主题：cc

    如果非要喝酒，那就喝红酒吧。因为红酒中葡萄皮的抗氧化物质多酚留存在酒液中，可以降低患心血管疾病的几率。
　　而且各种酒类相比较之下，红酒的普林（会使体内尿酸上升的物质）相当低。此外，红酒能提升抗氧化作用，以预防动脉硬化。最近根据研究结果得知，对于痴呆症也能发挥功效，是高龄社会所不可欠缺的饮品。但酒类依旧有热量，营养师建议每天还是控制在60cc以下。

　半断食减肥法的好处

　　“半断食”并不是不让人吃东西，而是要让人们有节制的吃，按照饮食计划吃。半断食食谱多为稀饭、酱菜、豆腐、味噌汤、蔬果汁、酸奶等清淡健康食物，多吃有利于身体排毒。

　　每两个月进行一次体内健康SPA，不仅可以调整肠胃、甩掉多余的脂肪和赘肉，还能促进新陈代谢，让你的身体更轻盈，一些常见的小毛病也会慢慢消失哦。当你体内堆积毒素轻松排出体外之后，瘦下来就不是难事了，1周让你减重9斤哦。

　　一周半断食减肥食谱

　　星期一

　　早餐：全麦吐司2片、水煮蛋一个、250cc咖啡一杯。

　　午餐：糙米饭3/4碗、炒青菜（蕃薯叶+橄榄油一茶匙）、红烧牛腩（牛腩一两、白萝卜60克、红萝卜30克）、丝瓜汤、莲雾2个。

　　晚餐：糙米饭3/4碗、炒四季豆（四季豆70公克、橄榄油一茶匙）、清蒸吴郭或虱目鱼（半条鱼、将少许姜）、白萝卜汤（白萝卜50克）、蕃茄一个。

　　星期二

　　早餐：三明治（吐司两片、洋火腿15克、蕃茄半个）、250cc咖啡一杯。

　　午餐：馄饨面（馄饨4个、鸡蛋面50克、小把小白菜）、凉拌海带丝、苹果一个。

　　晚餐：胚芽饭3/4碗、凉拌西洋芹菜、味噌汤（豆腐2格）、杨桃一个。

　　星期三

　　早餐：小餐包2个、白水煮蛋1个、250cc咖啡一杯、大蕃茄一个。

　　午餐：胚芽饭3/4碗、炒草菇、冬瓜汤一碗 、橙子一个。

　　晚餐：糙米饭3/4碗、烫绿花椰菜、洋葱炒蛋、金针汤（金针30克）、奇异果一个。

　　星期四

　　早餐：稀饭一碗、荷包蛋一个、烫青菜。

　　午餐：鲔鱼三明治（水煮鲔鱼30克、蕃茄半个、小黄瓜）、250cc咖啡一杯、色拉一盘、苹果一个。

　　晚餐：糙米饭3/4碗、烫青菜、清蒸虱目鱼半条、冬瓜汤 、梨一颗。

　　星期五

　　早餐：全麦吐司2片、水煮蛋2个、250cc咖啡一杯。

　　午餐：胚芽饭3/4碗、银芽鸡丝（豆芽菜50克、鸡腿半只）、小黄瓜炒蒟蒻、白萝卜汤、橙子一个。

　　晚餐：糙米饭3/4碗、烫蕃薯叶、凉拌竹笋、丝瓜汤、草莓10个。

　　星期六

　　早餐：玉蜀黍一条、小餐包一个、250cc咖啡一杯。

　　午餐：糙米饭3/4一碗、清蒸吴郭鱼半条 、凉拌西洋芹菜 、菠菜汤、杨桃1个。

　　晚餐：糙米饭3/4碗、炒芥兰菜（芥兰50克和橄榄油一匙）、卤豆干4片、清蒸排骨、苹果一个。

　　星期日

　　早餐：全麦吐司2片、蒸蛋（两个蛋）、250cc咖啡一杯。

　　午餐：茄汁牛肉面（熟面条100公克、牛肉2两 、小白菜100公克）、凉拌小黄瓜、木瓜半个。

　　晚餐：糙米饭3/4碗、蕃茄蛋、炒A菜、苦瓜汤、奇异果两个。

     肚子里藏着一粒粒铅球，身形沉、举步维艰...便秘苦恼不言可喻。能改善肠胃机能的清凉果醋饮，只要3天，宿便就会乖乖滚出去！

     香蕉凤梨醋奶昔...一般排便不顺者绝对要试

     材料：（1天份）凤梨醋 5cc、香蕉 半条、优酪乳 200cc、冷开水适量

     作法：香蕉去皮切小段，与优酪乳、凤梨醋、冷开水一起放入果汁机中打匀即可。

     最佳饮用时机：空腹饮用，如早或晚餐前30分钟。

     禁忌：胃病、肾功能异常、糖尿病或癌症患者不适合饮用。

     M emo：因优酪乳属于高嘌呤，饮用过量，易引起尿酸值升高，最好喝3天停1天！

     材料：（1天份）柠檬醋 5cc、甘蔗汁 200cc、小蕃茄 3粒

     作法：蕃茄洗净切半，放入碗中，再倒入柠檬醋和甘蔗汁，拌匀趁鲜饮用。

     最佳饮用时机：当餐与餐间点心，如上午10点、下午4点或晚上8点。

     禁忌：胃病、糖尿病或癌症患者不适合饮用。

     Memo：小蕃茄可用草莓代替，但草莓表皮的细毛需清洗干净，较不会引发过敏；甘蔗汁不宜久存，需趁新鲜饮用。

【摘要】  Chronic obstructive pulmonary disease (COPD) is a progressive disease associated with a cellular inflammatory response. CD8+ T cells are implicated in COPD pathogenesis, and their numbers significantly correlate with the degree of airflow limitation. Dendritic cells (DCs) are important sentinel immune cells, but little is known about their role in initiating and maintaining the CD8 T-cell response in COPD. To investigate the mechanisms for CD8+ T-cell recruitment to the lung, we used resected human lung tissue to analyze chemokine receptor expression by CD8+ T cells and chemokine production by CD1a+ DCs. Among 11 surveyed chemokine receptors, only CC chemokine receptor (CCR5), CXC chemokine receptor (CXCR) 3, and CXCR6 correlated with COPD severity as defined by criteria from the Global Initiative for Chronic Obstructive Lung Disease. The CD8+ T cells displayed a Tc1, CD45RA+ effector memory phenotype. CD1a+ DCs produced the respective ligands for CCR5 and CXCR3, CCL3 and CXCL9, and levels correlated with disease severity. CD1a+ DCs also constitutively expressed the CXCR6 ligand, CXCL16. In conclusion, we have identified major chemokine elements that potentially mediate CD8+ T-cell infiltration during COPD progression and demonstrated that CD1a+ mucosal-associated DCs may sustain CD8+ T-cell recruitment/retention. Chemokine targeting may prove to be a viable treatment approach.
--------------------------------------------------------------------------------
Chronic obstructive pulmonary disease (COPD) is a diagnostic umbrella that encompasses emphysema and chronic bronchitis. COPD is characterized by progressive airflow limitation associated with an abnormal inflammatory response. Neutrophils, macrophages, and CD8+ T cells have been implicated in COPD pathogenesis in a number of studies.1-6 More recently, airway infiltration by CD4 T cells and B cells has also been shown to associate with the progression of COPD.7,8 This cellular inflammation is associated with airway remodeling and destruction, but it is not clear which cell types are responsible for the damage. Neutrophils and macrophages are sources of reactive oxygen metabolites, inflammatory cytokines, metalloproteinases, and other tissue-damaging enzymes.9 By contrast, it is less clear how CD8+ T cells could destroy lung parenchyma, although in response to antigen, CD8+ T cells are able to lyse target cells, either through the release of cytotoxic proteins, such as perforin or granzyme, or by inducing apoptosis via the Fas ligand-Fas pathway.10 However, the nature of the antigen that could trigger this CD8+ T-cell response in COPD is unknown. One hypothesis suggests that intracellular pathogens, such as adenovirus or rhinovirus, may provide a foreign antigenic stimulus; and in fact, viral infections are a frequent occurrence in patients with COPD.11-13 Autoimmunity has also been postulated, thus far without supporting evidence.14
