral antigens, either through direct infection of the DCs themselves or after uptake and processing of fragments from infected epithelium (187).

    There are some tantalizing hints suggesting a possible role of DCs in the development of emphysema itself. An indirect indication is that LC histiocytosis (LCH) of the lung, which is also associated with smoking and involves a pathologic accumulation of LCs, is accompanied by destructive lesions in the alveolar zones. On a cellular and molecular level, it has been shown that, during the differentiation from monocytes, DCs show an impressive upregulation of transcripts for human macrophage elastase or MMP-12 (188). MMP-12 appears to be crucial for the induction of emphysematous changes in an animal model of tobacco smoke inhalation (189). In addition, transgenic animals with inducible TNF- have an increase in pulmonary MMP-12 levels and spontaneously develop emphysema (190). TNF- is increased in the sputum of patients with COPD (191) and is a strong activator of DC maturation and trafficking (192, 193). Recently, BALF macrophages from patients with COPD were found to display an increased elastolytic activity in vitro, together with increased expression of MMP-12 mRNA. However, as often in similar studies, BALF cell differentiation is performed on a simple morphologic basis, which precludes any accurate distinction between macrophages and DCs. An interesting observation is that elastin fragments generated through the proteolytic activity of MMP-12 are strongly chemotactic for monocytes (194). An interesting hypothesis would be that the continuous inflammatory transit of DCs through elastin-rich alveolar regions maintains or even amplifies itself by means of chemotactic degradation products. As an indication for such a phenomenon, DC migration into inflamed lung interstitium and BAL compartment is decreased in MMP-12eCdeficient mice (our unpublished observations).

    Further studies in animal models will be necessary to determine whether the pathogenesis of COPD relies in substantial amount on a pathologic response of DCs to tobacco smoke components. These investigations might lead to the discovery of several subentities within COPD, each based on the relative contribution of innate versus adaptive pathologic immune responses in the lung. It is hoped that these insights will help to extend the field of research toward novel targets in COPD treatment.

    Pulmonary LCH

    LCH is a disease characterized by an abnormal accumulation of Langerhans-like cells in one or several organs. Nowadays, the term LCH is used to encompass several clinical entities distinguished on the basis of organ involvement (solitary bone lesions in eosinophilic granuloma, as opposed to systemic involvement in Hand-Sche筶ler-Christian disease, Letterer-Siwe disease, and histiocytosis X). The link between histiocytosis X and Langerhans cells was suggested decades ago by the finding of Birbeck granules in the "histiocytes" accumulating in these lesions (195). The first detailed account of pulmonary involvement was published by Auld (196) in 1957. Classical LC markers, such as CD1a, S-100, and Langerin, are used for the immunohistologic diagnosis of LCH (Figure 10). Langerin has even been suggested as a diagnostic tool for the detection of LCs in the BAL of patients with pulmonary LCH (197). In addition to LCs, LCH granulomas contain macrophages, lymphocytes, and eosinophils (hence the occasional name of "eosinophilic granuloma").

    The disease initially involves granulomatous infiltrations around distal airways. Progression into alveolar zones leads to cystic lesions surrounded by a fibrous reaction. In contrast to the systemic forms of LCH, which typically develop in children, pulmonary LCH is mainly seen in adults and shows a clear association with smoking. Although pulmonary LCH is a rare disease (precise data on prevalence are currently unavailable), a dissection of the pathogenetic mechanisms involved could lead to useful insights on the homeostasis of DCs in the lung in general. There is still much speculation concerning initiating triggers. Tobacco smoke in itself induces an increase in LC numbers in the airway epithelium (182). However, the transition to unbridled accumulation of LCs likely requires additional factors, which could be genetic predisposition, acquired mutations (allelic loss at the level of tumor suppressor genes has been described in LCH lesions [198]), or maybe another environmental trigger, such as viral infection. The accumulation of LCH cells could be due to a local proliferation of putative LC precursors (199, 200), a proliferation that could even be clonal in nature (201). Nevertheless, LCH lacks true features of neoplasia because proliferation rate is very slow (202), lesions are often spontaneously remissive, and LCs are virtually absent from end-stage fibrocystic lesions. Alternatively, LCH lesions could grow through continuous recruitment of new cells. Indeed, the LCs of LCH were shown to express CCR6 as well as its ligand CCL20 (MIP-3) (203): this could imply a paracrine and autocrine mechanism for a self-perpetuating attraction and retention of LCs into the granuloma.

    There is still debate concerning the activation/maturation status of LCs in LCH lesions. In one study, LCH cells were described as having low levels of CD83 and B7-2, and weak T-cell stimulatory capacity (204). Combined with overexpression of CCR6, these findings suggest an immature phenotype, except in self-healing LCH lesions where the LCs appeared mature (204). The immature character of LCH cells seemed to correlate with the presence of numerous IL-10eCsecreting cells in the direct tissue environment. However, these observations were made in bone and skin LCH lesions; similar investigations performed in the lung show that LCs in pulmonary LCH are clearly mature, with very high expression of B7-1 and B7-2 (205). The mature/activated state of pulmonary LCH cells was clearly supported by the local presence of a DC-activating cytokine milieu with elevated IL-1 and low levels of IL-10. A robust increase in GM-CSF production has been documented in LCH lesions (206), whereas increased TNF- has been described in extrapulmonary LCH (207). Notably, both cytokines are known to be critical for the hematopoietic development of LCs (208). A recent study reported elevated levels of Flt-3L and stem cell factor (SCF) in the serum of patients with LCH (209), a combination of hematopoietic growth factors that was shown to sustain long-term expansion of primitive DC precursors (210). In addition, the abundant production of TGF- around LCH lesions (211), combined with GM-CSF, might allow freshly recruited monocytes to differentiate locally into LCs and thus contribute to lesion growth (212).

    It is still unclear why granulomatous LCH lesions at the level of the bronchioles evolve into fibrocystic scars in the alveolar zones. Additional studies on MMP production by LCH cells might shed some light on the process and possibly provide a link with the development of emphysema in smokers. Hayashi and colleagues (213) described overexpression of the collagenolytic MMP-2 in pulmonary LCH cells. Also, the cytokines that are overexpressed in LCH lesions are known to be strong inducers of MMPs in cells of monocytic/dendritic/macrophage lineage (214, 215). The role of non-LCs in LCH lesions is also worth examining. In particular, the interaction between LCH cells and T lymphocytes could be an important factor in the maintenance of the disease. Tazi and coworkers (205) reported a recruitment of CD40L-positive (i.e., activated) T cells around CD40-positive LCH cells, whereas T cells in uninvolved lung tissue were uniformly CD40L-negative. This finding, which was confirmed in pediatric cases of LCH (216), strongly suggest stimulatory, bidirectional interactions between LCH cells and T cells, possibly involving presentation of an as yet unidentified antigen. Tobacco glycoprotein might be a suitable candidate for driving the immune response in smokers with LCH. However, it was shown that lymphocytes from patients with LCH are hyporesponsive when stimulated with tobacco glycoprotein (217). In addition, engagement of CD40 on DCs or LCs leads to activation and cytokine production, even in the absence of cognate interactions (218). Hence, the presence of environmental or perhaps tissue-derived antigens may not be an absolute requirement for maintaining reciprocal LCeCT-cell activations in LCH granulomas. Another aspect of LCH is the frequent recruitment of eosinophils, suggesting some Th2-promoting mechanism. It would be interesting to examine whether TSLP, as a strong DC activator and inducer of Th2 responses, might be involved in the disease. It was recently shown that TSLP synergizes with CD40 engagement on DCs, leading to the induction of IL-5eC and IL-13eCsecreting cytotoxic T cells (219).

    As a whole, LCH remains a complex mix between reactive and neoplastic pathology. Nonetheless, ongoing investigations should continue to borrow knowledge gained in the field of experimental LC biology, in a process that will likely lead to promising therapeutic options for this puzzling disease.

    Lung Cancer

    Cancerous lesions elaborate an arsenal of tricks to evade immune defense: as such, they are perhaps the biggest challenge for the DC system. An insight in the way pulmonary malignancies affect the normal biology of DCs can help determine how best to improve the efficacy of cancer immunotherapy (however, it is by no means the purpose of this article to discuss the proliferation of DC-based cancer vaccines that are currently being developed or even clinically tested). Recent studies show that vascular-endothelial growth factor (VEGF), an important molecule for tumor-induced angiogenesis, is a suppressor of DC development and maturation (220, 221). In histologic analysis of noneCsmall cell lung cancer specimens, VEGF expression and the degree of DC infiltration are inversely related and of prognostic value, with the better prognosis in low-VEGF tumors with abundant DC infiltration (222). In contrast, GM-CSF expression in primary lung carcinoma (noneCsmall cell lung cancer and adenocarcinoma) correlates with increased infiltration of DCs and LCs (223, 224), but the prognostic significance of this finding has not been established. Overexpression of cyclooxygenase 2 (COX-2) by noneCsmall cell lung cancer has been documented as an indicator of poor prognosis (225). In addition, COX-2 and VEGF are both positively correlated in noneCsmall cell lung cancer specimens (226). Interestingly, animal experiments have shown that a whole range of DC functions, including antigen processing, expression of costimulatory molecules, and secretion of IL-12, are suppressed by tumor supernatant in a COX-2eCdependent fashion (227). PGE2, a major product of COX-2, inhibits development of DCs from bone marrow precursors, whereas EP2R knockout animals have longer survival times when inoculated with a lung carcinoma cell line (228). Neuropeptides secreted by small cell lung carcinomas, such as bombesin-like peptides, also interfere with normal DC function through inhibition of maturation and IL-12 production (229).

    An intriguing observation is the preferential infiltration of pDCs, rather than mDCs in noneCsmall cell lung cancer tissue specimens as well as draining lymph nodes (230). This is a finding of potential importance, because CD8+ T cells primed by mDCs display strong cytotoxicity and secrete large amounts of IFN- (232), both important assets for an efficient antitumor response (231). In contrast, CD8+ T cells primed by pDCs become tolerogenic T cells with poor cytolytic responses and elevated secretion of the immunosuppressive IL-10 (232). In addition, human pDCs express indoleamine 2,3-deoxygenase (IDO), an enzyme generating tryptophan catabolites, which are powerful suppressors of T-cell responses. Interestingly, experimental studies described IDO-positive pDCs in tumor-draining LNs, and these cells actively suppressed T-celleCdependent responses to tumor antigens (233). Finally, malignant tissues could disrupt the normal sentinel function of DCs by elaborating aberrant chemokine networks. In fact, several experimental cancer vaccination schemes already aim to improve intratumoral DC infiltration through local induction of DC-attracting chemokines (234, 235).

    Additional molecular mechanisms of lung tumoreCmediated DC suppression will surely be discovered in the future. It is hoped that this knowledge will help to refine DC-based vaccines that can finally "outsmart" the malignant cells and thus have a drastic impact on the poor prognosis of patients with lung cancer.

    Lung Transplant Rejection

    Lung transplantation is a vital therapeutic option in several end-stage lung diseases, such as cystic fibrosis, severe emphysema, idiopathic pulmonary fibrosis, or primary pulmonary hypertension. Unfortunately, compared with other vascularized allografts, lung transplants are prone to a higher incidence of life-threatening chronic rejection, which manifests itself clinically as bronchiolitis obliterans syndrome (affecting more than 10% of post-transplantation survivors per year [236]). Chronic rejection of allografts relies on a T-celleCmediated immune response against donor organ antigens. Increasingly, attention has been drawn to DCs as potential initiators of this reaction. There is a logical place for DCs in the so-called two-way paradigm of donor/acceptor immunologic interactions (237). On one hand, so-called central sensitization to the allograft is initiated in the lymphoid organs of the acceptor. This requires donor APCs emigrating from the donor organ into draining lymphoid tissues of the host. Donor-derived DCs, activated by profuse danger signals originating from the surgical trauma, should be good candidates for this task, provided that severed lymphatic connections have had enough time to reestablish themselves (238). The alternative mechanism for the initiation of rejection involves infiltration of the donor organ by host-derived APCs, a process known as "peripheral" sensitization. In this case, APCs and lymphocytes from the recipient would access the graft via the bronchial circulation and initiate a local T-celleCdriven immune response to donor antigens.

    A few clinical studies have examined the place of DCs in lung transplant rejection, by quantifying the infiltration of these cells in biopsies taken from allografts. Early reports have delivered conflicting results: depending on the methods used, there was either an increase or a decrease in DC numbers reported in the bronchial mucosa of lungs with post-transplantation bronchiolitis obliterans (239, 240). A recent study using more consistent markers (HLA-DR) and superior sampling techniques (endobronchial biopsy) reinforces the notion that DC infiltration is intensified in lung allografts, with chronic rejection as compared with stable transplants (241). Similar findings were made in a rat model, whereby host-derived DCs were seen accumulating in dense peribronchial aggregates for months after transplantation (242). These findings would favor the peripheral mode of allograft sensitization as the predominant process in cell-mediated lung transplant rejection. It should be noted, however, that trafficking of professional APCs to and from allografts is not necessarily detrimental to transplant acceptance. In several types of organ transplantation, including the lung, the establishment of microchimerism (i.e., the seeding of donor-derived leukocytes into acceptor lymphoid and nonlymphoid organs) is correlated with increased graft survival (243, 244). This paradox probably reflects the duality in DC function regarding induction of tolerance versus triggering of an active immune response. It has been proposed that the relatively better outcome of liver as opposed to lung transplants could rely on the hepatic cytokine environment strongly favoring the induction of tolerogenic DCs (245).

    There are several mechanisms through which DCs could induce tolerance. DCs expressing the proapoptotic receptor Fas-L are capable of deleting Fas-expressing T cells in vitro (246). Inspired by these findings, Min and coworkers (247) showed the induction of hyporesponsiveness to a vascularized allograft after injection of donor-derived, FasL-transfected DCs. However, this strategy should be used with great caution. Buonocore and coworkers (248) demonstrated that "FasL-DCs" can exacerbate allogeneic T-cell responses, and even provoke severe pulmonary granulomatous vasculitis after a single systemic injection (249). The reason for these discrepant results is obscure but might be related to differences in the degree of MHC mismatching. An alternative mode of tolerance induction is the generation of hyporeactive or "anergic" T cells by DCs displaying suboptimal levels of costimulatory molecules. Blockade of costimulatory pathways (e.g., using CD40L-blocking antibodies or CTLA-Ig fusion proteins) appears to be a viable strategy in rodent as well as primate transplantation models (250eC252). DCs can also suppress allogeneic T-cell proliferation by means of IDO (see LUNG CANCER). IDO-positive APCs appear to be present within the lung interstitium. Interestingly, overexpression of IDO in lung transplants is capable of abrogating graft rejection in an animal experiment (253). Finally, DCs appear to be involved in the generation of actively immunosuppressive or "regulatory" T cells. Currently, two main categories of regulatory T cells are distinguished (reviewed in Reference 254). On one hand, the inducible "Tr1" and "Th3" cells are immunosuppressive T cells that differentiate under the aegis of nonactivated DCs in the steady state. Tr1 and Th3 cells mediate immunosuppression through the secretion of IL-10 and TGF-, respectively. In turn, these cytokines suppress DC activation, thereby consolidating the tolerogenic climate. It remains to be investigated whether a relationship exists between DC-induced Tr1 or Th3 cell function and lung allograft survival. IL-10 has been shown to efficiently suppress T-cell proliferation and the production of inflammatory cytokines in alloreactions induced by human lung DCs (255). Interestingly, gene transfer of IL-10 is capable of preventing the development of airway fibroobliterating disease in an animal model of tracheal transplantation (256). The other class of immunosuppressive T cells is represented by the CD4+CD25+ "Tregs." These cells, which originate naturally from the thymus, maintain tolerance to self-antigen and also suppress allogeneic immune responses by cell contacteCdependent mechanisms (254). In addition, evidence exists that CD4+CD25+ Tregs need to collaborate with the DC-dependent Tr1 and Th3 cells to achieve optimal immunosuppression in vivo. Interestingly, a recent report showed a negative correlation between CD4+CD25+ cell counts in peripheral blood and the incidence of bronchiolits obliterans more than 3 years after lung transplantation (257). The Treg's high expression of CD25, which is the IL-2R subunit, warrants some caution with respect to using IL-2ReCblocking antibodies as immunosuppressive agents in transplantation medicine (depletion of CD25+ T cells in animals induces fulminant multiorgan autoimmune disease [258]) (259).

    Considering the experimental and clinical data available so far, it appears that DCs could be the key for controlling lung transplant rejection. A thorough knowledge of the anatomy and timing of DC-mediated allograft sensitization is necessary. Subsequently, more refined pharmaceutical interventions could be aimed at preventing activation of DCs in the graft as well as in the recipient, and/or actively support the function of tolerogenic molecules expressed on DCs.

    CONCLUSIONS AND PERSPECTIVES

    Insight into DC biology continues to expand at a furious pace, making it ever harder to define the "state of the art." The road from bench to bedside is particularly challenging when it comes to DC research. Luckily, scientists can nowadays draw from an ever-growing toolbox of techniques, such as gene transfections, RNA silencing, and so forth. However, as in other fields, the challenge will be to promote these cellular manipulations to clinically acceptable strategies. Because of the DC's powerful impact on the outcome of immune responses, any attempt to manipulate these cells for therapeutic purposes must walk the thin line between excessive immune suppression and sparking off autoimmunity. In addition, more fundamental research is needed before reaching the stage of clinical application. As a start, there is still a lot to be investigated in terms of cataloging different DC populations in the lung and documenting their respective function, origin, and relationship to other pulmonary cells. It can be expected that the continuing emergence of new DC markers will create a complex taxonomy that becomes as dendritic as the cells themselves. The next step will be to define with precision which molecular pathways are corrupted as DCs "switch" the normal pulmonary immune response to a pathologic one. Equally important is knowing which external factors divert the normal functions of pulmonary DCs and how genetic polymorphisms modulate the DC's susceptibility. As daunting as these tasks may seem, the reward in the field of pulmonary medicine will more than compensate for the efforts: DCs may hold the key to decisive breakthroughs in fighting asthma, COPD, and lung cancer, undoubtedly some of the world's major health issues in the present and in the foreseeable future.

    Acknowledgments

    The authors thank Prof. Dr. G. Joos, Prof. Dr. G. Brusselle, and Dr. I. Demedts for their critical reading of this manuscript. They also thank Dr. I. Demedts and Dr. S. Saeland for providing the micrographs of Langerin-stained pulmonary histiocytosis specimens and normal human bronchial tissue, respectively. In addition, the authors thank Greet Barbier, Eliane Castrique, Indra De Borle, Philippe De Gryze, Kathleen De Saedeleer, Marie-Rose Mouton, Ann Neessen, and Christelle Snauwaert for their technical contribution to this article.

    This article is dedicated to the memory of Prof. Dr. Romain Pauwels, who passed away January 3, 2005.

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    1 Laboratory of Virology and Infectious Disease, Center for the Study of Hepatitis C, The Rockefeller University, New York, NY 10021
    2 Cornell/Rockefeller/Sloan-Kettering Tri-Institutional MD-PhD Program, New York, NY 10021
    3 Institut Pasteur, Avenir, 75015 Paris, France
    4 Institut national de la santé et de la recherche médicale, Avenir, 75015 Paris, France

    The yellow fever (YF) 17D vaccine is one of the most successful live attenuated vaccines available. A single immunization induces both long-lasting neutralizing antibody and YF-specific T cell responses. Surprisingly, the mechanism for this robust immunity has not been addressed. In light of several recent reports suggesting flavivirus interaction with dendritic cells (DCs), we investigated the mechanism of YF17D interaction with DCs and the importance of this interaction in generating T cell immunity. Our results show that YF17D can infect immature and mature human DCs. Viral entry is Ca2+ dependent, but it is independent of DC-SIGN as well as multiple integrins expressed on the DC surface. Similar to infection of cell lines, YF infection of immature DCs is cytopathic. Although infection itself does not induce DC maturation in vitro, TNF-–induced maturation protects DCs from YF-induced cytopathogenicity. Furthermore, we show that DCs infected with YF17D or YF17D carrying a recombinant epitope can process and present antigens for CD8+ T cell stimulation. These findings offer insight into the immunologic mechanisms associated with the highly capable YF17D vaccine that may guide effective vaccine design.