To initiate antigen-specific CD8+ T-cell immune responses, it is necessary for the CD8 T-cell receptor to recognize foreign antigen in combination with a major histocompatibility class I molecule presented by an antigen-presenting cell (APC). It seems that dendritic cells (DCs) are the predominant APCs for the priming of naïve CD8 T cells15-17 and for initiating CD8 memory T-cell proliferative responses.15-18 Surprisingly, the role of dendritic cells has not been well studied in COPD. In murine models of cigarette smoke exposure, there are conflicting results as to whether DC numbers are increased or decreased in the lung in response to cigarette smoke.19,20 In the human lung, four subsets of pulmonary DCs have been identified: myeloid DC 1, myeloid DC 2, plasmacytoid DC, and CD1a+ DCs.21 In chronic asthmatics, CD1a+ DCs were present in greater numbers in the bronchial mucosa compared with healthy controls and were shown to play an important role in modulating the immune response.22 In view of the role of DCs in initiating and maintaining antigen-specific CD8 T-cell responses, their role in COPD deserves study.
Because CD8+ T cells potentially contribute to the pathophysiology of COPD, understanding how these cells are recruited to the lung may lead to novel treatment. The families of proteins known as chemokines and chemokine receptors are considered key mediators of recruitment. Chemokine receptors play an important role in the trafficking of immune cells to sites of injury, inflammation, and antigen encounter. Approximately 50 chemokines and 20 chemokine receptors have been identified. They are classified into four subgroups based on the position of critical cysteine residues: CXC, CC, C, and CX3C. Besides the ability of chemokines to drive leukocyte migration, they are also involved in proliferation, differentiation, retention, and survival.23 CC chemokine receptor 5 (CCR5) and CXC chemokine receptor 3 (CXCR3) have already been implicated in COPD because T cells infiltrating the lungs of COPD patients have been shown to express these chemokine receptors.24-26 However, many of these studies used immunoperoxidase or immunofluorescent analyses, which have limited sensitivity to detect the characteristically low surface expression of chemokine receptors and, moreover, may not reflect activity in the interstitium. In addition, previous studies did not correlate chemokine receptor expression with specific cell sources of chemokine ligands or disease severity.
In the present study, we performed a comprehensive chemokine receptor analysis of interstitial CD8+ T cells and DCs derived from clinically indicated lung resections from patients with COPD. We found that three chemokine receptors, CCR5, CXCR3, and CXCR6, demonstrated increased expression on lung CD8+ T cells that correlated with severity of COPD. These CD8+ lung T cells displayed a type-1 (Tc1) effector memory phenotype. Further analysis of CD1a+ DC chemokine transcripts revealed that CCL3 and CXCL9, respective ligands for CCR5 and CXCR3, likewise displayed a correlation with disease severity, whereas the ligand for CXCR6, CXCL16, was constitutively produced. Our findings suggest that CCR5 and CXCR3 may be involved in the recruitment of CD8+ T cells into the lungs of COPD patients, whereas CXCR6 may be more important in adhesion or retention events. These findings provide a potential rationale for the design of therapeutic agents to impact chronic CD8+ T cell-mediated inflammation in COPD patients.

【关键词】  chemokine receptor chemokine receptor expression correlates obstructive pulmonary severity

Materials and Methods

Specimens and Patient Population

Specimens were obtained from patients undergoing clinically indicated resection procedures for tumor nodules, lung volume reduction surgery, or lung transplant surgery at the VA Ann Arbor Healthcare and the University of Michigan Healthcare Systems. Studies and consent procedures were approved by Institutional Review Boards. Tissues and clinical data were de-identified before analysis. Only non-neoplastic lung tissue remote from tumor nodules and lacking postobstruction changes was collected. Histological sections were reviewed to confirm absence of pneumonia, lymphangitic neoplasm, or other unrelated interstitial diseases. The study population was composed of 30 subjects, all with a history of cigarette smoking. All subjects underwent two or more preoperative spirometric tests and full evaluation by a pulmonologist. A five-stage classification system derived from the Global Initiative for Chronic Obstructive Lung Disease (GOLD) was used to categorize patients.27,28 Stage 0 represents patients at risk, whereas stage 4 represents patients with the most severe cases of COPD. Classification is based on the forced expiratory volume in 1 second (FEV1) of the predicted value in combination with the ratio of FEV1 to forced vital capacity. Table 1 shows the FEV1 range for each GOLD stage and the number of patients in each stage along with age and smoking history ranges.

Table 1. Summary of Patient Age, Smoking History, and Disease Severity

For histological evaluation, samples of lung tissue from each patient were embedded in paraffin and sectioned for standard hematoxylin and eosin staining and immunohistochemical staining. Immunohistochemical staining for CD8 and CD1a was performed by the University of Michigan Health Systems Laboratory using an automated immunostainer (Ventana Medical Systems, Tucson, AZ).

Flow Cytometry

Lung sections weighing an average of 2.5 g were rinsed in RPMI 1640 medium (JRH Biosciences, Lenexa, KS) to remove excess blood and were homogenized in a Waring blender at low speed for 45 seconds. Cells were passed through a 70-µm nylon filter to remove debris and resuspended at 10 x 106 cells per ml of flow buffer (2% fetal bovine serum in phosphate-buffered saline) along with 20 µl/ml Human FC block (Miltenyi Biotec, Auburn, CA). Cells were incubated at 4??C for 10 minutes and then added in a volume of 100 µl to each flow tube. Monoclonal antibodies against CD8, CD1a, CCR5, CD45RA, CXCR3, CXCR1, CCR7, CD83 (BD Biosciences, San Jose, CA), CXCR6, CCR2, CCR3, CCR8, and CCR6 (R&D Systems, Minneapolis, MN) were used. Antibodies were conjugated to fluorescein isothiocyanate, phycoerythrin, or phycoerythrin-cyanine 5. Appropriate isotype-matched controls were used in all experiments. Cells were incubated with antibodies for 25 minutes at 4??C. After incubation, cells were washed twice and analyzed using a FACScan cytometer running CellQuest software (BD Biosciences).

CD8+ T-Cell and CD1a+ Cell Isolation

Lung samples were homogenized as described above, and cells were resuspended in 80 µl of phosphate-buffered saline containing 0.5% fetal bovine serum and 2 mmol/L ethylenediamine tetraacetic acid per 10 x 106 cells. Cells were divided into two samples and incubated with either human-specific CD8 or CD1a microbeads (Miltenyi Biotec) according to manufacturer??s instructions. MACS LS columns (Miltenyi Biotec) were used to separate cells, and the positively labeled CD8+ or CD1a+ cells were collected. Cells were lysed for RNA isolation and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analyses.

Real-Time RT-PCR

Micro Poly(A) Pure kits (Ambion, Austin, TX) were used to isolate RNA from lysed cells or from whole tissues. DNA-free (Ambion) was used to remove any contaminating genomic DNA. Each RNA sample was reverse-transcribed in a 20-µl reaction using SuperScript II RNase HC Reverse Transcriptase (Invitrogen Corporation, Carlsbad, CA). Analysis of the transcripts was performed by real-time PCR using the ABI Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, CA). Human glyceraldehyde-3-phosphate dehydrogenase, which acted as the endogenous reference, and primer-probe sets for target genes were purchased commercially (Applied Biosystems). Transcript levels were expressed as arbitrary units and were calculated using the comparative threshold cycle method, as recommended by the manufacturer.

Protein Analysis

Lung sections were snap-frozen in dry ice and stored at C80??C before use. For protein extraction, lobes were resuspended in 2 ml of phosphate-buffered saline and homogenized using a tissue homogenizer. Samples were centrifuged at 300 x g for 20 minutes. Supernatants were collected and stored at C80??C. Using the Luminex 200 (Luminex Corporation, Austin, TX), protein levels for CCL3 and CXCL9 were determined using Biosource Multiplex Assays (Invitrogen) according to manufacturer??s instructions. Total lung protein concentration was determined using a Micro BCA Protein Assay kit (Pierce Biotechnology, Rockford, IL), and chemokine levels were normalized to milligrams of lung protein.

Nonparametric (Spearman) correlation analysis was used to determine the correlation coefficient r. A two-tailed P value of <0.05 was considered to indicate significance.