    G. Barba-Spaeth and R.S. Longman contributed equally to this work.

    The yellow fever (YF) 17D vaccine is a live attenuated vaccine that has been used for >70 yr on more than 400 million people for vaccination against YF virus with a remarkable record of safety and efficacy (1). The vaccine strain YF17D was generated from the WT strain Asibi and differs from Asibi by only 32 amino acids, 12 of which are clustered in the envelope protein (2). Viscerotropism of the parental Asibi is markedly reduced in YF17D, but the basis of the attenuation remains unknown. Vaccination generates both long-lasting neutralizing antibodies and T cell responses (3, 4). The antibody response most likely accounts for the effectiveness of the vaccine; however, a role for cell-mediated immunity in generating an effective immune response has also been suggested (4). Only recently have YF-specific human CD8+ T cell responses been identified (5), but the immunologic mechanisms associated with YF-specific CD8+ T cell priming are still unknown.

    Given its safety and efficacy, YF17D has been engineered as a vaccine vector for antigens of other flaviviruses such as dengue, Japanese encephalitis, and West Nile viruses. Some of these chimeric viruses are in phase II clinical trials (6). We have recently reported that YF17D can also be engineered to deliver epitopes from unrelated pathogens, and it is able to induce protective immunity against heterologous agents (7). A better understanding of the mechanism underlying the efficacy of T cell priming by YF vectors would offer insight into vaccine design.

    DCs are potent APCs that play a crucial role in regulating the adaptive immune response (8). DCs may interact with pathogens in peripheral tissue via direct infection or via phagocytosis of either infected cells or viral particles. On exposure to inflammatory signals or certain pathogens, DCs undergo a programmed phenotypic maturation program coincident with CCR7 up-regulation and migration to the lymph node for engagement and activation of T cells. Recent studies have demonstrated DC interaction with flaviviruses. Specifically, DC-SIGN, a DC-specific C-type lectin present most robustly on immature DCs (iDCs), mediates entry of dengue virus into DCs (9, 10) and binding of the hepatitis C virus envelope protein (11).

    In this report, we explore the interaction of YF17D with human DCs. We investigate the ability of YF17D to infect DCs, the requirements for viral entry, and the cytopathic effect of YF17D in DCs. Furthermore, we evaluate the immunologic determinants of DC infection by monitoring T cell responses to endogenous and model antigens from YF17D vectors. This work provides new insight into the immunologic mechanisms associated with the highly effective YF vaccine and may provide clues relevant to effective vaccine design.

    Results and Discussion

    YF17D infects immature and mature DCs

    To test the hypothesis that the immune response induced by YF17D vaccination may be generated by direct infection of DCs, we exposed both immature and mature human monocyte-derived DCs with characteristic surface phenotypes (Fig. S1, available at http://www.jem.org/cgi/content/full/jem.20051352/DC1) to YF17D virus. FACS analysis for the intracellular nonstructural protein NS1 showed robust expression in both iDCs and mature DCs (mDCs) infected at 2 or 20 PFU/cell (Fig. 1 A). Detection of nonstructural viral proteins not present within the virion indicates productive infection. Infection was confirmed using an antibody to nonstructural protein NS4AB (NS4; Fig. 1 B). No staining was detected when UV-inactivated virus was used for infection. In addition, virus production in both the iDC and mDC cultures was monitored by plaque assay. Results showed a 3-log increase in infectious particles within 24 h, confirming productive infection (Fig. 1 C). Notably, no substantial difference in infectious particle production was seen between iDC and mDC cultures. Infection at 20 PFU/cell peaks 48 h after infection and, though infection at 2 PFU/cell may lag behind only slightly, considerable differences in the percentage of infected cells persist (Fig. 1 A). Induction of antiviral cytokines during infection may account for lower infectivity at 2 PFU/cell. To exclude antibody-mediated enhancement of infection, DCs were generated in plasma from naive donors and, in control experiments, blocking antibodies against the Fc receptor did not block YF17D infection (unpublished data). These data demonstrate that YF17D can infect and productively replicate in human DCs, and infection occurs irrespective of their maturation state.

    Other cell types were not as susceptible to YF infection. High-level virus production was not seen in T cells, B cells, or monocyte-enriched PBMCs (Fig. 1 D). In addition, infection of B cell lines (MC116, RAMOS, and Raji) did not result in productive infection (unpublished data).

    DC infection is Ca2+ dependent

    The cellular receptor involved in YF17D infection is unknown. Recent reports have identified the receptors for the related flaviviruses West Nile virus and dengue virus as v3 and DC-SIGN, respectively (9, 10, 12). In addition, the presence of an RGD motif in the ectodomain of the YF17D envelope protein suggests a mechanism for interaction with integrins (13). Both integrins and DC-SIGN require divalent cations for structural and functional integrity. Thus, to assess a possible requirement for divalent cations, iDCs were pretreated with 10 mM EDTA and exposed to YF17D for 1 h to evaluate their role in infection. NS4AB expression at 24 h was completely blocked by EDTA (Fig. 2 A), whereas EDTA-treated DCs were still competent for infection with influenza (Fig. 2 B). EGTA treatment also blocked infection, indicating a Ca2+-dependent mechanism (Fig. 2 A). Consistent with this finding, addition of Ca2+, but not Mg2+, was able to overcome the EDTA block (Fig. 2 A).

    In light of this result, we tested specific integrin-blocking antibodies, including 1, 3, v3, 5, v5, and v6, as well as RGD peptides, to evaluate the role of specific integrins in viral entry. Antibodies and peptides were used at concentrations reported to be sufficient for blocking West Nile virus (12). None of the integrin-blocking antibodies considerably inhibited infection as monitored by NS4AB expression 24 h after infection (Fig. 2 C and not depicted). The lack of inhibition by RGD peptides is consistent with previous reports suggesting YF17D infection is independent of the RGD motif (12, 13). These data, however, do not rule out the possibility that YF entry uses determinants not blocked by these antibodies or peptides.

    In addition, blocking antibodies against DC-SIGN (9) did not inhibit YF17D infection (Fig. 2 C). Antibodies were used at saturation conditions, and binding to DCs was confirmed by FACS (Fig. S1). These results suggest that, in contrast to dengue, YF17D infection of DCs is not mediated by DC-SIGN. This finding is consistent with our data showing equivalent virus production in iDCs and mDCs, which differ substantially in DC-SIGN expression (high in iDCs, low in mDCs; Fig. 1 C and Fig. S1), as well as a recent report showing the inability of DC-SIGN to permit infection in THP-1 cells (10). In contrast with dengue virus envelope protein, which contains one or two N-linked glycans, the envelope protein of YF17D-204 used in these experiments is not glycosylated (14). Although other YF17D substrains (YF17DD and YF17D-213) may be glycosylated, our data indicate that DC-SIGN interaction is not necessary for infection and that YF17D interacts with DCs via mechanisms distinct from related flaviviruses. Although YF17D infection of DCs is Ca2+ dependent, the receptor for YF17D remains elusive.

    YF17D infection does not alter DC maturation in vitro

    Several studies indicate that viral infection of DCs promotes or inhibits maturation (15–17). To evaluate DC maturation as a result of YF17D infection, surface expression levels of CD83, CD86, and MHC class II were assayed 36 h after infection. Interestingly, infection alone did not induce robust DC maturation. Although slight up-regulation of CD86 and MHC class II was observed (unpublished data), conventional maturation markers such as CD83 were not expressed (Fig. 3 A). However, in contrast to inhibition of maturation by several infectious pathogens (16, 17), YF17D infection did not inhibit TNF-–mediated DC maturation (Fig. 3 B). These results suggest, therefore, that in the context of YF17D vaccination, release of inflammatory cytokines in peripheral tissue may play an important role in triggering DC maturation. This will allow for DC migration to the lymph node and subsequent T cell engagement and priming. Consistent with this model, an increase in TNF- has been detected in subjects vaccinated with YF17D (18).

    Maturation protects DCs from YF17D-induced apoptosis

    Because YF17D has been shown to be cytopathic in mammalian cells, we evaluated potential cytopathogenicity in human DCs using a FACS-based assay for monitoring apoptotic cell death. Similar to infection by several other viruses, YF17D induces cleavage of caspase-3 in iDCs and triggers cell death. In a dataset representative of three individual experiments, we show 37% (2 PFU/cell) and 54% (10 PFU/cell) CaspaTag-positive iDCs at 48 h after infection (Fig. 4 A). These values were similar to the ones obtained in the SW13 cell line (29% at 2 PFU/cell and 69% at 10 PFU/cell). Interestingly, when we exposed infected iDCs to inflammatory stimuli capable of inducing maturation, there was a considerable reduction in the YF17D-induced cytopathogenicity: 9% (2 PFU/cell) and 13% (10 PFU/cell) of the total cells had activated caspase-3 48 h after infection (Fig. 4 A).

    To confirm that iDC cytopathogenicity was YF17D specific and that CaspaTag-negative mDCs were indeed YF17D infected, we stained the infected iDCs for CD83, YF NS4AB, and activated caspase-3. Again, in a representative dataset, gating on the YF-infected CD83– cells showed 21% caspase activation compared with 6% in the YF17D-negative population (Fig. 4 B). In contrast, mature YF17D-infected DCs (CD83+/NS4+) showed only 3% caspase activation (Fig. 4 B). Plaque assays performed on the supernatants from both cultures confirmed comparable levels of virus production (unpublished data). Based on this data, we conclude that the resistance to YF17D-induced cytopathogenicity is dependent on DC maturation. This resistance to cytopathogenicity could play an important role in allowing infected DCs to remain alive, permitting trafficking to lymph node and priming of T cell responses. This finding is consistent with data from the influenza model in which DC maturation confers resistance to cytopathogenicity, thereby facilitating T cell priming (15).

    Infection of DCs allows for processing and presentation of endogenous and model antigens

    In light of the direct infection of DCs and the resistance to YF17D-induced cell death on maturation, we evaluated the ability of mDCs to process and present endogenous antigens produced by YF17D. mDCs generated from subjects vaccinated with YF17D were infected and used to stimulate autologous CD8+ and CD4+ T cells. IFN- ELISPOT results showed that infected DCs were able to process and present YF17D antigen for antigen-specific CD8+ and CD4+ T cell stimulation (Fig. 5 A). Although the mechanism of processing and presentation of endogenous CD4 epitopes remains poorly characterized, antigen may be derived from the exogenous capture of the small percentage of dying infected cells or by alternative mechanisms of processing (19). No T cell response was detected in naive donors. Influenza was used as a positive control to show T cell responses in both immune and naive donors.

    Because of the efficacy of the YF17D vaccine, many groups have proposed its use as a vaccine vector for generating CD8+ T cell immunity. We propose that these vectors may directly infect DCs, allowing for processing and presentation of CD8+ T cell epitopes. We tested this hypothesis by constructing a YF17D vector carrying the immunodominant HLA-A2 M1 CTL epitope from influenza matrix protein. The M1 CTL epitope was inserted between NS2B and NS3 proteins. This site has been shown previously to tolerate a small insertion of foreign sequences (20). mDCs from naive donors were infected with YF17D or chimeric YF17D-M1 virus at 2 PFU/cell as described in Materials and methods and used to stimulate autologous CD8+ T cells. IFN- ELISPOT results showed that YF17D-M1–infected DCs stimulate robust M1-specific CD8+ T cell responses similar to M1 peptide–pulsed or influenza-infected DCs (Fig. 5 C), whereas DCs infected with YF17D alone did not stimulate IFN- secretion in the naive donor. Importantly, this is the first demonstration of a YF17D 2B/3 chimeric virus presenting an HLA A2 CTL epitope for T cell stimulation, and it offers proof of principle for a possible vaccine approach.

    These data indicate that the YF17D vector is capable of delivering endogenous and recombinant epitopes to the surface of the DC enabling specific T cell activation. In the presence of a maturation stimulus produced in infected tissue, we therefore propose that Ca2+-dependent direct infection of DCs may offer a mechanism for the robust and long-lasting immunity associated with the YF17D vaccine.

    Materials and Methods

    Isolation and preparation of cells

    PBMCs, DCs, and T cells were prepared as previously described (21). PBMCs were isolated from whole blood by sedimentation over a Ficoll-Hypaque gradient (GE Healthcare). T cell–enriched and T cell–depleted fractions were prepared by adherence to plastic in 1% single donor plasma. iDCs were prepared from the T cell–depleted fraction by culturing cells in the presence of 1,000 U/ml GM-CSF (Berlex) and 500–1,000 U/ml IL-4 (R&D Systems) for 6 d (22). Cultured cells consisted of >75% CD14– CD83– HLA-DR+ DCs, with contaminating cells being B cells and dying myeloid cells. To generate mature DCs, cultures were stimulated on day 6 with 50 ng/ml TNF- (Qbiogene) and 10 mM PGE-2 (Sigma-Aldrich) for 36–48 h (23). At that time, cells were >85% CD14– CD83+ HLA-DRHI DCs. Patient material was obtained as per protocol approved by the Institutional Review Board of the Rockefeller University Hospital (JKR-0397), and all patients gave informed written consent. Immunized donor 1 was vaccinated in 2000, and donor 2 was vaccinated in 2001. "Naive donor" refers to healthy blood donors that have not received any flavivirus vaccine.

    Preparation of virus stocks

    YF17D viral stocks were derived from pACNR/FLYF plasmid containing the full-length infectious YF17D genome under an SP6 promoter (24). In vitro–generated RNA transcripts were electroporated in SW13 cells as previously described (25). Virus stocks were harvested at 48 h after transfection with typical yields of 107–108 PFU/ml as determined by plaque assay on SW13. Single-use aliquots were stored frozen at –80°C until use. YF17D/M1 was constructed by inserting the influenza HLA-A2 CTL epitope of matrix protein (GILGFVFTL) (26) between YF NS2B and NS3 proteins. Two specific oligonucleotides containing the M1 epitope sequence (forward, 5'-aggggagcgcgcagaagtggaattttaggattcgtgttcacgctcggtcaccggagaagt-3' and reverse, 5'-acttctccggtgaccgagcgtgaacacgaatcctaaaattccacttctgcgcgctcccct-3') and two YF17D-specific primers were used to generate two PCR fragments containing the influenza M1 epitope and a portion of the NS2B or NS3 gene, respectively. 1/50 μl of each PCR reaction was mixed together and used as a template for PCR with YF17D-specific primers lying in NS2B and in NS3. The final product was digested with BssHII and BstEII and cloned in YF17D2B/3 as described previously (7). The recombinant virus was recovered 48 h after transfection of SW13 cells with infectious RNA and tested for stability by PCR and sequence analysis of the region overlapping the insertion. UV inactivation was performed with a UV chamber (GS Gene-Linker; Bio-Rad Laboratories) using the sterilizing program.

    Infection of cells

    DCs were washed in RPMI 1640 and infected for 1 h at 37°C using the PFUs indicated in the figures. UV-inactivated virus was used as a negative control. The infection was quenched with 5% pooled human serum and washed twice to remove excess virus. For EDTA/EGTA blocking, DCs were washed twice in PBS without Ca2+ and Mg2+, and 10 mM EDTA or EGTA was added before infection. For antibody blocking, DCs were washed in RPMI 1640 and resuspended in 1% FBS/PBS. DCs were incubated with 10 μg/ml of antibody (DC-SIGN [m612; R&D Systems] or v3 [Chemicon]) or 100 μg/ml RGD tripeptides (Sigma-Aldrich) for 15 min at room temperature before infection. After 1 h of infection, DCs were resuspended in conditioned media with or without TNF-/PGE-2 maturation stimulus, as indicated in the figures, and incubated 24 h before monitoring for infection.

    Immunostaining for FACS analysis

    Surface staining was done in serum containing media at 4°C. Anti-CD14, CD25, CD40, CD83, CD86, HLA-DR, and isotype control were obtained from BD Biosciences. Cytoperm/CytoFix Kit from BD Biosciences was used for fixation and permeabilization. mAb 1A5 is a mouse mAb against the nonstructural protein NS1 (27), and C12 is a rabbit polyclonal antisera that recognizes the nonstructural proteins NS4A and NS4B (28). Secondary antibodies used were PE (Jackson ImmunoResearch Laboratories) or APC (Invitrogen).

    Plaque assay

    For plaque titration, serial 10-fold dilutions were used to infect monolayers of SW13 for 1 h at 37°C. After infection, cells were overlayed with 0.6% agarose-containing medium, and plaques were allowed to develop at 37°C for 4 d. Plaques were fixed in 7% formaldehyde for 1 h and stained with crystal violet (1.25% in 20% ETOH) (25).

    Detection of influenza-specific T cells by ELISPOT

    DCs and T cells were plated in 96-well Millipore plates coated with 5 μg/ml of -IFN- mAb (Mab-1-D1K; Mabtech). Cultures were incubated for 24–36 h at 37°C, washed with mild detergent, and incubated with 1 μg/ml biotin-conjugated -IFN- mAb (Mab 7BG-1; Mabtech). Spots were visualized using Vectastain Elite Kit (Vector Laboratories). ELISPOT reagents were provided by R. Darnell (The Rockefeller University, New York, NY). Evaluation was performed in a blinded fashion by an independent service (Zellnet Consulting, Inc.) using an automated ELISPOT reader (Carl Zeiss MicroImaging, Inc.). Spots represent IFN- production by single cells and are reported as spot-forming cells/106 cells.

    Online supplemental material

    Fig. S1 shows purity and phenotype of iDC and mDC cultures. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20051352/DC1.

    Acknowledgments

    We would like to thank R. Darnell for reagents and helpful suggestions.

    This work was supported by the Greenberg Medical Research Institute and the Grand Challenges in Global Health. We are grateful to the donors who participated in this study.

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    1 Department of Molecular Preventive Medicine and Solution Oriented Research for Science and Technology (SORST), Graduate School of Medicine, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan
    2 Department of Anatomy (Macro) and SORST, Dokkyo University School of Medicine, Tochigi 321-0293, Japan
    3 Department of Pediatrics, Children's Hospital, Harvard Medical School, Boston, MA 02115

    Antiviral cell–mediated immunity is initiated by the dendritic cell (DC) network in lymph nodes (LNs). Plasmacytoid DCs (pDCs) are known to migrate to inflamed LNs and produce interferon (IFN)-, but their other roles in antiviral T cell immunity are unclear. We report that LN-recruited pDCs are activated to create local immune fields that generate antiviral cytotoxic T lymphocytes (CTLs) in association with LNDCs, in a model of cutaneous herpes simplex virus (HSV) infection. Although pDCs alone failed to induce CTLs, in vivo depletion of pDCs impaired CTL-mediated virus eradication. LNDCs from pDC-depleted mice showed impaired cluster formation with T cells and antigen presentation to prime CTLs. Transferring circulating pDC precursors from wild-type, but not CXCR3-deficient, mice to pDC-depleted mice restored CTL induction by impaired LNDCs. In vitro co-culture experiments revealed that pDCs provided help signals that recovered impaired LNDCs in a CD2- and CD40L-dependent manner. pDC-derived IFN- further stimulated the recovered LNDCs to induce CTLs. Therefore, the help provided by pDCs for LNDCs in primary immune responses seems to be pivotal to optimally inducing anti-HSV CTLs.

    Abbreviations used: Ab, antibody; BrdU, 5'-bromo- 2'-deoxyuridine; CFSE, carboxyfluorescein diacetate succinimidyl ester; HEV, high endothelial venule; mDC, myeloid DC; pDC, plasmacytoid DC; PDCA, pDC antigen; PLN, popliteal LN; rpm, revolutions per minute.