Distribution of CD1a+ and CD8+ Cells in the Lungs of COPD Patients

Initially, we determined the anatomical tissue compartment distribution of target cell populations in subject lung specimens. Paraffin-embedded tissues taken from COPD patients were sectioned and immunohistochemically stained for CD1a and CD8 (Figure 1) . Lung sections from individuals with a GOLD stage of 0, 1, 2, or 3 are depicted and are representative of the staining patterns seen for other individuals. The majority of CD1a+ cells were found within bronchial mucosal epithelium, whereas the CD8+ cells localized to peribronchial cuffs and epithelium. In general, these populations appeared to be increased in GOLD stages 2 and 3. These studies confirmed a close proximity of CD8+ and CD1a+ cells in a bronchocentric process. Similar findings for the localization of CD1a+ DCs21,29 and CD8+ T cells30 have been reported.

Figure 1. Immunohistochemical localization of CD1a+ (left) and CD8+ (right) cells in the lungs of smokers with GOLD stage 0 (A and B), stage 1 (C and D), stage 2 (E and F), and stage 3 (G and H) COPD. Stain is immunoperoxidase based with diaminobenzidine as the chromagen substrate. Arrows point to CD1a+ cells. Magnification: x200 (A, B, D, F, and H); x400 (C, E, and G).

CD8+ Cells Isolated from Lungs of COPD Patients Have an Effector Tc1 Phenotype

Previous studies have suggested that peripheral blood CD4+ T cells from COPD patients display a Th1 phenotype, evidenced in part by their production of interferon (IFN).31 However, recent studies have shown that CD8+ T lymphocytes from bronchoalveolar lavage fluid show a Tc2 profile.32,33 To address this issue in our study population, we analyzed transcript expression of IFN and interleukin (IL)-4 from isolated lung CD8+ T cells. We found that transcripts for IFN displayed a significant correlation to COPD severity, with an r value of 0.81 (Figure 2A) . However, there was little to no transcript expression for IL-4 (data not shown). To characterize these cells further, we examined CD45RA and CCR7 expression by flow cytometry. Human CD8+ cells can be classified as either naïve (CCR7+ CD45RA+), central memory (CCR7+ CD45RAC), or effector memory (CCR7C CD45RAC or CD45RA+) cells.34 Figure 2B demonstrates that the percentage of CD8 cells positively expressing CD45RA increased with COPD severity. These cells did not express CCR7 (data not shown), consistent with CD45RA+ effector memory (TEMRA) cells that apparently become more prevalent as COPD progresses. Our findings suggest that there is induction of an activated subpopulation of TEMRA cells in the lungs of COPD patients with a Tc1 effector phenotype that correlates with disease severity.

Figure 2. Lung CD8+ T cells from COPD patients express a Tc1 effector memory phenotype. CD8+ T cells isolated from human lung samples by immunomagnetic beads were used for RNA analysis. CD8+ T cells expressed transcripts for IFN (A). Transcripts are expressed as arbitrary units and were measured by quantitative real-time RT-PCR. Surface expression of CD45RA was determined by flow cytometry (B) and is shown as the percentage of CD8+ cells among total leukocytes. Correlation statistical analysis is shown.

CD1a+ DCs in the Human Lung Display Low CD83 Costimulatory Expression at All COPD Stages

CD1a has been shown to be a reliable marker for a subset of intramucosal DCs identified in the human lung.21 They have been implicated in modulating the immune response during chronic asthma,22 but little is known regarding their role in COPD. Because little is known regarding the maturation state of CD1a+ DCs in human lungs affected by COPD, we attempted to correlate DC costimulatory molecule expression to COPD stage. We analyzed expression of CD83, reportedly a marker of DC maturity.35 Figure 3 shows that on average, only 5% of total CD1a+ DCs were expressing CD83. Others have shown that lung DCs possess an immature phenotype, yet like mature DCs, they have the capacity to stimulate T-cell proliferation.36 Our findings are in accord with this notion and further suggest that disease severity is not associated with changes in the expression of CD83 by CD1a+ DCs.

Figure 3. Lung CD1a+ DCs do not express CD83. Surface expression of CD83 was determined by flow cytometry and is expressed as the percentage of CD1a+ cells among total leukocytes. Correlation statistical analysis is shown.

CCR5, CXCR3, and CXCR6 Expression Among Lung CD8+ T Cells Positively Correlates with Disease Severity

To determine the potential role of chemokine receptors in the infiltration of CD8+ T cells into the lungs of COPD patients, we analyzed the expression of 11 different chemokine receptors by flow cytometry and real-time PCR. For flow cytometric analyses, chemokine receptor-positive CD8+ T cells were expressed as the percentage of total CD8+ cells. To analyze transcript expression by real-time PCR, RNA was isolated from purified interstitial lung CD8+ cells. Of the examined receptors, CCR5, CXCR3, and CXCR6 were expressed by CD8+ T cells and demonstrated a positive correlation with disease severity using flow cytometric (Figure 4, ACC) and transcript (Figure 4, DCF) analyses. Representative histograms show the increased expression of CCR5, CXCR3, and CXCR6 in GOLD stage 4 versus GOLD stage 0 patients (Figure 5) . Our findings regarding CXCR3 expression by CD8+ T cells in COPD patients are in accord with the immunohistochemical study of Saetta et al.24 Table 2 lists the Spearman correlation coefficient r and the P value for all 11 chemokine receptors. Statistics were determined using flow cytometry data, except for CCR1, which was determined by RNA transcript levels. After observing the correlation between disease severity and CCR5 expression, CCR1 was included in the analysis because these chemokine receptors share some of the same ligands. However, CCR1 showed no correlation to COPD severity. In summary, we identified three chemokine receptors with the potential to mediate CD8+ T-cell infiltration into the lungs of COPD patients.

Figure 4. Chemokine receptors CCR5, CXCR3, and CXCR6 are expressed by lung CD8+ T cells and correlate with COPD severity. Chemokine receptor expression was profiled in dispersed lungs by flow cytometry (A, B, and C) and in preparations of enriched CD8+ T cells by real-time RT-PCR (D, E, and F). CCR5 (A and D), CXCR3 (B and E), and CXCR6 (C and F) expression was correlated to COPD severity, as determined by GOLD stage. Correlation analysis is shown.

Figure 5. Flow cytometric histograms of CCR5, CXCR3, and CXCR6 in GOLD stage 0 and GOLD stage 4 patients. Representative histograms show expression by gated CD8+ cells. The white profiles show receptor-specific antibody staining, and the shaded profiles show staining with isotype-matched control antibodies.

Table 2. Summary of Chemokine Receptor Expression on Lung CD8+ T Cells and Correlation with COPD Severity

Chemokine Ligand Expression in COPD Lung Tissues

To investigate further the potential chemotactic factors involved in CD8+ T-cell recruitment, we analyzed chemokine expression in the lung. Fresh lung specimens were homogenized, and RNA was isolated for real-time RT-PCR analysis. The expression of CCL3 (macrophage inflammatory protein 1), a CCR5 ligand; CXCL9 (monokine induced by interferon-), a CXCR3 ligand; and CXCL16, a CXCR6 ligand, are shown in Figure 6, ACC . Transcript expression of CCL3 and CXCL9 in the whole lung showed significant correlations to disease severity, with r values of 0.58 and 0.63, respectively. Although the transcripts for CXCL16 were highly expressed in the majority of samples, there was no correlation to COPD severity. To confirm the RNA data, we extracted protein from the whole lung. Using Luminex xMAP microsphere technology, we measured chemokine levels for CCL3 and CXCL9 (Figure 6, D and E) . Again, CCL3 and CXCL9 protein levels significantly correlated with COPD disease severity. Additional cytokine transcripts measured in the whole lung included CX3CL1, CCL17, CCL22, CXCL10, CXCL11, and granulocyte macrophageCcolony-stimulating factor (data not shown). These genes displayed either low transcription or no association with disease severity. Taken together, our results suggest that there is a coordinated expression of chemokine receptors and corresponding ligands in COPD patients that could mediate CD8+ T-cell recruitment or retention.

Figure 6. Chemokine expression in lungs of COPD patients. Real-time PCR was used to measure chemokine transcripts for CCL3 (A), CXCL9 (B), and CXCL16 (C) among mRNA isolated from whole lung samples. Results are expressed in arbitrary units. CCL3 (D) and CXCL9 (E) protein levels were measured using Luminex xMAP microsphere technology. Results are expressed as picograms of chemokine per milligram of total protein. Levels were correlated to COPD severity, as determined by GOLD stage. Correlation analysis is shown.

CCL3 and CXCL9 Are Produced by CD1a+ DCs

DCs are known to attract immune effector cells through chemokines.37-39 In view of their mucosal location, we determined whether CD1a+ DCs were a source of chemokines in COPD. To this end, CD1a+ DCs were isolated from lung tissue using immunomagnetic beads, and chemokine transcripts were measured by real-time PCR. As shown in Figure 7 , CCL3 and CXCL9 expression by CD1a+ DCs significantly correlated to COPD severity with respective r values of 0.72 and 0.71. CXCL16, although demonstrating high transcript levels in DCs, again showed no correlation to disease severity. Other chemokine transcript levels measured included CCL17, CCL22, CXCL8, CXCL10, CXCL11, and CX3CL1. Other than CXCL8, these chemokines were expressed by CD1a+ DCs at very low levels. CXCL8 was more strongly expressed but displayed no correlation to disease severity (data not shown). In our analysis, we also used flow cytometry to measure chemokine receptor expression by CD1a+ DCs. None of the receptors analyzed (CCR3, CCR4, CCR5, CCR6, CCCR7, CCR8, CXCR1, CXCR3, and CXCR6) displayed an association with disease severity (data not shown). Our results imply that CD1a+ DCs are active in COPD and may promote CD8+ T-cell recruitment to the lungs through the production of chemokines.

Figure 7. CCL3, CXCL9, and CXCL16 transcript expression by purified CD1a+ DCs. CD1a+ DCs were isolated from human lungs by immunomagnetic beads and used for RNA analysis. Chemokine transcripts for CCL3 (A), CXCL9 (B), and CXCL16 (C) are shown. Transcripts were measured by real-time PCR, and results are expressed as arbitrary units. Levels were correlated to COPD severity, as determined by GOLD stage. Correlation statistical analysis is shown.

This study is the first comprehensive and quantitative analysis of chemokine receptor expression by lung CD8+ T cells in COPD and the first study to examine the role of CD1a+ DCs in COPD. Our analysis of CD8+ T cells revealed that among 11 examined chemokine receptors, only CCR5, CXCR3, and CXCR6 correlated with COPD severity as measured by GOLD staging. The validity of our results was supported by concordant findings using two independent methods: flow cytometry and direct cell isolation with gene expression analysis. The chemokine receptors were likely expressed by a subpopulation of CD45RA+ effector CD8+ T cells. Moreover, our study further demonstrated that CD1a+ DCs might contribute to CD8+ T-cell recruitment through coordinated production of CCL3, CXCL9, and CXCL16, respective ligands for CCR5, CXCR3, and CXCR6. Production of CCL3 and CXCL9 correlated with COPD severity, whereas CXCL16 was constitutively expressed by CD1a+ DCs in all stages of COPD severity, possibly reflecting different functionalities.