    Viruses have evolved a variety of mechanisms to evade the immune system, invade host tissues, and, sometimes, establish persistent infections (1). One strategy by which hosts counter these assaults is the rapid response of the DC network to eliminate virally infected cells. In mice, there are at least three major functional subtypes of DCs in LNs: myeloid DCs (mDCs; CD11b+B220–CD11c+), CD8+ DCs (CD8+B220–CD11c+), and plasmacytoid DCs (pDCs; B220+CD11c+), which induce distinct types of antiviral T lymphocytes (2, 3). This heterogeneity in the DC network allows flexibility in the immune response, depending on the tissue environment and exogenous factors in LNs (2, 4). In HSV-2 infections of the vaginal submucosa, newly recruited mDCs induce virus-specific T helper cells (5), whereas in cutaneous HSV-1 infections, CD8+ DCs, which mainly reside in LNs, are responsible for cross-priming antiviral CTLs (6). In contrast, pDCs function poorly as APCs for naive T cells, but produce large amounts of antiviral IFN-. However, the contribution of pDCs to antiviral immunity in vivo remains controversial, as they have been reported to promote a variety of immune responses in vitro (3, 7).

    Recent investigations have revealed that tissue-resident mDCs and pDCs in a normal, infection-free animal maintain peripheral tolerance (7, 8). In response to inflammation, these DCs are activated to induce T cell–mediated immunity, but the mechanism by which the DC system is converted from "tolerogenic" to "immunogenic" remains controversial (3, 8). We are particularly interested in the role of inflammation-associated circulating DC precursors in this conversion (9–11). In a cutaneous HSV-1 infection model, we recently demonstrated that large numbers of both mDC and pDC precursors appear de novo in the circulation in response to TNF- (10). mDC precursors are preferentially recruited to sites of inflammation and remobilized into draining LNs, where they act as APCs (9, 10). In contrast, pDC precursors directly enter the LNs through activated high endothelial venules (HEVs) in a CXCL9- and E-selectin–dependent manner (10). This inflammation-dependent, CXCR3-driven trafficking pathway is distinct from that used by mDCs, but similar to that used by monocytes (12) and NK cells (13). This suggests that LN-recruited pDCs and mDCs have distinct functions, but the fate of pDCs after entering the LNs has not been established in vivo.

    We have studied the contribution of LN-recruited pDCs to anti-HSV CTL-mediated immunity and have shown that they are associated with LNDCs as well as 5'-bromo-2'-deoxyuridine (BrdU)–positive T cells in vivo, but act as poor APCs for IFN-+ CTLs. In vivo depletion of LN pDCs using three distinct methods and in vivo reconstitution assays using freshly isolated pDC precursors showed that LN-recruited pDCs are able to convert virally impaired LNDC networks into completely activated ones that are able to induce anti-HSV CTLs. Although the DC–virus interplay is dependent on the type and dose of virus, this study provides novel insights into the role of pDCs in antiviral CTL immunity.

    Results

    Recruitment and activation of pDCs in inflamed LNs

    We have traced the fate of pDCs (B220+CD11c+CD3––CD19–DX5–) in draining popliteal LNs (PLNs) after infecting mice with HSV-1 in the right footpad. The frequency and number of PLN pDCs peaked on day 2 and decreased thereafter (Fig. 1 A). The increase of PLN pDCs was largely caused by accelerated recruitment of pDC precursors directly from the circulation, as previously reported (10). Although blood pDC precursors (MHCII–B220+CD11c+CD3–CD19–DX5–) did not express CD86, CD40, and CD40 ligand (CD40L) on day 2 (Fig. 1 B), the expression of these activation markers was up-regulated on PLN pDCs on day 2 (Fig. 1 C). PLN pDCs isolated on day 2 and restimulated in vitro with irradiated HSV produced high levels of IFN-, but uninfected PLN pDCs and blood pDC precursors did not (Fig. 1 D). These results suggested that pDCs were activated after entering the draining LNs and produced cytokines on encountering HSV-1.

    Recruited pDCs contact physically with LNDCs

    Before infection, only small numbers of pDCs were found in the paracortex, a T cell zone of PLNs (Fig. 2 A). However, a new influx of pDCs appeared in the paracortex on day 2 and colocalized with B220– LNDCs (mDCs and CD8+ DCs; Fig. 2 B). To determine whether pDCs were attracted by LNDCs after entering LNs, we adoptively transferred carboxyfluorescein succinimidyl ester (CFSE)–labeled pDC precursors i.v. into HSV-infected mice on day 2. By 2 h, the transferred pDC precursors were in close contact with LNDCs in the paracortex, and some were associated with pDCs in the perifollicular zone (Fig. 2 C). 48 h later, transferred pDC precursors were still retained in these zones around HEVs (Fig. 2 D). The transferred pDCs did not always attach to CCL21+ stromal cells (Fig. 2 D), suggesting that other chemokines derived from LNDCs or pDCs might preferentially attract pDCs. Because LNDC-derived CXCL10 mediates DC–T cell interactions in inflamed LNs (14), the CXCL10–CXCR3 pathway must play an important role in inflammatory conditions. PLN pDCs on day 2 expressed high levels of CXCL10 (Fig. 2 E), which is known to attract Th1 precursor cells (14), as well as pDCs themselves (10).

    Blood pDC precursors constitutively express high levels of adhesion molecules, including VLA-4/4-integrin (97%) and CD11a/LFA-1 (99%; reference 10), but low levels of CD2/LFA-2. However, CD2 was up-regulated on day 2, but only on PLN pDCs (Fig. 2 F), indicating that activated pDCs up-regulate CD2 and CXCL10 rapidly after entering the LNs. To understand the mechanism by which recruited pDCs and LNDCs physically associate, we performed ex vivo clustering assays using pDCs and LNDCs isolated from PLNs. After 2 h of incubation at 40 revolutions per minute (rpm), the number of cluster-forming LNDCs was counted. Strong conjugates were seen only using DCs isolated from infected LNs, and CD40L was detected on the boundary (Fig. 2, G and H). Cell conjugates were significantly inhibited by treatment with anti-CD2 mAb alone or in combination with anti-CXCL10 mAb (P < 0.05; Fig. 2 H). This suggested that LNDC- and pDC-derived CXCL10 attracted pDCs, and that CXCR3 and CD2 on pDCs mediated their binding.

    In vivo depletion of LN-recruited pDCs impairs CTL-mediated virus eradication

    To investigate the contribution of LN-recruited pDCs to anti–HSV-1 CTL immunity, we used three independent approaches to deplete LN-recruited pDCs in vivo. We tested PLN CD8+ T cells on day 7 for CTL precursor frequency, using an IFN- ELISPOT assay, and for CTL function, using an in vitro cytotoxicity assay against HSV-1–infected syngeneic splenocytes.

    First, CXCR3-driven LN recruitment of pDCs was inhibited by the combined blockade of CXCL9 and E-selectin (10). A single injection of 100 μg of blocking anti-CXCL9 and anti–E-selectin antibodies (Abs) on day 0 almost completely blocked the increase in pDCs, but not LNDCs, in the PLNs (Fig. 3 A). This was mostly because of the selective inhibition of pDC precursor transmigration across inflamed LN HEVs (10). Both the induction of IFN-+ CTLs and CTL function were detected in CD8+ T cells isolated from PLNs from control mice, but reduced in mice treated with blocking Abs (Fig. 3, B and C). Control mice were able to eliminate HSV-1 to a significant extent, but the virus persisted in the anti-CXCL9 and anti–E-selectin Ab–treated mice (P < 0.05; Fig. 3 D). Although these findings showed that the early recruitment (Fig. 3 A) of pDC precursors into the inflamed LNs was crucial for generating functional anti–HSV-1 CTLs, we performed further experiments to rule out the possibility that a CXCL9 blockade was affecting late T cell responses, as well as early pDC effects.

    Our second approach used anti-Ly6G/C Ab to deplete the pDC population without affecting other APCs, B cells, and macrophages (15). Injecting mice twice with this antibody depleted 70% of PLN-resident pDCs (Fig. S1 A, available at http://www.jem.org/cgi/content/full/jem.20041961/DC1) and inhibited the influx of new pDCs during the experiment without affecting the numbers of LNDCs (Fig. 3 E) or the expression of CXCL9 and E-selectin on inflamed HEVs (Fig. S1 B). On day 7 after infection, mice treated with anti-Ly6G/C Ab showed impaired CTL induction and function (Fig. 3, F and G) and poor viral elimination (Fig. 3 H), compared with control mice.

    The third method used anti–pDC antigen (PDCA)-1 Ab, which is more specific for pDCs (16). Injecting mice twice with 100 μg of anti–PDCA-1 Ab on days –1.5 and 0.5 depleted 80% of PLN-resident pDCs (Fig. S1 C) without affecting LNDCs. This protocol also inhibited the increase in pDCs, but not LNDCs, in the PLNs (Fig. 3 I). The number of IFN-+ CTLs and the CTL activity were again reduced in anti–PDCA-1 Ab–treated mice, compared with control mice (Fig. 3, J and K).

    Impaired APC function of LNDCs in anti-Ly6G/C Ab–treated mice

    We next investigated the stage at which CTL generation was affected by the reduction in recruited pDCs in anti-Ly6G/C mAb–treated mice. To distinguish the APC activities of different LN DC subtypes, we isolated them from HSV-infected LNs and tested their abilities to stimulate IFN- production by HSV-infected LN T cells in vitro, without any further restimulation. To our surprise, LN pDCs alone poorly induced HSV-specific IFN- production (Fig. 4 A) by both CD4+ and CD8+ T cells, indicating that they were not themselves acting as APCs in vivo. In contrast, mDCs preferentially primed HSV-specific CD4+ T cells, and CD8+ DCs selectively primed HSV-specific CTLs (Fig. 4 A), which was consistent with previous reports (6, 7). However, mDCs and CD8+ DCs isolated from anti-Ly6G/C Ab–treated mice on day 2 failed to prime anti-HSV CD4+ or CD8+ T cells (Fig. 4 A).

    LNDCs from anti-Ly6G/C Ab–treated mice did not show reduced HSV uptake (5.3 ± 1.1% in anti-Ly6G/C Ab–treated mice vs. 5.9 ± 1.4% in control mice, on cytosmear preparations) or accumulation in LNs (Fig. 3 E). To examine the ability of LNDCs to attract T cells, we performed ex vivo clustering assays using B220–CD11c+ LNDCs and CD3+ T cells isolated from PLNs from infected mice. Cluster formation was significantly higher with cells isolated from mice treated with control Ab, compared with anti-Ly6G/C Ab (P < 0.05; Fig. 4 B). As a result, mDCs and CD8+ DCs from the anti-Ly6G/C Ab–treated mice also failed to form clusters with T cells and to induce IFN-–producing CD4+ or CD8+ T cells, respectively (Fig. 4 A).

    Reconstitution of anti-Ly6G/C Ab–treated mice with pDC precursors restores CTL induction by LNDCs in vivo

    To find out whether the impaired CTL responses observed in anti-Ly6G/C Ab–treated mice were actually dependent on LN-recruited pDCs, we performed in vivo reconstitution assays by transferring blood pDC precursors i.v. into HSV-infected, anti-Ly6G/C Ab–treated mice (Fig. 5 A). We used freshly isolated blood pDC precursors because maturing pDCs, restimulated in vitro, poorly enter LNs when transferred (10). The donor pDC precursors were characterized as nonantigen-pulsed, nonactivated cells (Fig. 1 B), which did not produce cytokines or chemokines unless they entered infected LNs (Fig. 1 D and Fig. 2 F). Blood pDC precursors from Propionibacterium acnes–primed mice were also used to see whether recruitment to LNs was antigen-dependent or not.

    In anti-Ly6G/C Ab–treated mice reconstituted with WT pDC precursors, significant numbers of pDCs were recruited to the paracortex of the PLNs (P < 0.05; Fig. 5 B) and made contact with DEC-205+ LNDCs (Fig. 5 C) on day 2 of infection. Recruited pDCs were still detectable on day 4 of the infection (48 h after transfer) and were associated with increased numbers of BrdU+CD4+ T cells in the T cell zone (Fig. 5 D). To investigate whether the function of LNDCs was restored, LNDCs from reconstituted mice were tested for in vivo APC activity and the ability to attract T cells. mDCs and CD8+ DCs isolated from reconstituted mice primed anti-HSV CD4+ or CD8+ T cells (compare Fig. 5 E with Fig. 4A). The ability of LNDCs to form clusters with T cells was also restored (Fig. 5 F). On day 7, the functional CTL responses were restored, and HSV was effectively eliminated after reconstitution with blood pDC precursors (Fig. 5 G). Interestingly, blood pDC precursors obtained from P. acnes–primed mice could restore the host responses (Fig. 5, B, F, and G), suggesting that pDC precursors could enter the inflamed LNs in an antigen-independent manner but flexibly respond to an antigen-bearing environment after entry.

    In contrast, when anti-Ly6G/C Ab–treated mice were reconstituted with blood pDC precursors from CXCR3–/– mice, donor pDCs did not increase in the LNs (Fig. 5 B), and neither LNDC function nor anti-HSV host responses were restored (Fig. 5, E–G). These results demonstrated that CXCR3-mediated recruitment of pDC precursors to LNs is required to induce an anti-HSV CTL response in vivo.

    LN-recruited pDCs restore CTL induction by impaired LNDCs in vitro

    The ability of LN-recruited pDCs to restore the impaired induction of anti-HSV CTLs by LNDCs was next examined in co-culture experiments (Fig. 6 A). LN pDCs from mice treated with control Ab (pDC) and LNDCs from anti-Ly6G/C Ab–treated mice (impaired LNDC) on day 2 of infection failed to induce anti-HSV CTLs in vitro (Fig. 6 B). Because pDCs are major producers of cytokines (references 7, 15, 17, 18; Fig. 1 D), we tested the effect of several cytokines on CTL induction by impaired LNDCs in this system. However, adding IFN- (Fig. 6 B) or other pDC cytokines, including TNF-, IL-10, and IL-12 (unpublished data) was not sufficient to restore the impaired LNDCs in co-culture. In fact, co-culture with LN pDCs was essential to restore the ability of impaired LNDCs to generate anti-HSV CTLs, and this process was dose dependent (Fig. 6 C). In addition, when pDCs and impaired LNDCs were co-cultured in separate compartments of transwells, anti-HSV CTL activity was not restored (Fig. 6 C), demonstrating that the help provided by LN-recruited pDCs to completely activate anti-HSV CTLs depends on cell–cell contact.

    To investigate which molecules are involved in functional recovery of impaired LNDCs in the presence of pDCs, we set up a two-step co-culture system (Fig. 6 D). In step one, impaired LNDCs and pDCs were co-cultured for 16 h. After washing, LN CD8+ T cells were added and co-cultured for a further 48 h as step two. Impaired LNDC-mediated CTL induction was restored in these culture conditions (Fig. 6 D, left), indicating that the initial interaction with pDCs provided sufficient signals to activate LNDCs to induce CTLs. When different blocking Abs were added during step one, pDC-mediated recovery was not inhibited by anti-CD86, -CD80, or -CD54 Abs (unpublished data), but was significantly inhibited by anti-CD2 and -CD40L Abs (P < 0.05; Fig. 6 D, middle). Although LN pDCs readily produce IFN-, even in short-term culture (Fig. 1 D), adding anti–IFN- Ab during step one did not affect the restoration of LNDC function (Fig. 6 D, middle). However, when anti–IFN- Ab was added during step two, CTL activity was inhibited (Fig. 6 D, right). Moreover, pDCs from CD40L–/– mice failed to restore the function of LNDCs (Fig. 6 E). Collectively, these results provide evidence that LNDC–pDC contact, involving the CD2 and CD40L pathways, is initially required to restore impaired LNDC function, and IFN- promotes CTL priming by LNDCs, once the provision of pDC help has converted their status from "impaired" into "recovered."

    Discussion

    Viruses, including HSV-1, can target DCs and disrupt the antigen-presenting pathway at multiple levels, leading to impairment of DC-mediated CTL immunity (1). Indeed, HSV-infected hosts have both activated DCs, responsible for T cell priming, and impaired DCs, showing reduced transport of MHC–peptide complexes, T cell stimulatory capacity, and cytokine production (1, 19–22). Therefore, the balance between the host DC networks and viruses, that is, the balance between DC activation and impairment, would determine the extent of antiviral host immunity. The mechanisms that skew the DC network toward activation are not completely understood but require the host to mount functional CTL immunity and to prevent viral escape. We have shown that pDCs contribute to the activation of the LNDC network after migrating to inflamed LNs. Although pDCs do not directly prime anti-HSV T cells, they help LNDCs to prime anti-HSV CTLs by multiple mechanisms.

    In response to local HSV-1 infection, pDC precursors are rapidly mobilized into the circulation and recruited to inflamed LNs (10). Here, pDCs up-regulate activation markers and T cell–attracting chemokines, change the integrins they express, and produce cytokines in response to encountering HSV. LN-recruited pDCs are retained for at least 48 h in the paracortex and associate with LNDCs. Thus, pDCs actively participate in ongoing T cell responses by creating appropriate cytokine fields in their vicinity and attracting immune cells by secreting chemokines. CXCR3 on pDCs plays an important role in mediating both trans-HEV migration (10) and DC–DC interactions in this model.

    To inhibit the early, short-term accumulation of LN pDCs (Fig. 1 A), we injected anti-CXCL9 and E-selectin Abs or anti-Ly6G/C Ab at the time of HSV inoculation. LN T cells isolated on day 2 from HSV-infected mice, treated with either blocking Ab regime, could proliferate when stimulated ex vivo with HSV-pulsed splenic DCs and migrate to HSV-infected PLNs when adoptively transferred (unpublished data). Although these findings suggested that the blocking Abs affected LN pDCs selectively at an early stage, we could not completely rule out the possibility that the blocking Abs had a direct affect on activated T cells at a later phase of infection. To rule this out, we performed in vivo reconstitution assays. Taking advantages of CXCR3-mediated trans-HEV migration of circulating pDC precursors (10), we intravenously transferred 2 x 106 freshly isolated blood pDC precursors per mouse, after treatment with anti-Ly6G/C Ab (Fig. 5 A). This was a greater number of blood pDC precursor cells than is present in an uninfected mouse (104) or an HSV-infected mouse (1–3 x 105; reference 10). Because depletion of pDCs by anti-Ly6G/C Ab treatment was incomplete, reducing the numbers by 70%, as described above, it is possible that sufficient numbers of adoptively transferred pDCs survived, even in the presence of anti-Ly6G/C Ab in the circulation. We actually detected a significant (P < 0.05) number of transferred pDCs in inflamed LNs (Fig. 5, B–D) from day 2 to 4 of infection, but this temporal presence seems to be enough for these cells to function, as accumulation of endogenous pDCs is also short-term (Fig. 1 A) in this model. In addition, a complete anti-HSV CTL response was mounted even in reconstituted, anti-Ly6G/C Ab–treated mice (Fig. 5, F and G), ruling out the possibility that this Ab directly affected activated CD8+ T cells. Furthermore, the impairment of the CTL response in mice treated with anti–PDCA-1 Ab (Fig. 3, I–K), strongly suggested that pDCs are required in anti-HSV, CTL-mediated immunity.

    One mechanism for the effect of pDCs may be the promotion of DC–T cell clustering (Fig. 4 B and Fig. 5F). Recent in vivo, real-time visualization techniques have provided new insights into DC–T cell interaction, showing that there are short, weak, and multiple interactions over approximately the first 8 h (23). It is timely to reevaluate the signals that induce DCs to form stable and specific clusters with T cells. We consider that pDCs deliver these signals to LNDCs and that CD2 plays a role in this process because LNDCs express some CD2 ligands (24). The formation of CD2-mediated LNDC–pDC conjugates (Fig. 2 H) is linked with the APC function of LNDCs for priming CTLs (Fig. 6 D). In this respect, in CD2-deficient mice, the induction of anti–lymphocytic choriomeningitis virus CTLs has been reported to require high doses of antigen (25, 26). It is possible that the extent of CD2's in vivo effect varies, depending on the type and dose of viral antigens. However, CD2 has also been reported to be involved in T cell responses against rare and cross-presented lymphocytic choriomeningitis virus antigens (25). In a cutaneous HSV-1 infection model, it is now clear that HSV-1 antigens are cross-presented to T cells (6). Thus, we consider that CD2 plays a significant role in cross-priming anti-HSV CTLs.

    The role of CD40L on pDCs is of interest for licensing LNDCs to prime CTL immunity. Previous studies have suggested that CD40L on activated CD4+ T cells generally provides this signal to DCs (2, 27). However, in a primary immune response, CD4+ T cells first need to be activated by antigen-primed mDCs (5, 14), and it is not clear how mDCs initially receive these signals before activated CD4+ T cells appear. We have shown that LN-recruited pDCs rapidly express cell surface CD40L (Fig. 1 C and Fig. 2 G). Although the CD40L+ pDCs were only 6.3% of the total LN pDCs, this was relatively high when compared with the CD40L+CD4+ T cells, which constitute only 0.28% of LN T cells (unpublished data) on day 2 of infection. We believe that the activated pDCs recruited to LNs rapidly provide the initial CD40L signals to LNDCs, to promote their ability to present antigen to both CD4+ and CD8+ T cells. This interaction of CD40L and CD40 on DCs involves cell–cell contact and leads to the translocation of lipid rafts, aggregation of MHC class II and CD80 to enhance antigen presentation (28), activation of TRAF6 to prime antigen-specific CD4+ T cells (29), up-regulation of transporter associated with antigen processing to augment antigen processing (30), and the promotion of cross-presentation on MHC class I to CD8+ T cells (31).

    Recent experiments have revealed that pDCs are potent producers of cytokines and chemokines in inflammation (7). IFN-, in particular, plays an important role by not only preventing viral replication and spread, but also by stimulating DCs and NK cells. In DC-mediated viral immunity, IFN- has been shown to promote the cross-priming of CTLs (32) and activate uninfected, bystander DCs (21), depending on the dose and timing. In contrast, in our culture conditions, impaired LNDCs failed to completely mature to induce CTLs, even in the presence of IFN- (Fig. 6 A). However, DCs used in previous studies were "conventional" DCs, which are distinct from the virally impaired DCs used in this study (Fig. 6). We propose that pDCs help LNDCs in at least two steps: first, pDCs contact impaired LNDCs through CD2–CD2L interactions, and engagement of the CD40L–CD40 interaction sets the LNDCs into a recovered condition; and second, recovered LNDCs can now prime T cells using the appropriate signals, such as IFN-. Inhibiting IFN- reduced the ability of LNDCs to induce CTLs only after the LNDCs received help from pDCs (Fig. 6 D), indicating that IFN- can stimulate only recovered/conventional LNDCs to prime CTLs, which is consistent with previous studies (21, 32). When we tested the in vivo effect of IFN- by injecting locally into anti-Ly6G/C–treated mice at the time of infection, there was a slight recovery of CTL numbers (Fig. S2, available at http://www.jem.org/cgi/content/full/jem.20041961/DC1) with undefined mechanism. However, recovered LNDCs need additional IFN- signals to prime T cells at the second stage (Fig. 6 D). Because pDCs were not completely depleted by anti-Ly6G/C Ab, it is plausible to speculate that the residual activity of remaining pDCs might have been compensated by exogenous IFN- at the second stage.

    Our observations demonstrate a novel role for LN-recruited pDCs as "immune networkers" in antiviral CTL immunity, in addition to their known function as a source of IFN-. Although distinct subsets of DCs can induce the corresponding effector T cells ex vivo (2, 5, 6), in vivo LNDC–T cell networks are more interactive, both temporally and spatially. The creation of appropriate cytokine fields and the mediation of cell communication by pDC-derived cytokines, and chemokines are essential to antiviral host defense. Viral escape creates a major problem in immunotherapy, and some viruses evade immune surveillance even in hosts vaccinated with antigen-primed DCs or CTLs generated ex vivo (4, 24). DC dysfunction is also seen in cancer patients and is a major factor impairing the generation of anti-tumor CTLs (4, 24). Our study suggests the potent ability of pDCs to improve the activity and communication of impaired DC networks and may provide a novel strategy for the development of DC-based vaccination, to improve CTL induction against persistent viral infection, such as hepatitis and AIDS, and also in cancer.

    Materials and Methods

    Mice

    Specific pathogen-free mice were used in all experiments. Female C57BL/6 mice (8–9-wk old) were obtained from CLEA Japan Inc. CXCR3-deficient mice were generated as previously reported (33) and backcrossed into C57BL/6 mice for eight generations. B6 background CD40L-deficient mice (provided by R. Abe, Tokyo University of Science, Tokyo, Japan) were bred as previously reported (34). All animal experiments complied with the guidelines set by the University of Tokyo Graduate School of Medicine.

    HSV-1 and P. acnes infection

    The KOS strain of HSV-1 (provided by T. Kaburaki, University of Tokyo, Tokyo, Japan) was propagated and titered using Vero cells grown in MEM with 10% FCS. Mice were infected with 5 x 104 PFU in the right hind foot, and the right PLNs were removed for examination at the times indicated in the figures. Virus titers were determined on day 7 in standard serial dilution plaque assays. Mice were infected with heat-killed P. acnes (1 mg/100 μl PBS; 11828; American Type Culture Collection) via tail vein injections (9–11, 14). In some experiments, mice were injected with BrdU (500 μg/100 μl PBS; Sigma-Aldrich) 1 h before death (9, 14).

    Cell sorting and culture and flow cytometry

    DC and T cell populations were isolated using a cell sorter (EPICS ELITE ESP; Beckman Coulter) or a MACS system (Miltenyi Biotech) as previously described (9–11, 14). For cell sorting, MHCII-, CD19-, CD3-, and DX5-depleted blood cells or CD19-, CD3-, and DX5-depleted LN cells were stained with anti-CD11c–FITC mAb and anti-B220–PE mAb (BD Biosciences). Analysis of the sorted populations showed purities >95%. For DC phenotyping, blood and LN cells were stained with biotinylated mAbs against CD2, CD40, CD40L, CD86 (BD PharMingen), and Cy-Chrome–conjugated streptavidin, followed by incubation with anti-CD11c–FITC mAb and anti-B220–PE mAb.

    Purified B220+CD11c+ cells (105 cells/200-μl well) were incubated for 16 h in round bottom 96-well culture plates in RPMI 1640 with 20% FCS, 100 U/ml penicillin G, and 100 μ/ml streptomycin in the presence or absence of irradiated HSV (30 PFU/well). IFN- (PBL-Biomedical) in the culture supernatants was assayed using an ELISA (10).

    pDC depletion and reconstitution experiments

    To inhibit pDC precursor recruitment to LNs, mice were injected i.v. with 100 μg anti-CXCL9 (R&D Systems) plus E-selectin (10) or control (goat IgG; Sigma-Aldrich) Abs immediately before HSV infection. To deplete the pDC fractions, mice were injected i.p. with 200 μg anti-Ly6G/C mAb (clone RB6-8C5; BD Biosciences), 100 μg of anti–PDCA-1 mAb (clone JF05-1C2.4.1, functional grade; Miltenyi Biotec), or control Ab (rat IgG; Sigma-Aldrich) 1.5 d before and 0.5 d after infection (Fig. 3 and Fig. S1 C).

    For short-term reconstitution assays, 2 x 106 blood pDC precursors, collected on day 2 from HSV-infected WT or CXCR3–/– mice or from P. acnes–primed mice, were injected i.v. on day 1.5 into anti-Ly6G/C Ab–treated mice infected with HSV. pDC-reconstituted mice were killed 0.5, 1.5, and 5.5 d later (Fig. 5 A). Before cell transfer, 90–92% pure MACS-sorted pDCs were labeled with 2.5 μM CFSE (Molecular Probes) for 10 min at 37°C or with 5 μM 5-(and-6)-([(4-chloromethyl)benzoyl]amino) tetramethylrhodamine (CMTMR; Molecular Probes) for 15 min at 37°C (9, 10).

    Antibodies and immunohistochemistry

    Immunohistochemical staining was performed as previously reported (9, 14). The following anti–mouse mAbs were used: CD2 (clone RM2-5), CD3 (145-2C11), CD4 (RM4-5), CD8 (53-6.7), CD11c (HL3), CD19 (1D3), CD40 (HM40-3), CD45R/B220 (RA3-6B2), CD86 (GL1), CD154/CD40L (MR1) and Pan–NK cell (DX5; all from BD Biosciences), DEC-205 (BMA), and CD11c (N418; Serotec). Rabbit pAb to CCL21 was made in house (9). As secondary Abs, we used Alexa 488–labeled anti–rat Ig, Alexa 564–labeled anti–hamster, rabbit, and rat Igs, Alexa 647–labeled anti–rat Ig, and streptoavidin labeled with Alexa 564 or 647 (Molecular Probes). For control Abs, we used rat IgG, rabbit IgG, and hamster IgG (Sigma-Aldrich).

    ELISPOT assay

    Nitrocellulose 96-well plates (Millipore) were coated with 2 μg/ml of purified anti–IFN- mAb (clone R4-6A2; BD Biosciences) overnight at 4°C and washed five times in RPMI 1640 with 10% FCS. For CTL precursor frequency assays (Fig. 3, B, F, and J), 92–96% pure MACS-isolated CD8+ T cells obtained from HSV-infected PLN on day 7 were plated at 106 cells/well in the presence or absence of irradiated HSV (30 PFU/well). For ex vivo APC assays (Fig. 4 A and Fig. 5 E), MACS-isolated CD4+ or CD8+ T cells obtained from HSV-infected PLN on day 2 were plated at 106 cells/well without restimulation by exogenous antigens. The indicated numbers of sorted DC populations (mDCs, CD8+ DCs, and pDCs) from HSV-infected PLN on day 2 were separately added, in triplicate, to the wells in 100-μl aliquots. After overnight incubation at 37°C, the plates were washed eight times with 0.05% Tween–PBS and incubated with 2 μg/ml of biotinylated anti–IFN- mAb (clone XMG1.2; BD Biosciences) for 2 h, followed by streptavidin–horseradish peroxidase conjugate (Zymed Laboratories) for 2 h and aminoethyl carbazole substrate (Vector Laboratories). The red spots were counted using a dissecting microscope (SZX9; Olympus). In CTL precursor frequency assays, no spots were detected when CD8+ T cells were cultured in the absence of HSV.

    Cluster formation assay

    An ex vivo cluster formation assay was performed as described previously (35), with slight modifications. For DC–T cell clusters (Fig. 4 B and Fig. 5 F), 105 MACS-separated B220– LNDCs from the indicated mice and 106 MACS-separated CD3+ T cells from the PLNs of HSV-infected mice at day 2 were co-cultured for 2 h in 500 μl of medium, rotating at 40 rpm at 37°C. For LNDC–pDC conjugates (Fig. 2, G and H), 5 x 104 MACS-separated B220– LNDCs and 5 x 104 pDCs from the PLNs of uninfected or HSV-infected mice at day 2 were incubated for 2 h in 500 μl of medium, rotating at 40 rpm at 37°C with anti-CD2, anti-CD2 plus CXCL10, or 100 μg/ml of control Abs. Cluster formation was estimated by directly counting 1,000 free and clustered LNDCs on cytosmear preparations. In DC–T cell clusters, an LNDC cluster was judged as three or more T cells. In DC–DC conjugates, an LNDC conjugate was judged as containing one or more B220+ pDCs after immunostaining for B220. In some experiments, MACS-sorted LNDCs or pDCs were prelabeled with 10 or 2.5 μm CFSE, respectively, to determine the distribution of CD40L (Fig. 2 G). Clustered LNDCs were expressed as a percentage of the total number of cells counted.

    CTL assay

    To investigate DC-mediated CTL activity (Fig. 6, A–C), 106 PLN CD8+ T cells, obtained from HSV-infected mice on day 2, were co-cultured with 105 mitomycin C–treated d2 DCs of the indicated subsets for 48 h without any restimulation in vitro. Four sets of culture experiments were performed using (a) LNDCs from anti-Ly6G/C Ab–treated mice (impaired LNDC) supplemented with or without 1,000 U/ml of recombinant IFN- (Hycult Biotechnology); (b) impaired LNDCs plus pDCs at ratios of 1:1, 3:1, and 10:1; (c) LNDCs from control Ab–treated mice; and (d) pDCs from control Ab–treated mice. To exclude the T cell–derived factors, a two-step co-culture system was also set up (Fig. 6 D). Equal numbers (105) of impaired LNDCs and pDCs were co-cultured for 16 h, supplemented with a neutralizing anti–IFN- (Hycult Biotechnology), anti-CD2, anti-CD40L mAb, or 100 μg/ml of control Abs. After washing, these DCs were co-cultured for a further 48h with 106 PLN CD8+ T cells obtained from HSV-infected mice on day 2. Cytotoxicity was measured in standard 4-h LDH release assays using a Cytotoxicity Detection Kit (Boehringer) as described previously (11, 36). Specific cytotoxicity was determined using HSV-infected splenocytes as target cells and calculated relative to LDH release in medium alone. In some experiments (Fig. 6 E), we used pDCs from HSV-infected, CD40L-deficient mice. Because CD40L–/– mice show reduced number of LN cells as reported (37), we pooled right inguinal, popliteal, and lumbar LNs to collect a sufficient number of cells. To investigate whether CTLs were generated in vivo (Figs. 3, C, G, and K and Fig. 5G), 106 PLN CD8+ T cells obtained from HSV-infected mice on day 7 (and day 0 for negative control) were tested without adding DCs.

    Online supplemental material

    Fig. S1 provides data for the effect of anti-Ly6G/C Ab (A and B) and anti–PDCA-1 (C) on the percentage of PLN pDCs. Fig. S2 shows the in vivo effect of recombinant IFN- on anti-HSV CTL generation in anti-Ly6G/C Ab–treated mice. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20041961/DC1.

    Acknowledgments

    We are grateful to Dr. R. Abe for providing CD40L–/– mice and to Dr. T. Kaburaki for providing KOS-HSV.

    This work was supported in part by Solution Oriented Research for Science and Technology, the Japan Science and Technology Agency, and a grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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1Laboratory of Cellular Immunobiology,

2Hematology Service, Division of Hematologic Oncology,

3Department of Medicine,

4Memorial Sloan-Kettering Cancer Center, New York, New York, USA.

5Weill Medical College of Cornell University, New York, New York, USA.

6Biostatistics Service, Department of Biostatistics and Epidemiology, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.

7MacroGenics, Rockville, Maryland, USA.

8Leonard Wagner Laboratory of Molecular Genetics and Immunology, The Rockefeller University, New York, New York, USA.

9Allogeneic Bone Marrow Transplantation and Clinical Immunology Services, Division of Hematologic Oncology, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.

    Abstract

Human monocyte-derived DCs (moDCs) and circulating conventional DCs coexpress activating (CD32a) and inhibitory (CD32b) isoforms of IgG Fc receptor (FcR) II (CD32). The balance between these divergent receptors establishes a threshold of DC activation and enables immune complexes to mediate opposing effects on DC maturation and function. IFN- most potently favors CD32a expression on immature DCs, whereas soluble antiinflammatory concentrations of monomeric IgG have the opposite effect. Ligation of CD32a leads to DC maturation, increased stimulation of allogeneic T cells, and enhanced secretion of inflammatory cytokines, with the exception of IL-12p70. Coligation of CD32b limits activation through CD32a and hence reduces the immunogenicity of moDCs even for a strong stimulus like alloantigen. Targeting CD32b alone does not mature or activate DCs but rather maintains an immature state. Coexpression of activating and inhibitory FcRs by DCs reveals a homeostatic checkpoint for inducing tolerance or immunity by immune complexes. These findings have important implications for understanding the pathophysiology of immune complex diseases and for optimizing the efficacy of therapeutic mAbs. The data also suggest novel strategies for targeting antigens to the activating or inhibitory FcRs on human DCs to generate either antigen-specific immunity or tolerance.

    Introduction

mAbs are among the most rapidly growing therapies for the treatment of cancer (1) and autoimmunity (2). Antibodies either fix complement or engage cells of the innate immune system to mediate target cell lysis. The latter process, known as antibody-dependent cellular cytotoxicity (ADCC), requires that the Fc portion of a mAb ligate activating IgG Fc receptors (FcRs), e.g., FcRI (CD64), FcRIIa (CD32a), FcRIIc (CD32c), or FcRIII (CD16), on monocytes, NK cells, neutrophils, or DCs (3). Recent evidence suggests a more indirect effector mechanism, in which FcRs on DCs mediate phagocytosis and enhance cross-presentation of antibody-coated antigens, leading to effective stimulation of both CD4+ Th1 and CD8+ CTL effector responses (4-7). Studies in mice show that coligation of the unique inhibitory FcRIIb (CD32b) abrogates all of these effects (7, 8).

The activating and inhibitory FcRs on DCs offer rational targets for immunotherapy based on the unique capacity of DCs to play critical roles in both immunity and tolerance (9). Studies in mice have been very promising (7), though translation into the human system has been lacking. Investigators have not been able to distinguish surface CD32a and CD32b when coexpressed on human cells, given their highly homologous extracellular domains (3). In addition, a common genetic polymorphism of CD32a caused by an arginine (R) to histidine (H) amino acid substitution at position 131 yields divergent avidities for mouse and human IgG ligands (10), which further confounds studies of FcR function in the human system.

We have used a recently developed mAb that, unlike any other available reagent, can specifically bind the inhibitory CD32b isoform, as well as block its interaction with IgG, on intact human cells (M.C. Veri et al., unpublished observations). We have evaluated the relative expression of the activating CD16, CD32a, and CD64, in addition to the inhibitory CD32b, on circulating DCs and their precursors as well as on cytokine-induced monocyte-derived DCs (moDCs). We have demonstrated the phenotypic and functional sequelae of ligating either or both the activating CD32a and inhibitory CD32b on immature moDCs. We have also identified factors that modulate the balanced expression of these receptors, which in turn affect the IgG-mediated changes in maturation and function of the DCs themselves. Our findings have important implications for understanding the pathophysiology of diseases mediated by immune complexes and for developing and optimizing antibody- and DC-based therapies for antigen-specific immunity or tolerance. The data also suggest the need for further studies to define the cell biology of enhanced processing and presentation conferred by antigen opsonization.

    Results

Specific mAbs identify CD32 isoforms and CD32a allelic variants by flow cytometry.

We first validated the specificity of mAbs for this study using neutrophils and B cells that express only CD32a or CD32b, respectively, on the cell surface. The novel clone 2B6, which binds extracellular CD32b exclusive of CD32a (M.C. Veri et al., unpublished observations), stained B cells but not neutrophils (Figure 1A). Clone FL18.26 is not isoform specific (11) and stained neutrophils and B cells (Figure 1B). In contrast, Fab fragments of IV.3 are CD32a specific (12, 13) and detected neutrophils but not B cells (Figure 1C). These data confirm the specificity of 2B6 for CD32b and Fabs of IV.3 for CD32a, thus enabling a clear distinction between activating and inhibitory isoforms of CD32 expressed on the cell surface.

   Figure 1

2B6 is a novel mAb that specifically detects an extracellular domain of CD32b. Neutrophils and PBMCs were isolated from peripheral blood samples. Cells were stained with various anti-CD32 mAbs and counterstained with anti-CD66b to define neutrophils (N) or anti-CD20 to define B cells (B). (A) mAb 2B6 detected CD32b on B cells but not CD32a on neutrophils. (B) mAb FL18.26 detected CD32a or CD32b, and it stained neutrophils as well as B cells. (C) In contrast, mAb IV.3 (Fab) detected CD32a on neutrophils but not CD32b on B cells. (D) Some mAbs were able to distinguish between the common polymorphic variants of CD32a. mAbs FL18.26 (B) and IV.3 (C) bind both the R131 and H131 subtypes of CD32a and stain neutrophils from HH homozygotes, RR homozygotes, and HR heterozygotes equally. mAbs 3D3 and 41H16 recognize only the R131 subtype (16), shown staining neutrophils from CD32a131RR individuals but not from CD32a131HH individuals, with intermediate staining of neutrophils from heterozygous (CD32a131HR ) individuals.