Our study implicates CCR5 and CXCR3, along with their respective ligands, in mediating recruitment of CD8+ T cells to the lung. CCR5 could have an important role in CD8 activity because CCR5 stimulation is known to induce production of IL-2 and IFN by T cells.40 Persistent production of IFN could provide an amplification loop to continuously drive T-cell accumulation into the lungs by promoting additional chemokine synthesis. In addition, CCR5 has been shown to be important for selective leukocyte migration in response to chemotactic stimuli.41 CCR5 is the receptor not only for CCL3 but also for CCL4 (macrophage inflammatory protein 1ß) and CCL5 (regulated on activation normal T cell expressed and secreted). We have not yet investigated whether these other CCR5 agonists are expressed in the lungs of COPD patients. However, CCL4 is reportedly increased in the bronchoalveolar lavage fluid of patients with chronic bronchitis and mild to moderate airflow limitation.42 Our finding that CCR5 expression correlated with disease severity did not agree with a previous study reporting decreases in CD3+ CCR5+ cells in severe COPD.43 However, that study was based on immunohistochemical observations in small biopsy fragments and did not determine expression specifically by CD8+ T cells, nor were patients stratified by GOLD stage. This apparent discrepancy could also be due to the fact that severe COPD may represent a more complicated disease state.44 We cannot exclude that end-stage COPD may differ from mild to moderate stages. Patients with end-stage COPD (stage 4) are seldom candidates for lobectomies, from which we most readily recover sufficient cells for analysis. Although these patients are candidates for lung volume reduction surgery or lung transplants, the tissues removed in these operations have proven far less amenable to isolation of adequate cell number. Hence, we obtained fewer specimens; therefore further evaluation may reveal more subtle differences. Nevertheless, for therapeutic purposes, it is more important to identify the chemotactic factors involved at earlier stages of COPD. In addition, it is important to recognize that in human studies, a high degree of experimental variation is not uncommon. Thus, our ability to reveal underlying correlations gains in significance. In the current GOLD classification of disease severity based on spirometric tests, there is an imperfect relationship between the degree of airflow limitation and the presence of symptoms. More precise classification using parameters such as chemokine receptor expression may help better define prognostic groups.

Our analysis of transcripts for all known CXCR3 ligands extends a previous observation that lung CXCR3+ T cells, most coexpressing CD8, are increased in smokers with COPD.24 That study also detected immunoreactivity for CXCL10 in the bronchiolar epithelium of COPD patients. Using a quantitative approach, we found that CXCL9 (monokine induced by interferon-), and not the other known CXCR3 ligands, CXCL10 (interferon-inducible protein 10) and CXCL11 (interferon-inducible T-cell -chemokine), correlated with disease status. Transcripts for CXCL10 were detected in the lungs, in agreement with the above study, but showed no correlation to disease severity. However, CXCL11 displayed low transcript levels, even though it reportedly has the highest affinity for CXCR3.45

Our finding that CXCL16 transcripts were constitutively expressed by CD1a+ DCs suggests that CXCR6 and CXCL16 could be involved in homeostatic and inflammatory cell recruitment and retention events. CXCL16 and its receptor CXCR6 are relatively new additions to the chemokine family. Importantly, CXCL16 exists in membrane-bound and in secreted forms. Although studies have suggested that CXCR6 facilitates recruitment of activated CD8+ T cells to sites of inflammation,46-48 our results imply that this is not its role in COPD. Because CXCL16 has been shown to function as a membrane-bound adhesion molecule for cells expressing CXCR6,49 one possibility is that CXCR6 might allow binding of CD8+ T cells to CD1a+ DCs to promote more efficient stimulation of cell-activating chemokine receptors such as CCR5. Coexpression of CXCR6 and CCR5 has been demonstrated on peripheral blood T cells.50 This would allow DCs and CD8+ T cells to interact, facilitating antigen presentation and allowing the time for T cells to activate or up-regulate additional genes. Our immunohistochemical study indicated that CD1a+ and CD8+ cells are in proximity to each other with the potential for contact. Another possibility is that CXCL16 may be expressed constitutively in its membrane-bound form but that under inflammatory conditions is released in soluble form. Such a functional change would not be detected by transcript analysis and merits further study.

Our analysis of lung CD8+ T cells in COPD supports a previous report of Grumelli et al26 in favor of a Tc1 phenotype. Like them, we found that lung CD8+ T cells expressed IFN but little IL-4. Furthermore, the receptors that we identified, CCR5, CXCR3, and CXCR6, have been reported to be associated with Tc1 cells.46,51 This area remains controversial, for although CD4+ cells were shown to display a Th1 phenotype in COPD,31 two recent studies found CD8+ T cells in COPD to be Tc2-like, IL-4 producers.32,33 We did not identify such a population, possibly because those investigators examined bronchoalveolar lavage rather than interstitial cells.

The detection of CCR7C CD45RA+ CD8+ lung T cells revealed the presence of an effector memory (TEMRA) population. This population was probably expressing chemokine receptors and IFN and, as such, is a candidate for COPD-related effector cells. CCR7C CD45RA+ TEMRA cells are associated with respiratory viral infections.52,53 Viruses have been implicated in promoting COPD by inducing CD8+ T cells to differentiate into IFN-producing effector cells and establishing a cycle of chronic inflammation. It should be noted that not all CD8+ T cells had an effector phenotype. Although not detected by flow cytometry, RNA transcript analysis revealed that there was low expression of CCR7 among CD8+ T cells, suggesting that a population of naïve or central memory CD8+ T cells was present in the lung (data not shown).