The R and H variants of CD32a have differing avidities for mouse and human IgG subtypes. Compared with the CD32a131R allotype, CD32a131H has a higher avidity for complexed human IgG2 and IgG3 (14) and, to a lesser extent, human IgG1 (15). Unlike CD32a131H, however, CD32a131R binds complexed mouse IgG1 (10). We therefore determined the CD32a phenotypes of all samples used in functional studies, based on staining of neutrophils that express abundant CD32a only. mAbs 3D3 and 41H16 recognize only the R131 variant of CD32a (16), whereas mAbs FL18.26 and IV.3 recognize both the R131 and H131 variants (11, 14). As shown in Figure 1D, FL18.26 stained neutrophils from all CD32a phenotypes with equal intensity. In contrast, 3D3 stained cells homozygous for CD32a131R, did not detect cells homozygous for CD32a131H, and displayed intermediate staining of heterozygous samples. We confirmed that this method accurately typed U937, THP-1, and K562 cell lines expressing known CD32a subtypes (17, 18) (data not shown). These data validate this quick method for distinguishing the polymorphic phenotypes of CD32a by flow cytometry (14).

Subtypes of freshly isolated DCs and DC precursors have distinct Fc  R expression profiles.

We studied the differential expression of the activating and inhibitory FcRs on freshly isolated populations of DCs and their precursors in peripheral blood. We identified "myeloid" or conventional DCs from freshly isolated PBMCs as being lineage (CD3, CD14, CD20, and CD56)negative, HLA-DRbright, CD11c+, and CD123low, whereas plasmacytoid DC precursors were lineagenegative, HLA-DRbright, CD11cnegative, and CD123bright (19).

Consistent with previously published results, we found that CD11c+CD123low conventional DCs in peripheral blood expressed the activating FcRs CD32a and CD64 but did not express CD16 (4). Almost all circulating conventional DCs also expressed the inhibitory CD32b (n = 10; mean ± SD, 92% ± 3.3%) (Figure 2A). We confirmed that freshly isolated plasmacytoid DCs, however, did not express detectable surface levels of CD16, CD32b, or CD64 and expressed minimal to no CD32a (Figure 2A) (20). All CD14+ monocytes, which are circulating precursors of moDCs, expressed CD32a and CD64 (Figure 2B). Monocyte coexpression of the inhibitory CD32b was highly variable among 30 healthy volunteers, however, ranging from 1% to 48% (mean, 18.1%, SD = 8.3). A separate, but partially overlapping, small subpopulation of CD14low monocytes expressed CD16 (Figure 2B) (21, 22). Immature moDCs expressed a balance of CD32a and CD32b similar to that of conventional DCs circulating in fresh blood. On average, 56% ± 10.7% of immature moDCs expressed CD32a (n = 17) and 64% ± 9.4% expressed CD32b (n = 17) (Figure 2B). Most often, these divergent receptors were coexpressed on the same population of cells (Figure 2C). Unlike their monocyte precursors, moDCs lost expression of CD16 and CD64 by day 1–2 of differentiation. Immature moDCs thus provide an excellent model for studying the modulation and function of CD32a and CD32b on the same cells.

   Figure 2

Monocytes, circulating conventional DCs, and cytokine-induced moDCs all express a range of FcRs, whereas freshly isolated plasmacytoid DCs lack detectable surface expression of all FcRs. Freshly isolated PBMCs were labeled with fluorochrome-conjugated mAbs. (A) After gating on HLA-DRbright PBMCs that were lineage marker negative, CD32a (left) and CD32b (right) were detected on CD123low conventional DCs (conv. DCs) but not on CD123bright plasmacytoid DCs (pDCs). (B) Monocytes were identified as CD14+ PBMCs. moDCs were studied as immature cells, gated according to characteristic phenotype (48) but lacking the surface CD83 expression of mature moDCs. Open histograms correspond to isotype controls, and filled histograms represent staining of the indicated FcR. Most often, CD32a and CD32b were coexpressed on the same subpopulation of moDCs, as shown by a representative sample in C.

Various stimuli modulate the balanced expression of CD32a and CD32b on immature moDCs.

We tested the effects of several immune modulators on the balance of activating and inhibitory FcRs on immature moDCs. Figure 3 shows mean fold changes in the percentage of cells expressing the respective FcRs (Figure 3A) and the relative changes in FcR density on the cell surface (Figure 3B) compared with untreated moDCs from the same donors (n = 6 independent experiments). FcR density was measured as the number of anti-FcR detection mAbs bound per cell. Mean fluorescence intensities (MFIs) and shifts in MFIs were identical for F(ab')2 and whole IgG 2B6 (anti-CD32b), which indicates that FcR staining was mediated by Fab-specific binding and not by interactions with the Fc portions of the detection mAbs. Upregulation of FcRs by the agents tested did not increase nonspecific Fc-mediated binding (Figure 3C).

   Figure 3

Various stimuli modulate the balanced expression of CD32a and CD32b on immature moDCs. The indicated reagents were added to cultures of immature moDCs from day 3 to day 6. Expression of FcRs was measured by flow cytometry (CD16 and CD64 not shown). Analyzed cells were immature or specifically gated for the absence of CD83 in cultures where there was a small amount of maturation (PGE2 and TNF-). The mean fold changes (± SD) in the frequency of cells expressing a given FcR induced by each reagent, compared with untreated cells, are shown in A. Density was calculated on the FcR+ cells as the number of anti-FcR antibodies bound per cell using a commercially available kit. The mean fold changes (±SD) in FcR density induced by the reagents in 5 independent experiments, compared with the averaged FcR densities on untreated/control moDCs, are shown in B. Sample histograms for untreated immature moDCs and IFN-–treated immature moDCs are shown in C. Open histograms correspond to isotype controls, and filled histograms show staining by the indicated anti-FcR mAbs.

All factors affected only CD32a and/or CD32b expression, or neither, except IL-10 and IFN-, which also induced expression of CD16 and CD64, respectively (data not shown). IL-10, IL-6, and dexamethasone all led to proportional increases in expression of CD32a and CD32b without a clear shift favoring either isoform. IFN- most potently shifted the balance in favor of activating FcRs by inducing expression of CD64, increasing the frequency and density of CD32a expression, and exerting opposing effects on CD32b. Conversely, antiinflammatory concentrations of soluble monomeric IgG (0.15 mM), approximating the levels achieved in vivo after administration of intravenous Ig (IVIG), decreased CD32a expression and yielded little to no increase in CD32b expression. Among all factors tested, this led to the greatest relative shift in favor of the inhibitory FcRs. CD32a and CD32b did not bind soluble monomeric IgG, which was not detected on the surface of moDCs using anti-human IgG antibodies (data not shown). Hence, receptor occupancy could not account for any change in detection of CD32 isoforms. Culturing cells in the presence of 10% FCS or TNF- potently reduced the frequency and density of FcR expression. All tested maturation stimuli decreased the frequency of CD32a- and CD32b-expressing cells. However, LPS and CD40L, but not the combination of IL-1?, IL-6, TNF-, and PGE2 (23), shifted the balance of remaining FcR-expressing cells in favor of CD32a.

We tested the effects of factors on circulating conventional and plasmacytoid DCs enriched from whole blood after negative selection and cultured in Teflon beakers in 10% normal human serum–RPMI (NHS-RPMI). We added IL-3 to support the viability of plasmacytoid DCs in culture (24). IFN-, IFN-, TGF-?, IL-10, and soluble IgG modulated CD32a and/or CD32b on circulating conventional DCs, which was similar to their effects on cytokine-induced moDCs. Unlike freshly isolated cells, plasmacytoid DCs in culture expressed CD32a, which was modulated by these factors in the same manner as on moDCs (data not shown).

CD32a and CD32b have opposing effects on DC maturation.

We studied the effects of ligating human IgG to CD32a, CD32b, or both on immature moDCs, using immobilized IgG to mimic complexed IgG (25). Selective ligation of CD32a matured a subpopulation of moDCs, as evidenced by upregulation of the DC maturation marker CD83 (26) and the costimulatory molecule CD86 (average 58% CD83+ and 79% CD86+; n = 8 experiments; Figure 4A, filled histograms). The frequency of maturation was proportional to the percentage of cells that expressed CD32a. Blocking CD32b in the absence of a ligand for CD32a (recultured in complete medium with 1% NHS, GM-CSF, and IL-4 but without immobilized IgG) did not promote DC maturation (9% CD83+; Figure 4A, open histograms) compared with untreated controls (8% CD83+; Figure 4C, open histograms) in a total of 8 independent experiments. Ligation of CD32b was associated with a minimal increase in maturation (average 10% increase in CD83; Figure 4B). When the 2 FcRs were targeted simultaneously, CD32b limited CD32a-induced maturation (Figure 4, C vs. A, filled histograms; P = 0.001). Table 1 summarizes the average increases in the percent of total moDCs expressing CD83 and CD86 after coculture with immobilized IgG, compared with that of similarly treated moDCs cultured without immobilized IgG.

   Figure 4

Ligation of CD32a or CD32b on immature moDCs has opposing effects on maturation phenotype. (A–E) MoDCs were cultured on plates with immobilized (Imm.) human IgG to ligate FcRs (filled histograms). CD32a (A) or CD32b (B) was specifically ligated by first incubating moDCs with blocking antibodies against either CD32b or CD32a, respectively. CD32a and CD32b were ligated simultaneously (C) by preincubating moDCs without blocking antibodies. DCs with or without blocking antibodies were also cultured on untreated plates as negative controls (open histograms). Cells were harvested at 48 hours, and DC phenotype was assessed by flow cytometry. Histograms from 1 representative experiment of 8 that used CD32a131HH or -HR samples are shown in A–C. Immature IFN-–treated moDCs (D) and soluble IgG–treated moDCs (E) were washed to remove these factors and recultured with (filled histograms) or without (open histograms) immobilized human IgG. Cells were harvested at 24–48 hours and phenotype was assessed by flow cytometry. Representative histograms from 1 of 5 separate experiments are shown. (F) In contrast to results obtained from CD32a131HH or -HR samples (C, filled histograms), CD32a131RR samples were not matured to the same extent after coculture with immobilized human IgG (F, filled histograms; n = 4 experiments). Immobilized mouse IgG1 (F, open histograms), which ligates CD32a but not CD32b in CD32a131RR individuals (10), led to maturation that was similar to conditions specifically targeting CD32a on CD32a131HH or -HR samples (A, filled histograms). Averaged changes in CD83 and CD86 expression are summarized in Table 1.

   Table 1

Average changes in total moDCs expressing CD83 and CD86

Cytokine- or soluble IgG–induced shifts in the balance between activating and inhibitory Fc Rs affect susceptibility to immobilized IgG-mediated maturation.

We pretreated moDCs with factors that modulated the balance in favor of either activating or inhibitory FcRs, washed the cells to remove these factors, then recultured the moDCs in complete medium with 1% NHS, GM-CSF, and IL-4, in the presence or absence of immobilized IgG. Culturing IFN-–treated DCs in the presence of immobilized IgG led to increased DC maturation compared with cultures lacking immobilized IgG (62% CD83+ vs. 13% CD83+ in 4 experiments; Figure 4D). The relative increase in maturation was similar to conditions in which CD32b was blocked before coculture with immobilized IgG (Figure 4A). Conversely, culturing soluble IgG–treated moDCs with immobilized IgG led to only a 6% increase in CD83 expression (Figure 4E). These data indicated that immobilized IgG ligated predominantly activating FcRs on IFN-–treated moDCs or CD32b on IgG-treated moDCs. Pharmacologic modulations of the balance between CD32a and CD32b could therefore affect IgG-mediated maturation.

Differences in affinity for IgG ligand cause a functional shift in the balance between CD32a and CD32b.

Coculturing untreated moDCs from CD32a131HH or CD32a131HR individuals with immobilized IgG (Figure 4C, filled histogram) yielded a greater increase in maturation compared with moDCs from RR individuals (average absolute increase in CD83+ cells: 27% vs. 7%; filled histograms in Figure 4, C and F, respectively; P < 0.001). Immobilized mouse IgG1 in the same RR samples, however, led to substantial maturation (Figure 4F, open histograms) and a loss of the discrepancy between the different CD32a131 phenotypes. In the context of a fixed quantity of FcRs, differences in ligand avidity can thus cause a shift in the functional balance between CD32a and CD32b.

Coligation of CD32b limits CD32a-mediated cytokine release.

After ligating IgG to CD32a, CD32b, or both, as outlined above, we collected cell-free supernatants at 24–48 hours and measured a panel of cytokines using a multiplexed bead assay. Samples from CD32a131HH and -HR individuals are shown in Figure 5A. Simultaneous ligation of CD32b and CD32a (no pretreatment with blocking mAbs) led to suppressed levels of inflammatory cytokines compared with targeting CD32a alone. Differences were statistically significant for TNF- (P < 0.001), IL-6 (P = 0.02), and IL-8 (P = 0.002) secretion (n = 6 experiments). Significant levels of IL-1? and IL-12p70 were not detected under any condition, and therefore results are not shown. These data offer further functional evidence of the inhibitory role of CD32b.

   Figure 5

Coligation of CD32b limits CD32a-mediated cytokine release. Immature moDCs were cocultured with immobilized IgG to target FcRs. After 2 days, supernatants were collected and cytokines measured using a flow cytometry–based multiplexed bead assay. Mean cytokine levels (picograms per milliliter) are plotted on the y axis. (A) For samples derived from CD32a131HH or -HR donors, CD32a, CD32b, or both were ligated on immature moDCs after first blocking or not with the mAb to the other isoform (n = 6 independent experiments). P values for each cytokine reflect differences between CD32a-targeted DCs and DCs on which CD32a and CD32b were targeted simultaneously. (B) IFN-– and soluble IgG–mediated shifts in the expression of CD32a and CD32b were also assessed (n = 4 independent experiments). P values for IFN-–treated DCs (top) and IgG-treated DCs (bottom) reflect differences between DCs from respective conditions cocultured with or without immobilized IgG. (C) Immobilized mouse IgG was used as the ligand for FcRs from CD32a131RR samples (n = 3 independent experiments).

We also tested cytokine release after ligation of immobilized human IgG to FcRs on IFN-– and soluble IgG–treated immature moDCs (CD32a131HH and -HR samples), which favor the respective expression of either CD32a or CD32b. Soluble IgG–treated moDCs cocultured with immobilized IgG did not increase secretion of TNF-, IL-6, or IL-8, compared with soluble IgG–treated moDC controls (Figure 5B). In contrast, ligating FcRs on IFN-–treated DCs led to enhanced secretion of TNF- (P < 0.001), IL-6 (P < 0.001), and IL-8 (P = 0.01) compared with IFN-–treated DCs cultured without immobilized IgG (Figure 5B). The results were similar to those for DCs on which CD32a alone was ligated (Figure 5A).

The HH/HR versus RR phenotypes have functional significance in cytokine release assays.

Compared with samples from CD32a131HH and -HR individuals (Figure 5A), coculturing moDCs from CD32a131RR individuals with immobilized human IgG did not significantly enhance cytokine production (Figure 5C). Because the CD32a131R subtype has a high affinity for murine IgG1 (10), however, immobilized mouse IgG (Figure 5C) led to marked increases in TNF- (P = 0.02), IL-6 (P = 0.04), and IL-8 (P = 0.01) levels that were similar to those in CD32a-ligated DCs from CD32a131HH or -HR individuals (Figure 5A).

Taken together, the results indicate that shifts in the expression of CD32a and CD32b have functional consequences for moDC cytokine secretion. CD32a variants also determine moDC susceptibility to human or mouse Ig and hence the efficacy of certain mAb therapies (27).

Targeting CD32a versus CD32b has opposing effects on DC stimulatory capacity in the allogeneic mixed leukocyte reaction.

We targeted CD32a, CD32b, both, or neither, on immature moDCs and measured effects on DC immunogenicity using allogeneic mixed leukocyte reactions (alloMLRs). Results from 5 experiments using CD32a131HH and -HR samples are shown in Figure 6A. CD32a-targeted DCs were the most potent stimulators of allogeneic T cells (P < 0.001; Figure 6A, triangles) because of the maturation effect of ligating CD32a. Coligation of CD32b (circles) limited the absolute increase in stimulatory capacity otherwise mediated by targeting CD32a alone (P < 0.001). Specifically ligating CD32b (inverted triangles) did not support moDC maturation, so the stimulatory activity was no different than that in untreated immature DCs (squares). This is further evidence of the opposing functions of CD32a and CD32b on immature moDCs. These results also indicate that CD32b can actively inhibit CD32a-mediated DC activation and immunogenicity, even in the context of a very strong stimulus like alloantigen.

   Figure 6

Targeting CD32a or CD32b affects DC allostimulatory capacity in the MLR. Two days after ligating CD32a, CD32b, both, or neither on immature moDCs, the moDCs were harvested and washed. These moDCs were then recultured at varying doses with 105 allogeneic T cells in triplicate round-bottomed 96-microwell plates without additional cytokines. DC doses ranged from 3,000 to 300 cells per well, yielding DC:T cell ratios from 1:30 to 1:300. TdR uptake by proliferating allogeneic T cells over the last 12 hours of a 4–5 day culture was measured as an index of DC immunogenicity. (A) The averaged triplicate values (mean ± SEM) for TdR incorporation by T cells stimulated in 4 independent experiments using samples derived from CD32a131HH or -HR donors are depicted logarithmically (log2) on the y-axis. (B) Experiments using samples from CD32a131RR donors were performed in parallel, with immobilized mouse or human IgG ligating FcRs on the immature moDCs. The averaged triplicate values (mean ± SEM) from 3 independent experiments are depicted logarithmically (log2) on the y axis. Differences between conditions in A and B were tested using a stratified (by DC:T cell ratios) permutation t test.

The HH/HR versus RR phenotypes also affect reactivity to alloantigens in the alloMLR.

Ligating immobilized human IgG to FcRs on immature moDCs from CD32a131RR individuals did not significantly enhance immunogenicity (Figure 6B, triangles) compared with untreated immature moDCs (Figure 6B, squares). Coculturing these samples with immobilized mouse IgG (Figure 6B, circles), however, led to a marked increase in allostimulation (P < 0.001). This again confirms the functional significance of the CD32a131 polymorphism and exemplifies how differences in the avidity of CD32a for IgG can create a functional shift in the overall balance between activating and inhibitory FcRs.

    Discussion

Previous studies of CD32 expression and function on human DCs (4, 25, 28) could not distinguish between activating CD32a and inhibitory CD32b. We believe this is the first distinct characterization of CD32a and CD32b expression, modulation, and function on circulating human DCs, their precursors, and cytokine-induced moDCs. Nearly all circulating conventional DCs and a major population of moDCs express both CD32a and CD32b, which have opposing effects on IgG-mediated maturation and function of DCs. Ligating complexed human IgG to CD32a matures and activates moDCs in proportion to the frequency of CD32a expression, without any apparent bystander effect. This supports potent DC function in the alloMLR, a standard assay of DC immunogenicity, and in the release of proinflammatory cytokines. Ligation of CD32b significantly limits these immunogenic functions.

Cross-linking CD32a by complexed IgG induces phosphorylation of a cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM) (29). In contrast, CD32b bears a cytoplasmic immunoreceptor tyrosine-based inhibition motif (ITIM). Coligation of CD32b initiates tyrosine phosphorylation of the ITIM that inhibits ITAM-mediated events (29). Coexpression of these divergent receptors, which share almost identical ligand-binding domains, therefore establishes a threshold of DC activation.

CD32b plays a major role in regulating immune responses in mice. Genetic deletion of CD32b predisposes to autoimmunity (30) and results in pathologically enhanced immune responses (31). We have studied the expression and modulation of CD32a and CD32b on the surface of live human cells with an antibody that detects an extracellular domain of CD32b. Unlike previously available reagents, this mAb does not cross-react with CD32a and finally permits an assessment of surface-expressed FcRs. This is more physiologically relevant than prior methods that measured total CD32b protein or mRNA from cell lysates, which include the large intracellular compartment in addition to surface FcRs.

Monocytes have emerged as the predominant effectors of ADCC against tumor cells in vivo (32). In contrast to their constitutive expression of the activating FcRs, CD32a and CD64, monocyte expression of the inhibitory CD32b is surprisingly diverse, ranging from 1–48% among 30 healthy volunteers. This previously unrecognized variability in the balance between activating and inhibitory FcRs in the resting state may influence the quality and/or magnitude of antibody-based immune responses.

Perturbations in the balance between activating and inhibitory FcRs may affect the pathophysiology of autoimmune diseases (33) or the efficacy of antibody-based therapies (8, 34). For example, TNF-–mediated suppression of CD32a and CD32b may lead to decreased immune complex clearance in vivo. This may be one mechanism by which TNF- contributes to an immune complex–mediated disease like rheumatoid arthritis (35). While dexamethasone increases CD32a expression, it also increases CD32b and could shift the balance in favor of the inhibitory FcRs, accounting for a treatment effect of steroids on autoimmune diseases. This may also underlie the negative effect of steroids on ADCC against tumors (36). Increased CD32b expression in other settings may actually improve therapeutic responses to IVIG (34). The addition of any adjunctive treatment to mAb therapy must therefore consider the consequences this has on therapeutic efficacy, owing to alterations in the ratio of activating to inhibitory FcRs.

In a murine model of immune thrombocytopenic purpura, Samuelsson et al. showed that IVIG requires the presence of CD32b to mediate protective antiinflammatory effects and that IVIG increases CD32b expression on splenic macrophages (34). We found that antiinflammatory concentrations of soluble monomeric IgG decrease CD32a expression on immature moDCs without significantly affecting CD32b. Accordingly, soluble IgG–treated DCs do not gain allostimulatory capacity (data not shown) or increase production of inflammatory cytokines after coculture with immobilized IgG. The net effect on the balance between activating and inhibitory FcRs on murine macrophages and human DCs is similar, however, and yields the same functional sequelae. Our findings are therefore consistent with those from mouse studies showing that the overall balance between activating and inhibitory FcRs is pertinent to the mode of action of IVIG (34).

Several lines of evidence show that optimum immune rejection of tumors or infectious pathogens requires coordinated cellular and humoral immune responses (37, 38). Though most often studied for their ability to stimulate antigen-specific T cells, DCs can also potently affect innate (39) and humoral (40) immune responses. We found that ligating CD32a on moDCs leads to secretion of IL-10 and IL-6, which stimulate B cells and plasma cells, and TNF- and IL-8, which serve as chemoattractants for other innate effector cells like neutrophils. Despite the apparent Th2 type of response, and the absence of IL-12p70, CD32a-targeted DCs were able to stimulate more potent allogeneic T cell proliferation compared with control DCs. This reveals a unique type of maturation through which DCs can play a key role in recruiting a diversified immune response against antibody-coated pathogens or tumors.

Similar findings regarding the expression and function of CD32b on human moDCs were recently published (41). We did not find, however, that CD32b blockade increased secretion of IL-12p70. Our results are consistent with previous reports of absent or diminished IL-12p70 production after triggering CD32a on human monocytes (42) and moDCs (25, 43). This discrepancy may derive from differences in methodology, e.g., the specific conditions of FcR blockade or subsequent removal versus inclusion of excess blocking mAb from DC cultures. We also used immobilized IgG to ligate the unblocked activating FcRs, instead of relying on circulating immune complexes in serum that were diluted 100-fold in culture (41). This may have led to differences in the extent of FcR cross-linking and hence the divergent biological responses. We also blocked CD32b and recultured moDCs in the presence of 1% serum without immobilized IgG, and we did not find a statistically significant increase in moDC maturation or activation. We do not know whether blockade of CD32a in the other system would have prevented maturation and IL12p70 production or whether an alternative activation pathway might have been operative.

Blockade of cell activation has unique implications for DCs. While mature DCs are the most potent stimulators of immunity, immature DCs can mediate the opposite task of generating antigen-specific tolerance (9). We have shown that engaging CD32b, by first blocking CD32a or pretreating DCs with soluble IgG, limits IgG-mediated maturation and activation of moDCs, even in the context of a very strong immunogen such as alloantigen. CD32b helps maintain B cell tolerance (3), but we believe this is the first evidence that CD32b may play an active role in limiting DC maturation and hence in inducing T cell tolerance as well. That CD32b can mediate the uptake of immune complexes (44) suggests a rationale for targeting antigens to this receptor to induce antigen-specific tolerance, and such studies are underway. It also implies that tumor cells coated with therapeutic mAbs could inadvertently initiate an inhibitory pathway with undesirable results.

In the setting of fixed numbers of CD32a and CD32b surface epitopes, differences in functional avidity for IgG can shift their functional balance. In our studies using human IgG as ligand, the maturation and functional sequelae are most evident in samples from donors displaying the high-binding CD32a131 alleles. Samples with low-affinity receptors are less affected by immobilized human IgG but are instead matured and activated by mouse IgG1. This indicates one of the mechanisms by which CD32a polymorphisms may affect immune responses to self or pathogenic/tumor antigens (27, 45). It also highlights how the species of IgG ligand can shape laboratory (6) or clinical studies (46) involving human FcRs.

These data have shown that subsets of human DCs express a pair of receptors that share a common ligand but mediate opposing functions. These can elicit divergent immune responses. The balance of available activating versus inhibitory FcRs determines the net response. We have altered this balance by using blocking mAbs, modulating the expression of activating and/or inhibitory FcRs, or using ligands of differing affinities for the respective FcRs. These findings have important implications for elucidating the pathophysiology of autoimmune diseases, optimizing mAb therapies, engineering mAbs to target specific FcRs (47), and rationally targeting Ags to FcRs on DCs in vitro or in vivo.

    Methods

Media, sera, and Igs.

Complete RPMI 1640 was supplemented with 10 mM HEPES, 1% penicillin/streptomycin (Media Laboratory, Memorial Sloan-Kettering Cancer Center ), 50 μM 2-ME (Invitrogen Corp.), 1% L-glutamine (Invitrogen Corp.), and heat-inactivated NHS (Gemini Bio-Products). All media and reagents were endotoxin free. Stock solution of therapeutic-grade sterile 10% pooled human IgG (Gamunex; gift of Bayer HealthCare, Biological Products Division) was 100% monomeric by fast protein liquid chromatography (data not shown). Pooled human IgG, mouse IgG1 (Sigma-Aldrich), and mouse IgG Fabs (Jackson ImmunoResearch Laboratories Inc.) were diluted immediately before use in sterile PBS.

Cytokines.

Sterile recombinant, endotoxin-, pyrogen-, carrier-, and mycoplasma-free human cytokines used to support generation of moDCs in vitro and/or to modulate FcR expression included GM-CSF (Berlex); IL-1?, IL-2, IL-3, IL-4, IL-6, IL-10, TGF-?1, TNF- (all from R&D Systems); IFN-2b (INTRON A; Schering Corp.); IFN--1b (Actimmune; InterMune); and PGE2 (Calbiochem).

Cell purification and generation of DCs.

Informed consent was obtained for research specimen collection using protocols approved by the Institutional Review and Privacy Board of Memorial Hospital, MSKCC. PBMCs were obtained from whole blood or leukocyte concentrates (Blood Bank, MSKCC) by density centrifugation over Ficoll-Paque PLUS (endotoxin-free, no. 17-1440-03; Amersham Pharmacia Biotech). Neutrophils were purified from the rbc pellet after hypotonic lysis of erythrocytes.

Circulating blood DCs were enriched from buffy coats by negative immunomagnetic selection according to the manufacturer’s instructions (StemSep Human Dendritic Cell Enrichment Cocktail; StemCell Technologies). Enriched populations were cultured in 10% NHS with IL-3 (10 ng/ml) to support CD11c–CD123bright plasmacytoid DCs (24) or without IL-3 for CD11c+CD123low "myeloid" or conventional DCs. Immature moDCs were generated over 5–6 days from tissue culture plastic-adherent PBMCs (CD14+ monocyte precursors) in the presence of GM-CSF (1000 IU/ml) and IL-4 (500 IU/ml) in complete RPMI–1% NHS (48). Fresh medium and cytokines were replenished every 2 days.

Antibodies and secondary reagents for staining and blocking.

Direct FITC-, PE-, allophycocyanin-, PE-cyanine-5– (PC-5–), and PC-7–conjugated mouse anti-human mAbs included anti-CD3, anti-CD11c, anti-CD14, anti-CD16, anti-CD19, anti-CD20, anti-CD56, anti-CD64, anti–CD83(-FITC), anti-CD86, anti-CD123, anti–HLA-DR (BD Biosciences — Pharmingen); and anti–CD83(-PE) (Immunotech). Anti-human CD32 mAbs included clone 41H16 (kind gift of Jan G.J. van de Winkel, University Medical Center, Utrecht, The Netherlands); clone IV.3 (HB-217; ATCC), with Fab fragments produced by the MSKCC Monoclonal Antibody Core Facility; clone 2B6 ; PE-conjugated clone CIKm5 (CALTAG Laboratories); and FITC-conjugated clones FL18.26 and 3D3 (BD Biosciences — Pharmingen). Isotype controls included the appropriate fluorochrome-conjugated or unconjugated mouse IgG1, IgG2a, or IgG2b (DakoCytomation and BD Biosciences — Pharmingen). Unconjugated primary Igs were secondarily stained with FITC- or PE-conjugated F(ab')2 fragments of goat anti-mouse IgG (Jackson ImmunoResearch Laboratories Inc.). Flow cytometry studies used a Cytomics FC 500 (Beckman Coulter), gating for collection and analysis of at least 10,000 live cells.

Production of 2B6 mAb against CD32b.

Human CD32a-transgenic mice were immunized with a recombinant protein containing the complete extracellular domain of human CD32b. Spleen cells from immunized mice were fused with a proprietary myeloma cell line (MacroGenics Inc.). Resulting mAbs secreted by the hybridomas were screened by ELISA for differential binding of the inhibitory and activating isoforms of CD32. Specificity and selectivity were further confirmed by surface plasmon resonance analysis and flow cytometry of stained Burkitt lymphoma–derived cell lines and human peripheral blood leukocytes. 2B6 was a CD32b-blocking clone selected among several anti-CD32b–secreting hybridomas for its ability to react with CD32b at high titers with no binding to CD32a. Furthermore, clone 2B6 blocked binding of aggregated human IgG to CD32b (M.-C. Veri et al., unpublished data).

Modulation of Fc Rs.

The following specific factors were added to immature moDC cultures starting on day 3 or 4: IL-1? (2 ng/ml), IL-2 (6,000 IU/ml), IL-3 (10 ng/ml), IL-6 (1,000 IU/ml), IL-10 (25 ng/ml), IFN- (1,000 U/ml), IFN-2b (1,000 U/ml), PGE2 (5 μM), TGF-? (10 ng/ml), and TNF- (5 ng/ml). IFN-–treated DCs were very adherent and required 5 minutes incubation with 0.4 μM EDTA for harvesting. Pooled human IgG was added to yield a final concentration approaching that achieved after therapeutic administration of IVIG (0.15 mM). Sterile therapeutic grade dexamethasone (American Regent Laboratories) was added to yield a final concentration of 0.1 μM. All conditions used only 1% NHS, except the one condition in which FCS was evaluated. Cells were harvested for phenotypic or functional studies on day 6–7.

To assess the effects of different maturation stimuli on FcR expression, the following were added to separate cultures of immature moDCs on day 6–7: cocktail of inflammatory cytokines (IL-1? , IL-6 , TNF- ) and PGE2 (5 μM) (23), LPS (10 ng/ml), or CD40L-transfected murine fibroblasts (5 DCs per 1 CD40L-expressing fibroblast; kind gift of Jacques Banchereau, Baylor Institute for Immunology Research, Dallas, Texas, USA) (49). Cells were harvested 2 days later and evaluated for effects on FcR expression. Maturation was confirmed by upregulation of cell-surface CD83 on more than 90% of cells. Gating on HLA-DRbright cells during cytofluorographic analysis excluded CD40L-transfected fibroblasts.

Quantitative flow cytometry.

The frequencies of respective FcR+ cells per population, and relative changes thereof, were calculated as the number of viable immature (CD14–CD83–HLA-DR+) DCs with more intense staining than that of the isotype control, divided by the number of viable moDCs in the analysis gate. To quantify receptor density per cell, we used standardized antibody-binding beads (Quantum Simply Cellular; Bangs Laboratories Inc.), which comprise 4 populations with varying capacities to bind mouse IgG (e.g., detection mAbs) and 1 nonbinding blank population. The beads were stained and evaluated in parallel with moDCs using the same anti-FcR detection reagents. A standard calibration curve was generated by plotting the known antibody-binding capacity of each bead population against the measured MFI of the bound detection mAb. We then calculated the number of detection mAbs bound per cell from the MFI of each sample using the standard curve. Whole IgG1 formulations of clones CIKm5 (anti-CD32a) and 2B6 (anti-CD32b) were used at saturating concentrations to quantify CD32a and CD32b. Staining with Fab fragments of clone IV.3 and F(ab')2 fragments of 2B6 yielded the same changes in the frequency of CD32a+ or CD32b+ DCs and shifts in MFI.

Ligation of Fc Rs with immobilized IgG.

We immobilized IgG on plastic plates to mimic the effect of complexed IgG (25). Pooled human or mouse IgG (both 1.0 mg/ml) was added to each well of a 96-well round-bottom tissue culture–treated plate. Plates were washed 4 times with PBS after overnight incubation to yield immobilized IgG. For selective ligation of CD32a, harvested day 5–6 immature moDCs were washed with PBS; blocked with anti-CD32b (clone 2B6, 5 μg/ml) by incubation at 4°C for 30 minutes; washed with PBS 3 times to remove excess blocking mAb; resuspended in complete medium supplemented with 1% NHS, GM-CSF, and IL-4; and recultured in 96-well plates with immobilized IgG (105 cells/0.1 ml/well). For selective ligation of CD32b, cells were blocked with mAb IV.3 (10 μg/ml) before reculture with or without immobilized IgG. Preincubation without mAbs allowed simultaneous ligation of CD32a and CD32b. For each condition, cells added to plates lacking immobilized IgG served as negative controls. For experiments that used immobilized mouse IgG1, plates with immobilized Fab fragments of mouse IgG served as negative controls to rule out an effect of xenogeneic protein. Where indicated, treated moDCs were washed to remove excess soluble IgG or IFN- before reculture with or without immobilized IgG.

T lymphocytes.

T cells for alloMLRs were obtained from tissue culture plastic-nonadherent PBMCs, then further purified by nonadherence and elution from nylon wool columns (Polysciences Inc.). Purity was greater than 90–95% based on CD3 expression.

Cytokine measurement by cytometric bead array.

Supernatants of DCs were collected 2 days after FcR ligation, immediately frozen, and thawed once for assay. Levels of IL-1?, IL-6, IL-8, IL-10, IL-12p70, and TNF- were measured simultaneously using a Human Inflammation Cytometric Bead Array (CBA) Kit (BD Biosciences — Pharmingen) according to the manufacturer’s instructions.

AlloMLRs.

DCs were harvested 48 hours after FcR ligation, extensively washed, then cocultured with 105 purified allogeneic T cells in triplicate round-bottomed microwells (Costar 3799; Corning Inc.) at variable ratios from 1:30 to 1:300 (DC:T cells), in complete RPMI 1640 supplemented with 10% NHS. Methyl-thymidine (TdR; 1 μCi/well; New England Nuclear, PerkinElmer) was added to each well on day 4–5, and plates were harvested 10–12 hours later using a Harvester 96 Mach III (Tomtec). Proliferation of responder T cells was based on TdR incorporation, which was measured using a microplate scintillation counter (TopCount NXT; PerkinElmer).

Biostatistics.

The 2-sample t test was used to compare expression of CD83 and CD86, and concentrations of IL-10, IL-6, and TNF- between experimental conditions. Due to truncation of values at 5,000, the Mann-Whitney U test was used to compare concentrations of IL-8. We analyzed differences in TdR uptake between conditions in the alloMLR across a range of DC:T cell ratios. The differences were tested using a stratified (by DC:T cell ratios) permutation t test, where the subject was the permutation unit, in order to maintain the correlation structure between T cell doses. P values less than or equal to 0.05 were considered statistically significant.

    Acknowledgments

This work was supported by grants from the Charles A. Dana Foundation and the Mortimer J. Lacher Fellowship Fund, MSKCC (to A.M. Boruchov); the National Cancer Institute, NIH (R01 CA 83070 to J.W. Young; P01 CA 59350 to J.W. Young and G. Heller; and P01 CA 23766 to J.W. Young and G. Heller); the National Institute of Allergy and Infectious Diseases, NIH (P01 AI 51573 to J.V. Ravetch); the Alliance for Lupus Research (to J.V. Ravetch); and William H. Goodwin and Alice Goodwin of the Commonwealth Foundation for Cancer Research through The Experimental Therapeutics Center of MSKCC (to A.M. Boruchov and J.W. Young). We appreciate the technical assistance of Michael Basedow.

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1Department of Internal Medicine III, Division of Nephrology and Dialysis, Medical University Vienna, Vienna, Austria.

2Intercell AG, Vienna, Austria.

3Department of Internal Medicine III, Clinical Division of Endocrinology and Metabolism, Medical University Vienna, Vienna, Austria.

4Ludwig Boltzmann Institute for Rheumatology, Vienna, Austria.

5Institute of Immunology, Medical University Vienna, Vienna, Austria.

6Center of Molecular Medicine (CeMM), Austrian Academy of Sciences, Clinical Division of Endocrinology and Metabolism, Medical University Vienna, Vienna, Austria.

7Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan.

    Abstract

Tamm-Horsfall glycoprotein (THP) is expressed exclusively in the kidney and constitutes the most abundant protein in mammalian urine. A critical role for THP in antibacterial host defense and inflammatory disorders of the urogenital tract has been suggested. We demonstrate that THP activates myeloid DCs via Toll-like receptor-4 (TLR4) to acquire a fully mature DC phenotype. THP triggers typical TLR signaling, culminating in activation of NF-B. Bone marrow–derived macrophages from TLR4- and MyD88-deficient mice were nonresponsive to THP in contrast to those from TLR2- and TLR9-deficient mice. In vivo THP-driven TNF- production was evident in WT but not in Tlr4–/– mice. Importantly, generation of THP-specific Abs consistently detectable in urinary tract inflammation was completely blunted in Tlr4–/– mice. These data show that THP is a regulatory factor of innate and adaptive immunity and therefore could have significant impact on host immunity in the urinary tract.

    Introduction

Professional APCs, such as DCs, are pivotally positioned at the interface of innate and adaptive immunity (1). Recognition of pathogens leads to a typical maturation program including upregulation of costimulatory and MHC molecules, migration of DCs to adjacent lymph nodes, and subsequent priming of naive antigen-specific T cells. For pathogen recognition, DCs are specifically equipped with distinct pattern recognition receptors such as Toll-like receptors (TLRs) to detect pathogen-associated molecular patterns (2, 3). Microbial components recognized by TLR have been identified for TLR2 (e.g., peptidoglycan), TLR3 (e.g., double-stranded RNA), TLR4 (e.g., LPS), TLR5 (bacterial flagellin), TLR6 (e.g., mycoplasmal macrophage-activating lipopeptide 2 kDa), TLR9 (e.g., unmethylated bacterial CpG DNA), and TLR11 (uropathogenic bacteria) (4, 5). Moreover, various conserved sugar residues have been shown to be involved in accessory cell stimulation in concert with TLR-dependent cellular activation by putative lectin-binding receptors (6-8). Recently, distinct host-derived molecules such as fibronectin, hyaluronic acid, or ?-defensin 2 have also been shown to signal through TLR (9-11). Hence, TLRs are thought to essentially contribute to transduce signals by exogenous as well as endogenous molecules to activate distinct immune cells or to control tissue integrity, respectively (12).

Tamm-Horsfall glycoprotein (THP), or uromodulin, is the most abundant protein in normal human urine, present at 30–50 mg/24 hour (13). It is expressed only in the thick ascending limb of Henle’s loop in the kidney, and it is cleaved from its GPI anchor to be secreted into the urine. The relative abundance and specific localization of THP indicates an important physiological role in the urinary tract, which still remains to be elucidated. Numerous clinical and experimental studies have indicated an involvement of THP in several forms of inflammatory kidney disease (13-15). Importantly, anti-THP Abs are consistently found in the peripheral blood of patients with interstitial nephritis or urinary tract infection (15, 16). THP constitutes the matrix of all urinary stones, and there is a well-known relationship between interstitial stone deposition and surrounding leukocytes (17). Recent studies have shown a proinflammatory role of THP through triggering of inflammatory molecule induction in human monocytes as well as in granulocytes (18, 19). Significantly, recent data indicated a role for THP in the antibacterial defense system of the urinary tract, since THP KO mice were highly susceptible to severe urinary bladder and kidney infection (20, 21).