Together, our findings suggest a means by which the CD1a+ DC and CD8+ TEMRA interaction could contribute to lung pathology as COPD progresses. Although the absence of CD83 indicates that most CD1a+ DCs in the lungs are immature, more work is necessary to confirm the activation status of these DCs. However, some studies have demonstrated that immature DCs stimulate T cells.36,54 Dumortier et al54 showed in vivo that immature antigen-presenting DCs stimulate naïve CD8+ T cells to acquire cytotoxic T-cell function and take on a central memory phenotype. Activated CD8+ T cells might cause the lung damage observed in COPD via their high levels of perforin and Fas ligand.55 We did not examine perforin, but its expression would be predicted. Sputum CD8+ T cells from COPD patients have been shown to secrete perforin.56 CXCR6/CXCL16 interactions could also contribute to lung damage. Interestingly, CXCR6+ Tc1 cells reportedly contain preformed granzyme A and are cytotoxic. Constitutive CXCL16 expression might contribute to the creation of an environment that promotes apoptosis of structural cells.57

In conclusion, we have demonstrated the potential participation of the chemokine receptors CCR5, CXCR3, and CXCR6 in the infiltration of CD8+ Tc1 effector memory cells into the lungs of COPD patients. We further show a role for CD1a+ DCs in the production of complimentary chemokines. The identified receptors represent possible therapeutic targets to ameliorate CD8+ T cell-mediated inflammation during COPD.

【参考文献】
  Keatings VM, Collins PD, Scott DM, Barnes PJ: Differences in interleukin-8 and tumor necrosis factor- in induced sputum from patients with chronic obstructive pulmonary disease or asthma. Am J Respir Crit Care Med 1996, 153:530-534

Saetta M, Turato G, Facchini FM, Corbino L, Lucchini RE, Casoni G, Maestrelli P, Mapp CE, Ciaccia A, Fabbri LM: Inflammatory cells in the bronchial glands of smokers with chronic bronchitis. Am J Respir Crit Care Med 1997, 156:1633-1639

Di Stefano A, Turato G, Maestrelli P, Mapp CE, Ruggieri MP, Roggeri A, Boschetto P, Fabbri LM, Saetta M: Airflow limitation in chronic bronchitis is associated with T-lymphocyte and macrophage infiltration of the bronchial mucosa. Am J Respir Crit Care Med 1996, 153:629-632

Grashoff WF, Sont JK, Sterk PJ, Hiemstra PS, de Boer WI, Stolk J, Han J, van Krieken JM: Chronic obstructive pulmonary disease: role of bronchiolar mast cells and macrophages. Am J Pathol 1997, 151:1785-1790

Saetta M, Di Stefano A, Turato G, Facchini FM, Corbino L, Mapp CE, Maestrelli P, Ciaccia A, Fabbri LM: CD8+ T-lymphocytes in peripheral airways of smokers with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1998, 157:822-826

O??Shaughnessy TC, Ansari TW, Barnes NC, Jeffery PK: Inflammation in bronchial biopsies of subjects with chronic bronchitis: inverse relationship of CD8+ T lymphocytes with FEV1. Am J Respir Crit Care Med 1997, 155:852-857

Hogg JC, Chu F, Utokaparch S, Woods R, Elliott WM, Buzatu L, Cherniack RM, Rogers RM, Sciurba FC, Coxson HO, Pare PD: The nature of small-airway obstruction in chronic obstructive pulmonary disease. N Engl J Med 2004, 350:2645-2653

Turato G, Zuin R, Miniati M, Baraldo S, Rea F, Beghe B, Monti S, Formichi B, Boschetto P, Harari S, Papi A, Maestrelli P, Fabbri LM, Saetta M: Airway inflammation in severe chronic obstructive pulmonary disease: relationship with lung function and radiologic emphysema. Am J Respir Crit Care Med 2002, 166:105-110

O??Donnell R, Breen D, Wilson S, Djukanovic R: Inflammatory cells in the airways in COPD. Thorax 2006, 61:448-454

Kägi D, Ledermann B, Burki K, Zinkernagel RM, Hengartner H: Molecular mechanisms of lymphocyte-mediated cytotoxicity and their role in immunological protection and pathogenesis in vivo. Annu Rev Immunol 1996, 14:207-232

Saetta M, Turato G, Maestrelli P, Mapp CE, Fabbri LM: Cellular and structural bases of chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2001, 163:1304-1309

Retamales I, Elliott WM, Meshi B, Coxson HO, Pare PD, Sciurba FC, Rogers RM, Hayashi S, Hogg JC: Amplification of inflammation in emphysema and its association with latent adenoviral infection. Am J Respir Crit Care Med 2001, 164:469-473

Seemungal TA, Harper-Owen R, Bhowmik A, Jeffries DJ, Wedzicha JA: Detection of rhinovirus in induced sputum at exacerbation of chronic obstructive pulmonary disease. Eur Respir J 2000, 16:677-683

Cosio MG, Majo J: Inflammation of the airways and lung parenchyma in COPD: role of T cells. Chest 2002, 121:160S-165S

Jung S, Unutmaz D, Wong P, Sano G, De los Santos K, Sparwasser T, Wu S, Vuthoori S, Ko K, Zavala F, Pamer EG, Littman DR, Lang RA: In vivo depletion of CD11c+ dendritic cells abrogates priming of CD8+ T cells by exogenous cell-associated antigens. Immunity 2002, 17:211-220

Probst HC, van den Broek M: Priming of CTLs by lymphocytic choriomeningitis virus depends on dendritic cells. J Immunol 2005, 174:3920-3924

Tian T, Woodworth J, Skold M, Behar SM: In vivo depletion of CD11c+ cells delays the CD4+ T cell response to Mycobacterium tuberculosis and exacerbates the outcome of infection. J Immunol 2005, 175:3268-3272