We hypothesized that THP governs the performance of professional APCs such as DCs, thereby affecting local immune responsiveness. We show here that immature DCs exposed to THP develop into fully mature DCs. The molecular action of THP was dependent on TLR4 signaling and resulted in NF-B activation. In vivo application of THP induced a prominent anti-THP Ab response that was fully blunted in TLR4 KO mice. The immunostimulatory effects of THP by TLR4 could represent an important host defense mechanism employed in the human urinary tract system.

    Results

THP induces phenotypic maturation of human monocyte-derived DCs.

We first sought to determine whether THP is able to induce maturation of immature DCs as reflected by phenotypic changes. DCs exposed to THP for 48 hours expressed the typical DC maturation markers CD83 and CD25 (Figure 1A). Furthermore, the costimulatory molecules CD80 and CD86, as well as the antigen-presenting molecule HLA-DR, were upregulated. These phenotypic features exactly matched the DC maturation profile induced by LPS (Figure 1A).

   Figure 1

THP activates professional APCs. (A) Immature monocyte-derived human DCs were stimulated with THP or LPS. Profiles with fine lines represent staining patterns with an isotype-matched control Ab, and profiles with bold lines represent staining with mAb of the indicated specificity. Data are representative of 5 independent experiments. Numbers indicate mean fluorescence intensity of specific Ab staining. (B) Immature human DCs were stimulated with different concentrations of THP or LPS. Cell-free supernatants were collected 18 hours after addition of THP or LPS and were analyzed by ELISA. Med, medium control. (C) Intracellular cytokines were stained in human DCs 18 hours after stimulation and then analyzed by FACS. Results are representative of 3 independent experiments. (D) Mouse RAW264.7 macrophages were exposed to THP, LPS, and CpG for the indicated time periods. Cell-free supernatants were analyzed for TNF- by ELISA. Similar results were obtained in 3 other independent experiments and data are expressed as means ± SD of triplicate cultures in a representative experiment.

In addition, maturation of DCs as reflected by IL-12p40 and TNF- production was examined in THP- or LPS-stimulated DCs. THP was a potent inducer of proinflammatory cytokine secretion (Figure 1B) and production (Figure 1C). We also studied whether THP had effects on murine myeloid cells. THP induced cytokine production in splenocytes, bone marrow–derived immature DCs, and the murine macrophage cell line RAW264.7 in a dose-dependent manner (Figure 1D and data not shown). These data indicate that THP potently stimulates myeloid immunocompetent cells leading to cytokine production and final maturation of immature monocyte-derived DCs.

LPS at concentrations as low as 50 pg/ml has been described to activate myelomonocytic cells (22). To exclude the possibility that LPS contamination of the THP preparation had caused immune cell stimulation, we first tested for their endotoxin content the materials used for DC culturing and found them to be endotoxin free (<0.06 U/ml) as confirmed by Limulus amebocyte lysate assay (data not shown). Furthermore, we preincubated the THP probe with polymyxin B, a specific inhibitor for LPS, before adding it to immature DCs. DC maturation induced by THP was independent of LPS, since polymyxin B did not affect THP-induced DC maturation but completely prevented activation by LPS (Figure 2A). Similarly, absorption over a polymyxin B column, which effectively eliminates LPS, did not affect TNF- production induced by the THP preparation but abolished the response to LPS (Figure 2B). Conversely, proteinase K treatment did not inhibit LPS-induced TNF- production but restrained activation by THP.

   Figure 2

DC maturation induced by THP is not due to LPS or protein contamination. (A) THP and LPS were left untreated or pretreated with polymyxin B (PMB) for 2 hours and then added to human immature DCs. After 48 hours, cells were harvested and analyzed by FACS. Profiles with fine lines represent staining patterns with an isotype-matched control Ab, and profiles with bold lines represent staining with a mAb of the indicated specificity. Data are representative of 5 independent experiments. (B) THP was incubated with PMB beads overnight. The supernatant was analyzed for the presence of THP by SDS-PAGE. Additionally, THP was incubated with proteinase K (Prot.K) for 45 minutes as described (11). The PMB-purified and the proteinase-treated samples were incubated with RAW 264.7 macrophages for 18 hours and analyzed for TNF-. LPS as a control was treated similarly. Similar results were obtained in another independent experiment. (C) Effect of THP and LPS on the induction of TF activity in HUVECs. HUVECs were pretreated with or without IFN- and then exposed to THP or LPS. A 1-stage clotting assay was used to determine TF activity. The results are representative of 3 independent experiments. (D) LPS, Pam3Cys (P3C), THP isolated by standard NaCl precipitation (THP), THP isolated by NaCl precipitation and ultracentrifugation (THP-UC), and THP isolated by NaCl precipitation, ultracentrifugation, and diatomaceous earth filter (THP-DEF) were added to C57BL/6 splenocytes for 20 hours. Cell-free supernatants were analyzed for TNF- by ELISA.

Furthermore, we sought to determine a potential functional effect of possible LPS contamination in the THP preparation. Recent data demonstrated that THP, in contrast to LPS, is not able to stimulate the expression of tissue factor (TF) in human umbilical vein endothelial cells (HUVECs) (18). LPS induced TF expression in HUVECs, whereas THP was not able to induce TF in endothelial cells even in IFN-–pretreated cells that are highly sensitive to LPS (Figure 2C). These results suggest a functional difference in LPS and THP signaling. Collectively, these experiments rule out the possibility that the effects of THP on DCs are due to contamination with LPS.

To exclude contaminating molecules other than LPS, the standard THP preparation obtained by repeated NaCl precipitation from urine was further purified by 2 sequential procedures taking advantage of the fact that THP forms a gel when monovalent ions above 60 mM are present. After induction of gel formation by 70 mM sodium phosphate, THP was purified by ultracentrifugation (23). The pellet fraction containing THP was fully active on C57BL/6 splenocytes for TNF- and IL-6 production (Figure 2D and data not shown). Moreover, the ultracentrifuged THP was further purified over a diatomaceous earth filter, which is known to purify THP to homogeneity (24). This THP preparation highly purified by 3 different methods was still able to induce TNF- production (Figure 2D). These data strongly indicate that THP but not other possible contaminants stimulates myeloid cells to induce cytokine production.

Functional consequences of THP-induced DC maturation.

Another hallmark of mature DCs is their ability to trigger potent T cell stimulation. THP-activated human DCs were used as stimulator cells in a standard mixed lymphocyte culture (MLC). Interaction of THP with immature DCs led to a dramatic enhancement of their stimulatory capacity (Figure 3A). Further analysis of T cell cytokine production revealed that high levels of IL-2 and IFN- existed in allogeneic T cell cultures challenged with THP-treated DCs (Figure 3B). These data demonstrate a strong T cell responsiveness that occurs after activation of immature DCs by THP.

   Figure 3

Induction of T cell stimulatory capacity and type 1 immune responsiveness by THP. (A) Immature human monocyte-derived DCs were stimulated with THP or LPS, washed extensively, irradiated, and subsequently cocultured with highly purified allogeneic T cells at the indicated ratios. After 48 hours, the cells were used as allogeneic stimulators as described above. DNA synthesis was assessed on day 5 and is expressed as mean counts per minute of a representative experiment. SD of triplicates were generally below 20%. Similar results were obtained in 4 other experiments. (B) For T cell cytokine production, immature DCs were stimulated with THP at the indicated concentrations or LPS, extensively washed, irradiated, and subsequently cocultured with highly purified allogeneic T cells at a DC/T cell ratio of 1:2. Cell-free supernatants were collected and analyzed by ELISA for IL-2 and IFN- secretion. Data are expressed as mean ± SEM of 4 different donor combinations.

THP induces activation of p38-MAPK, ERK-1/2, and Akt.

DC maturation has been shown to be associated with phosphorylation of distinct tyrosine kinases, including ERK, p38, and Akt (25). Addition of THP to human DCs (Figure 4A) or murine macrophages (Figure 4B) led to rapid phosphorylation of p38-MAPK and ERK within 15–30 minutes. Interestingly, we consistently found delayed kinetics of p38 and ERK phosphorylation compared with LPS. Next, we analyzed the PI3K/Akt pathway, which is involved in activation and survival of many cell types, including DCs (25). The peak of Akt phosphorylation in LPS-stimulated DC cultures was detected after 30 minutes, whereas THP-stimulated DCs also exhibited a kinetic shift as seen in the MAPK activation profile (Figure 4C). Highly selective inhibitors of ERK-1/2 (PD98059 and UO126) or p38 (SB203580) effectively blocked THP-induced TNF- production in immature DCs, demonstrating a requirement of MAPK activation for cytokine production (Figure 4D). These results indicate that THP engages DC-signaling pathways similar to those of LPS and point to a critical involvement of MAPK phosphorylation in THP-mediated DC activation.

   Figure 4

Immunostimulatory effects of THP are dependent on p38-, ERK-1/2–MAPK kinase and NF-B signaling. Immature human DCs (A) or murine macrophages (B) were incubated with THP, LPS, or medium. Subsequently, phospho-p38 (p-p38) and phospho–ERK-1/2 (p-ERK) were determined by immunoblotting; p38 and ERK-2, respectively, were detected from stripped membranes. Blots are representative of 4 independent experiments. (C) Immature DCs were incubated with THP, LPS, or medium, and phospho-Akt (p-Akt) as well as total Akt were determined from whole cell lysates by immunoblotting. Data are representative of 3 independent experiments. (D) Immature human DCs preincubated with or without the indicated MAPK inhibitors were exposed to THP, LPS, or medium. Cell-free supernatants were collected after 18 hours and were analyzed for TNF- by ELISA. Data are expressed as mean ± SEM of 4 different donor combinations. *Significantly different from the values for stimulated control; P < 0.05. (E) Immature DCs were incubated with THP, LPS, or medium, and immunoblotting was performed from whole-cell lysates using Abs against IB- and ERK-2. (F) THP, LPS, or medium was added to immature human DCs. Oligonucleotides labeled with 32P, containing a NF-B consensus sequence, were incubated with nuclear extracts followed by nondenaturing gel electrophoresis. Similar results were obtained in 2 independent experiments. (G) Immature human DCs preincubated with or without the NF-B inhibitor SN50 were exposed to THP, LPS, or medium. Cell-free supernatants were collected after 18 hours and were analyzed for cytokines by ELISA. Data are representative of 4 independent experiments. *Significantly different from the values for stimulated control; P < 0.05.

THP activates the NF-B–signaling pathway in immature DCs.

Transactivation of NF-B is essential for proper DC maturation (26). Degradation of IB- in THP-stimulated DCs indicated activation of the NF-B–signaling pathway (Figure 4E). To directly assess nuclear translocation of NF-B, electrophoretic mobility shift assays (EMSAs) of nuclear extracts were performed. THP induced NF-B translocation in a time-dependent manner, detecting prominent signals 40 minutes after stimulation (Figure 4F). Inhibition of cytokine production by the specific NF-B inhibitor SN50 that was recently shown to prevent LPS-mediated NF-B transactivation in DCs (25) further corroborated these findings (Figure 4G). These data indicate that activation of NF-B is a prerequisite for successful activation of immature DCs by THP.

Involvement of TLR4 in the immunostimulatory effects of THP.

The overlapping activation pattern accomplished by THP and LPS prompted us to assess a possible role of the TLR-signaling pathway in the immunostimulatory effects exerted by THP. Here we show that IL-1 receptor–associated kinase 1 (IRAK-1), a serine/threonine kinase involved in early TLR-mediated NF-B activation (27), was degraded in THP-stimulated immature human DCs in a time-dependent manner (Figure 5A). Furthermore, selective inhibition of TIRAP, a critical adaptor protein in the signaling pathway downstream of TLR1, TLR2, TLR4, and TLR6, but not TLR5, TLR7, and TLR9 (28), by a TIRAP/Mal inhibitory peptide abrogated THP-induced cytokine production in murine DCs (Figure 5B). To investigate a role of TLR4 in THP-induced immune cell activation, human DCs were preincubated with the anti-TLR4 Ab, mAb HTA-125, and a significant impairment of cytokine production was observed (Figure 5C). These results indicate a critical role of TLR4 in THP-mediated immunostimulatory effects on DCs.

   Figure 5

Involvement of TLR signaling in APC activation by THP. (A) Immature human DCs were incubated with THP, LPS, or medium, and immunoblotting was performed from whole-cell lysates using antibodies against IRAK-1 and ERK-2, which served as loading control. A representative of 4 independent experiments is shown. (B) Bone marrow–derived murine DCs (BM-DCs) were treated with or without TIRAP/Mal peptide followed by stimulation with THP or LPS. After 18 hours, cell-free supernatants were collected and analyzed by ELISA. (C) Immature human DCs were pretreated with a mAb against TLR4 (HTA125) or a control mAb before addition of LPS or THP. Cell-free supernatants were then collected and analyzed by ELISA. Data are representative of 3 independent experiments. mDCs, monocyte-derived DCs. *Significantly different from the values for stimulated control; P < 0.05.

Impaired cytokine production upon stimulation with THP in MyD88- and TLR4-null, but not in TLR2- and TLR9-null, mice.

To obtain direct evidence of TLR activation upon THP engagement, we isolated bone marrow–derived macrophages from MyD88 and TLR2, TLR4, and TLR9 KO mice as well as from WT mice. While THP potently stimulated TNF- production in APCs obtained from WT, TLR2–/–, and TLR9–/– mice, cells from TLR4–/– and MyD88–/– mice were completely resistant to THP or LPS stimulation (Figure 6A). Importantly, myeloid cells from TLR4–/– mice were functionally active, since they fully responded to the TLR2 agonist Pam3Cys (Figure 6, A and B).

   Figure 6

The immunomodulatory effects of THP are exerted by a TLR4-dependent mechanism. Bone marrow–derived murine macrophages (BM-Mo) (A) or murine splenocytes (B) from C57BL/6 WT, TLR2, TLR4, TLR9, or Myd88 KO mice were stimulated with THP, LPS, or Pam3Cys. Cell-free supernatants were collected 18 hours after addition of LPS, THP, or Pam3Cys and analyzed for TNF- by ELISA. nd, not determined. *Significantly different from the values for activated controls in WT mice; P < 0.001. (C) Analysis of in vivo cytokine production in serum of C57BL/6 mice or TLR4 KO mice after i.v. administration of LPS or THP. One-hour postinjection serum levels of TNF- were analyzed by ELISA. bd, below detection limit. Similar results were obtained in another independent experiment. (D) THP was injected i.v. on days 0, 1, 2, and 7 into C57BL/6 mice or TLR4 KO mice, and the subsequent THP-specific Ab response (IgG) was determined by ELISA. Ab response is shown at a serum dilution of 1:500.

To analyze cytokine production in vivo, WT and TLR4–/– mice were challenged with intravenous administration of THP. One hour after THP or LPS injection, high levels of TNF- were detected in WT mice (Figure 6C). In contrast, no detectable amounts of TNF- were observed in TLR4–/– mice challenged with THP, indicating a complete unresponsiveness toward the immunostimulatory effects of the glycoprotein. Thus, the prominent proinflammatory cytokine induction by THP in vivo occurs by a TLR4-dependent mechanism.

THP-mediated Ab production in vivo is abrogated in TLR4–/– mice. Increased production of THP-specific autoantibodies is a consistent finding in several inflammatory disorders and infections of the urinary tract (13, 29, 30). Furthermore, THP has previously been shown to be a powerful autoantigen when rats, mice, or rabbits were challenged with autologous THP (31-33). We therefore were interested in the involvement of the TLR4-signaling pathway in the induction of an Ab response against THP. We detected a prominent IgG response in C57BL/6 mice within 7 days after intravenous THP delivery (Figure 6D). While WT mice displayed a typical Ab kinetic over a period of 35 days, TLR4–/– mice were dramatically impaired in their ability to produce specific Abs in response to THP challenge (Figure 6D). LPS was not able to induce any Ab response in both mouse strains (Figure 6D). These data show that TLR4 plays a pivotal role not only in THP-induced proinflammatory cytokine production, but also in the emergence of an anti–THP-directed immune response.

    Discussion

DCs link the innate with the adaptive immune system by their ability to sense different microbial stimuli and conveying this information to lymphocytes. This process is accomplished by pathogen recognition and subsequent induction of the DC maturation program comprising increased surface expression of costimulatory molecules, cytokine production, and potent antigen-presenting function. In this study the unique property of the abundant urinary glycoprotein THP to activate professional APCs such as DCs is demonstrated to involve functional TLR4 signaling. Furthermore, THP-specific humoral immune responsiveness was found to be severely impaired in the absence of intact TLR4 signaling, indicating a failure to implement APC-driven specific immune cell activation.

Numerous clinical and experimental studies have indicated an involvement of THP in several forms of inflammatory kidney disease. While THP is normally expressed at the luminal surface of renal tubular epithelial cells and excreted into the urine, its aberrant presence was also detected at the basolateral surface and in interstitial infiltrates in several inflammatory kidney diseases (34-36). Earlier studies indicated a role of THP in the pathogenesis of interstitial nephritis, since intravenous challenge of animals with THP resulted in the induction of a tubulointerstitial inflammatory response and microscopic scarring localized to the distal nephron segments (31-34). Furthermore, cytotoxic T cells with specificity for THP and anti-THP Abs were present in affected animals. Ab responses to THP are a well-known phenomenon in patients with infections of the urinary tract such as interstitial cystitis (37, 38) and acute pyelonephritis (30, 39, 40), where THP has been discussed to be involved in kidney scarring leading to overt renal insufficiency (29, 41). Moreover, elevated autoantibody levels against THP could be involved in chronic pyelonephritis and subsequent renal damage (15, 16). Abnormal deposition of THP and ensuing inflammatory reactions have been consistently observed in cast nephropathy and urolithiasis (13, 16). In acute renal transplant rejection THP deposits were found to be surrounded by mononuclear cell infiltrates (42), similar to kidneys affected by immunoglobulin light chain deposition in patients with multiple myeloma (43). Despite the apparent participation of THP in a large variety of inflammatory kidney diseases, the underlying mechanisms that would explain how THP contributes to inflammatory reactions have largely remained obscure.

In the present study we clearly show that THP is able to induce maturation of immature DCs by a TLR4-dependent mechanism. Moreover, our study is in line with previous findings demonstrating that intravenous challenge with THP or autologous urine results in rapid induction of THP Abs. Remarkably, TLR4 is essential for the THP-specific Ab response in the present model as Tlr4–/– mice were severely impaired in their THP-specific humoral immune responsiveness. In this respect, THP might be regarded as both adjuvant and antigen because it stimulates its own Ab production. Presumably, healthy mammals do not raise Abs against THP because the exclusive localization of THP at the luminal surface of tubular epithelial cells keeps the protein from the Ab-producing machinery. Segregation could be abolished in kidney diseases by loss of cell integrity or of luminal/basolateral tubular polarization. The physiological function of THP, therefore, could come from its ability to immediately activate innate immune responses, also recruiting components of the adaptive arm of the immune system. THP could thereby provide a critical danger signal to prevent host invasion of potentially harmful bacteria in case of local injury or increased epithelial permeability. While considered as a glycoprotein with direct antimicrobial effects, as has been suggested in recent studies employing THP–/– mice (20, 21), our data indicate a further role of THP as an endogenous activator of local immune responsiveness.

Various investigators have pointed to the problem of sufficiently high purity of potential immunostimulatory molecules acting through TLR. Therefore, it is of note that a number of data in the present as well as in previous studies (18, 44) confirmed that the results obtained with THP are not due to contaminating LPS. First, polymyxin B, which binds to lipid A and thereby blocks LPS responses, did not affect activation of both human and mouse APCs at all. Second, we found that HUVECs, which respond to very low amounts of LPS with TF production, were completely insensitive toward THP. Finally, the complete lack of response to THP in Tlr4–/– cells rule out possible contaminations with lipoproteins or lipopeptides. We cannot, however, completely eliminate the possibility that THP may act as a potentiator of subthreshold amounts of LPS or some other bacterial cell wall components that are tightly bound within the THP molecule and that might directly stimulate TLR4. Even in this case, however, THP could play a significant antibacterial role by amplifying potential danger signals derived from microbes entering the urinary tract and thereby inducing an appropriate antibacterial host response.

Recent studies have identified various sugar structures such as zymosan (7, 8), hyaluronic acid (10), or phospholipomannan (6) as effective activators of myeloid cells. In these studies an absolute requirement was demonstrated for the engagement of TLR2, TLR4, or both receptors for proper immune cell activation. Therefore, it is currently believed that bacterial recognition and subsequent immune cell activation might depend on both a functional TLR complex and a specific sugar-recognizing receptor supporting a model in which specific host responses are mediated by a combination of molecules (3). It was recently shown that zymosan activates immune cells only by engagement of both dectin-1, its respective receptor, and TLR2 (7, 8). The highly complex glycomoiety of THP, which consists of n-linked glycans of the polyantennary-type sialylated, fucosylated, and sulphated sugar residues that are evolutionarily conserved across species (13), might similarly contain ligands for a sugar-recognition receptor. Therefore, further investigations will focus on the identification of the active moiety and a putative cell surface receptor for THP.

In conclusion, we demonstrate here that the abundant urinary glycoprotein THP is a strong activator of professional APCs, including DCs. Its immunostimulatory potential and its ability to induce an anti-THP Ab response depends critically on TLR4 and its subsequent signaling pathway. We hypothesize that THP might serve as an endogenous activator of a molecular machinery typically associated with host responsiveness against bacterial infections. Similar data were recently reported for ?-defensin 2, which is also produced by tubular epithelial cells. While similarly considered as peptide with direct antimicrobial activity, ?-defensin 2 possesses DC stimulatory properties involving a TLR4-dependent process (11). Our findings thus extend the understanding of THP’s function in antibacterial defense mechanisms and may have broad implications for the regulation of immune responses in the urinary tract.

    Methods

Media and reagents.

LPS (Escherichia coli 0111:B4) was purchased from Sigma-Aldrich Chemie GmbH. Human THP isolated from sterile urine from healthy individuals, purified by 3 repetitive cycles of precipitation in 0.58 M NaCl and centrifugation as described (45), was purchased from Accurate Chemical & Scientific Corp. Purity was assessed through a single homologous band at 92 kDa in SDS-PAGE. Mouse THP was purified from sterile urine from female C57BL/6 mice by precipitation in 0.58 M NaCl as described (46) or by dialysis against PBS using a 50-kDa Spectra/Por Biotech RC dialysis membrane from Spectrum Laboratories Inc. To further purify THP, the THP precipitation was diluted in sodium phosphate buffer, pH 6.3, to a concentration of 70 mM to induce THP gel formation and was incubated for 1 hour at room temperature. Afterward, the sample was ultracentrifuged at 109,000 g for 30 minutes at 10°C, and the pellet was dissolved in H2O and dialyzed against water (23). The pellet fraction contained the majority of THP as assessed by SDS-PAGE. The pellet fraction was further purified by bringing the solution to 140 mM NaCl and 250 mM phosphate buffer, pH 7.5, which induces gel formation. The THP sample was then added onto a column containing diatomaceous earth, washed with PBS, and eluted by addition of H2O as described (24). The MAPK inhibitors SB203580, PD98059, and UO126 and the NF-B inhibitor SN50 were purchased from Calbiochem. Polymyxin B was from Sigma-Aldrich Chemie GmbH. Affi-Prep Polymyxin Support was from Bio-Rad Laboratories Inc.

All reagents, media, and buffers used in our assays were checked by Limulus amebocyte lysate assays (BioWhittaker Inc.) and contained less than 0.06 U/ml of endotoxin in accordance with the European Community standard value of water injection. The TIRAP/Mal inhibitory peptide (28), consisting of a Drosophila antennapedia sequence positioned at the N-terminal end of TIRAP amino acids 138–145, was synthesized by Fmoc-chemistry. Tripalmitoyl cysteinyl (Pam3Cys) lipopeptide was purchased from EMC Microcollections. Mouse mAb against human TLR4 (HTA125) was from eBioscience. PE-labeled IL-12 and TNF- mAbs were from BD Biosciences. RPMI-1640 (Invitrogen Corp.) supplemented with 2 mM L-glutamine, 100 μg/ml streptomycin, 100 U/ml penicillin, and 10% FCS (HyClone Laboratories Inc.) was used as culture medium. Recombinant human GM-CSF (rhGM-CSF) was obtained from Schering-Plough and rhIL-4 was from Strathmann Biotech GmbH.

Cell separation and preparation of human DCs.

Human PBMCs were isolated by density-gradient centrifugation over endotoxin-free Lymphoprep (Nycomed Pharma A/S). For monocyte enrichment, PBMCs were depleted of T cells by sheep erythrocyte rosetting. Resting T cells were isolated by sheep erythrocyte rosetting and subsequent lysis of remaining erythrocytes at 4°C.

For DC differentiation, enriched monocytes were cultured in 50 ng/ml rhGM-CSF and 10 ng/ml rhIL-4 for 5–7 days. DC activation was induced by THP or LPS addition for 48 hours.

Mouse studies.

Female C57BL/6 mice were obtained from Harlan Winkelmann GmbH. Tlr2–/–, Tlr4–/–, Tlr9–/–, and MyD88–/– mice in a C57BL/6 background were housed under specific pathogen-free conditions. Mice were used at age 6–8 weeks. All animal experiments were approved by the veterinary magistrate (MA58/60) of Vienna, Austria. Bone marrow–derived macrophages were prepared by incubation of 107 bone marrow cells with 1 ml conditioned M-CSF medium. For in vivo studies, 50 μg THP or 50 ng LPS in 100 μl PBS were injected intravenously in the tail vein on days 0, 1, 2, and 7. Blood was taken on the indicated days, and Ab levels against THP were measured by ELISA. Briefly, 5 μg/ml THP or OVA were coated on MaxiSorp plates (Nunc A/S) overnight and serial dilutions of blood serum were applied. Specific Ab binding was proven using a peroxidase-conjugated F(ab')2 fragment of a rabbit anti-mouse Ab provided by Jackson ImmunoResearch.

Phenotypical characterization of DCs by FACS analysis.

For evaluation of surface marker expression, cells were incubated with FITC- or PE-conjugated mAb for 30 minutes at 4°C. For control, nonbinding isotype-matched FITC- and PE-conjugated mouse IgGs were employed. Cells were analyzed on a FACScalibur flow cytometer (BD Biosciences). FITC-conjugated CD25 (2A3), anti–HLA-DR (L243), and anti-CD83 (HB15e), as well as PE-labeled anti-CD80 (L307.4) and anti-CD86 (IT2.2) mAb, were from BD Biosciences.

Measurement of cytokine production and T cell proliferation.

For evaluation of TNF- and IL-12p40, 5 x 105 DCs were stimulated with THP (10 μg/ml), LPS (100 ng/ml), or CpG (10 μM) in 24-well plates (final volume 1 ml). Cell-free supernatants were harvested 48 hours after addition of the bacterial stimulus. Cytokines were measured by sandwich ELISA using matched-pair Abs. Capture as well as detection Abs against human IL-12p40 were obtained from R&D Systems Inc. Abs against human TNF- were from BD Biosciences. Standards consisted of human recombinant material from R&D Systems Inc. Assays were set up in duplicates and were performed according to the recommendations of the manufacturers. The lower limit of detection was 20 pg/ml for all cytokines.

Cytoplasmic staining for cytokine production was performed as described (47). Briefly, monensin (5 μM) was added during the last 12 hours of stimulation. DCs were harvested and fixed for 20 minutes at room temperature by adding 100 μl of FIX solution (An der Grub Bio Research GmbH). Subsequently, cells were washed once with 4 ml PBS, resuspended in 100 μl PBS, and permeabilized by the addition of 100 μl PERM solution (An der Grub Bio Research GmbH). The indicated PE-conjugated anti-cytokine mAb was incubated for 20 minutes at room temperature. After extensive washing, cells were analyzed by flow cytometry.

Mouse RAW 264.7 macrophages (5 x 104) were stimulated with THP (10 μg/ml), LPS (10 ng/ml), or CpG (10 μM) in 96-well plates (final volume 200 μl). Supernatants were harvested after 4 and 16 hours, and cytokines were measured by sandwich ELISA kits from R&D Systems Inc.

For MLC, stimulator cells were irradiated (30 Gy, 137Cs source) and added at increasing cell numbers to 105 allogeneic T cells in 96-well culture plates in RPMI-1640 medium supplemented with 10% FCS (total volume 200 μl/well). After 5 days, cells were pulsed with 1 μCi -thymidine (ICN Pharmaceuticals Inc.). After another 18 hours, the cells were harvested on glass-fiber filters (Topcount; Packard Instrument Company) and DNA-associated radioactivity was determined using a microplate scintillation counter (Packard Instrument Company). DNA synthesis was expressed as mean counts per minute of triplicate cultures. Supernatants from the respective MLC were obtained 24 and 48 hours after culture initiation and analyzed for IL-2 and IFN- by ELISA (capture as well as detection Abs were obtained from R&D Systems Inc.). The lower detection limit for these cytokines was 20 pg/ml.

Biochemical analysis of signal-transduction events.

Immature DCs or mouse RAW 264.7 macrophages were stimulated with 30 μg/ml THP, 100 ng/ml LPS, or medium alone for 15–60 minutes. Activation was stopped by addition of ice-cold washing buffer (RPMI-1640). Cells were pelleted by short centrifugation (12,000 g for 20 seconds) and lysed on ice for 30 minutes in TBS, pH 7.4, containing 1% NP-40 (Pierce Biotechnology Inc.), phosphatase (1 mM sodium orthovanadate, 10 mM NaF, 5 mM sodium pyrophosphate, 25 mM ?-glycerophosphate, 5 mM EDTA), and protease inhibitors. Nuclei were removed by short centrifugation; supernatants are referred to as whole-cell lysates. Proteins were separated using SDS-PAGE and were blotted onto nitrocellulose membranes (Hybond ECL; Amersham Biosciences). Abs against IRAK-1 (Santa Cruz Biotechnology Inc.), Akt (BioVision Inc.), and ERK-2, p38, IB- (Santa Cruz Biotechnology Inc.) were used to detect the respective antigens. Protein phosphorylation was assessed using Abs directed against phosphorylated isoforms of Akt (BioVision Inc.), ERK-1/2 (Santa Cruz Biotechnology Inc.), and p38 (Cell Signaling Technology Inc.). Peroxidase-labeled secondary Abs (Bio-Rad Laboratories Inc.) were used to detect primary Ab binding. Chemiluminescence generated from enhanced chemiluminescence substrate (Roche Diagnostics GmbH) was detected on a Lumi-Imager (Roche Diagnostics GmbH) (48).

Clotting assay determination of TF activity.

HUVECs were seeded in 6-well plates at 80–90% confluence and grown overnight. Cells were scraped from the plates and analyzed for TF activity as described (49). HUVECs were left untreated or treated with 200 U/ml IFN- (R&D Systems Inc.) for 2 hours and then stimulated with LPS or THP at the indicated concentrations for 6 hours. The cells were washed twice and scraped in 1 ml clotting buffer (12 mM sodium acetate, 7 mM diethylbarbitate, and 130 mM sodium chloride; pH 7.4); 100 μl of resuspended cells were mixed with 100 μl of citrated plasma, and clotting times were measured after recalcification with 100 ml of 20 mM CaCl2 solution at 37°C. TF equivalents were determined using a standard curve obtained from rabbit brain thromboplastin.

EMSA.

Nuclear extracts from DCs were prepared as described (50, 51). Oligonucleotides resembling the consensus binding site for NF-B (5'-AGTTGAGGGGACTTTCCCAGGC-3') and CRE (5'-AGAGATTGCCTGACGTCAGAGAGCTAG-3') were purchased from Santa Cruz Biotechnology Inc. The double-stranded oligonucleotides used in all experiments were end-labeled using T4 polynucleotide kinase and -ATP. After labeling, 5 μg of nuclear extract was incubated with 120,000 cpm of labeled probe in the presence of 3 μg of poly(dI-dC) at room temperature for 30 minutes. This mixture was separated on a 6% polyacrylamide gel in Tris/glycine/EDTA buffer at pH 8.5. Control experiments were performed as described (51). For control, competition experiments using unlabeled NF-B or CRE oligonucleotides were performed.

Statistics.

Cytokine levels were compared using Student’s t tests.

    Acknowledgments

This work was supported in part by grants (P14874-B08, to G.J. Zlabinger; P16788-B13, to T.M. Stulnig) from the Fonds zur F?rderung der Wissenschaftlichen Forschung, ?sterreich, as well as by grants from the Center of Molecular Medicine (CeMM), a basic research institute within the companies of the Austrian Academy of Sciences (to T.M. Stulnig). Moreover, we are grateful to Wolfgang Zauner for synthesis of the TIRAP/Mal inhibitory peptide, and to Bianca Weissenhorn, Alessandra Mathe, and Margarethe Merio for excellent technical assistance.

Conflict of interest: The authors have declared that no conflict of interest exists.

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　　【摘要】目的 探讨对32例股骨髁上及髁间骨折DCS内固定治疗临床效果。方法 按AO分类：33-A1型6例，33-A2型10例，33-A3型4例，33-C1型6例，33-C2型4例，33-C3型2例，经股骨下段外侧切口入路整复骨折，并用DCS固定骨折。结果  经1年随访，骨折愈合，平均愈合时间6个月，关节功能优良率93.75%，其中33-C3型骨折1例术后出现膝内翻。结论  DCS对股骨髁上及髁间骨折是较理想的内固定物。

　　【关键词】  股骨髁上及髁间骨折；DCS内固定
   
　　股骨髁上骨折发生于股骨髁与股骨干干骺端的连结部，是松质骨与密质骨的移行部位，为较常见的骨折，常合并髁间骨折，治疗方法多，疗效不甚满意，我科2000年1月～2004年12月收治了56例，其中32例行动力髁螺钉（DCS）内固定，经1年以上随访，取得满意临床疗效，现报告如下。

　　1  临床资料

　　1.1  一般资料  本组共32例，男26例，女6例，年龄26～67岁，平均42岁。受伤原因：交通事故伤16例，高处坠落伤10例，重物砸伤6例；按AO法分开：33-A1型6例，33-A2型10例，33-A3型4例，33-C1型6例，33-C2型4例，33-C3型2例［1］，均为新鲜闭合性骨折，合并其他部位骨折6例。入院到手术时间2～7天，平均4.5天，住院天数7～62天，平均26天。

　　1.2  手术方法  采用持续硬膜外阻滞麻醉，患者取仰卧位，患侧臀下垫枕，大腿中上段上气压止血带，压力0.08kPa，取股骨下段外侧切口入路暴露股骨下段外侧，直视下整复骨折，于股骨外侧髁部距关节面上方2cm处做一与股骨干纵轴垂直的直线，股骨干纵轴与直线交点即为髁螺钉进针点，并用2根克氏针来引导方向，一根沿两髁插入标明膝关节轴，另一根置于髌股关节面标明髌股关节的倾斜度，X线检查克氏针位置、方向准确后用导向器钻入导向针、测深、扩孔、攻丝、拧入髁螺钉，将DCS钢板套入髁螺钉上并锁定，配合加压螺钉固定DCS钢板，检查固定牢固、膝关节被动活动良好，缝合切口术毕［2］。全部病人平安经过手术关。

　　1.3  术后处理  术后生命体征平稳及麻醉反应消失后半卧位，鼓励咳嗽，预防肺不张和坠积性肺炎，并用抗生素5～7天预防感染，抬高患肢促进肢体肿胀消退，术后3天指导患者进行股四头肌等长收缩锻炼及屈伸膝关节锻炼，术后2～4周扶双拐患肢不负重下地，根据骨折愈合情况逐渐恢复术肢完全负重。

　　1.4随访结果本组病例均获1年以上随访，骨折愈合，平均愈合时间为6个月，无内固定松动、折断。术后膝内翻1例，扶单拐行走，为33-C3型骨折，并有过早下地负重情况。参照Marshall测试评定法提出的关节功能评定标准［3］，关节功能优良率93.75%。见表1。

　　表1  32例股骨髁上及髁间骨折DCS内固定疗效（略）

　　2  讨论

　　2.1  局部解剖特点与骨折关系  膝关节前方关节囊被股四头肌肌腱、髌韧带所覆盖及保护，髌骨及髌韧带两侧为阔筋膜及股四头肌肌腱的扩张部所加强，后方关节囊则由半腱肌附着点之一向外上反折部分有所加强；腓肠肌分内外两个头，外侧头在腘肌腱及膝关节腓侧副韧带附着点上方，起自股骨外上髁，内侧头较高，起自股骨内上髁。由于上述解剖结构、受伤机制的不同，加上腓肠肌牵拉等因素的影响，股骨髁上及髁间骨折常常发生移位，易波及髁间窝而发生粉碎性骨折，股骨膝关节面不平整，骨折远端移位可损伤腘动静脉和坐骨神经。因此股骨髁上及髁间骨折有以下几个特点：（1）粉碎性；（2）易移位、不稳定；（3）易波及关节内成分；（4）多发性损伤多；（5）易致邻近血管、神经损伤；（6）骨折要求内固定牢固可靠，以便早期进行康复训练［1］。

　　2.2  股骨髁上及髁间骨折内固定物的比较选择  股骨髁上及髁间骨折常采用加压钢板、AO髁钢板、L-型髁钢板及逆行交锁钉等内固定方法。加压钢板较厚，塑形困难，使钢板与股骨远段外侧不能完全贴合，且弯曲处压力大，固定力线上存在偏移，骨折两端折弯力与扭曲力易使螺钉松动，固定不够牢固，不利于早期康复训练；AO髁钢板亦难以塑形，入点选择困难，三维空间定位不易准确，骨折块间无加压作用，稳定性差，术后易出现膝关节内、外翻及旋转畸形等并发症，且切口长、创伤大、广泛剥离软组织和骨膜，易发生骨折延迟愈合、不愈合和膝关节功能障碍，常发生螺钉脱出和钢板断裂；L-型髁钢板其L端打入时对骨结构破坏大，并有切割作用，固定不易使髁间锁固，对髁部粉碎性骨折及内髁骨折不易固定，致使膝外翻角度丢失，不利于早期康复训练；逆行交锁钉固定较牢固，但术后膝关节疼痛、髌股关节退行性变等并发症时有发生［2，4，5］。相比之下，DCS固定有如下优点：（1）手术操作简单，容易掌握，手术创伤小、时间短，术中出血量少；（2）DCS的拉力髁螺钉作用可使骨块间加压，增加其稳定性；（3）外行设计符合股骨远端的解剖要求，符合力学原理，固定牢固；（4）容易正确的选择髁螺钉的入点，使钢板轴与股骨干轴一致；（5）髁螺钉植入时不需像L-型髁钢板、AO髁钢板那样敲击，对骨结构破坏小；（6）患者可早期下床活动，功能康复快，从而防止了长期卧床导致的并发症［2，6］；因此DCS是股骨髁上骨折及髁间骨折治疗中较理想和应用较广泛的内固定物，本组仅有的1例33-C3型骨折术后出现膝内翻，说明33-C3型骨折应慎用；同时DCS亦不适用于骨骺尚未愈合的青少年和骨质疏松的患者。

　　2.3  DCS手术要点  髁螺钉导针入点的确定和方向的准确是手术成功与否的关键，需以两根关键的克氏针来引导方向以确保准确性，否则可能偏离预定的方向，甚至进入关节腔，如有条件最好术中使用C型臂或X线摄片证实定位准确，因为一旦打好孔后再来修正，即使勉强修复，其固定的牢固性亦会大大降低；另外如有较大骨缺损或关节面有塌陷时需植骨充填修复，关节面要尽可能平整，膝关节外翻角应维持在5°～8°之间，固定要牢固而坚强［6］。

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　　(编辑：夏  琳)

　　作者单位： 638001 四川广安，广安市人民医院骨科