Zammit DJ, Lefrancois L: Dendritic cell-T cell interactions in the generation and maintenance of CD8 T cell memory. Microbes Infect 2006, 8:1108-1115

Robbins CS, Dawe DE, Goncharova SI, Pouladi MA, Drannik AG, Swirski FK, Cox G, Stampfli MR: Cigarette smoke decreases pulmonary dendritic cells and impacts antiviral immune responsiveness. Am J Respir Cell Mol Biol 2004, 30:202-211

D??hulst AI, Maes T, Bracke KR, Demedts IK, Tournoy KG, Joos GF, Brusselle GG: Cigarette smoke-induced pulmonary emphysema in scid-mice: is the acquired immune system required? Respir Res 2005, 6:147

Demedts IK, Brusselle GG, Vermaelen KY, Pauwels RA: Identification and characterization of human pulmonary dendritic cells. Am J Respir Cell Mol Biol 2005, 32:177-184

Hoogsteden HC, Verhoeven GT, Lambrecht BN, Prins JB: Airway inflammation in asthma and chronic obstructive pulmonary disease with special emphasis on the antigen-presenting dendritic cell: influence of treatment with fluticasone propionate. Clin Exp Allergy 1999, 29(Suppl 2):116-124

Panina-Bordignon P, D??Ambrosio D: Chemokines and their receptors in asthma and chronic obstructive pulmonary disease. Curr Opin Pulm Med 2003, 9:104-110

Saetta M, Mariani M, Panina-Bordignon P, Turato G, Buonsanti C, Baraldo S, Bellettato CM, Papi A, Corbetta L, Zuin R, Sinigaglia F, Fabbri LM: Increased expression of the chemokine receptor CXCR3 and its ligand CXCL10 in peripheral airways of smokers with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2002, 165:1404-1409

Panina-Bordignon P, Papi A, Mariani M, Di Lucia P, Casoni G, Bellettato C, Buonsanti C, Miotto D, Mapp C, Villa A, Arrigoni G, Fabbri LM, Sinigaglia F: The C-C chemokine receptors CCR4 and CCR8 identify airway T cells of allergen-challenged atopic asthmatics. J Clin Invest 2001, 107:1357-1364

Grumelli S, Corry DB, Song LZ, Song L, Green L, Huh J, Hacken J, Espada R, Bag R, Lewis DE, Kheradmand F: An immune basis for lung parenchymal destruction in chronic obstructive pulmonary disease and emphysema. PLoS Med 2004, 1:e8

Pauwels RA, Buist AS, Ma P, Jenkins CR, Hurd SS, : GOLD Scientific Committee: Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease. National Heart, Lung, and Blood Institute and World Health Organization Global Initiative for Chronic Obstructive Lung Disease (GOLD): executive summary. Respir Care 2001, 46:798-825

Fabbri L, Pauwels RA, Hurd SS: Global Strategy for the Diagnosis, Management, and Prevention of Chronic Obstructive Pulmonary Disease: GOLD Executive Summary updated 2003. COPD 2004, 1:105-141discussion 103C104

van Haarst JM, de Wit HJ, Drexhage HA, Hoogsteden HC: Distribution and immunophenotype of mononuclear phagocytes and dendritic cells in the human lung. Am J Respir Cell Mol Biol 1994, 10:487-492

Maestrelli P, Saetta M, Mapp CE, Fabbri LM: Remodeling in response to infection and injury: airway inflammation and hypersecretion of mucus in smoking subjects with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2001, 164:S76-S80

Majori M, Corradi M, Caminati A, Cacciani G, Bertacco S, Pesci A: Predominant TH1 cytokine pattern in peripheral blood from subjects with chronic obstructive pulmonary disease. J Allergy Clin Immunol 1999, 103:458-462

Barcel? B, Pons J, Fuster A, Sauleda J, Noguera A, Ferrer JM, Agusti AG: Intracellular cytokine profile of T lymphocytes in patients with chronic obstructive pulmonary disease. Clin Exp Immunol 2006, 145:474-479

Barczyk A, Pierzchala W, Kon OM, Cosio B, Adcock IM, Barnes PJ: Cytokine production by bronchoalveolar lavage T lymphocytes in chronic obstructive pulmonary disease. J Allergy Clin Immunol 2006, 117:1484-1492

Geginat J, Lanzavecchia A, Sallusto F: Proliferation and differentiation potential of human CD8+ memory T-cell subsets in response to antigen or homeostatic cytokines. Blood 2003, 101:4260-4266

Zhou LJ, Tedder TF: CD14+ blood monocytes can differentiate into functionally mature CD83+ dendritic cells. Proc Natl Acad Sci USA 1996, 93:2588-2592

Cochand L, Isler P, Songeon F, Nicod LP: Human lung dendritic cells have an immature phenotype with efficient mannose receptors. Am J Respir Cell Mol Biol 1999, 21:547-554

Dieu-Nosjean MC, Vicari A, Lebecque S, Caux C: Regulation of dendritic cell trafficking: a process that involves the participation of selective chemokines. J Leukoc Biol 1999, 66:252-262

Cyster JG: Chemokines and cell migration in secondary lymphoid organs. Science 1999, 286:2098-2102

Weninger W, von Andrian UH: Chemokine regulation of naive T cell traffic in health and disease. Semin Immunol 2003, 15:257-270

Loetscher P, Uguccioni M, Bordoli L, Baggiolini M, Moser B, Chizzolini C, Dayer JM: CCR5 is characteristic of Th1 lymphocytes

Springer TA: Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell 1994, 76:301-314

Capelli A, Di Stefano A, Gnemmi I, Balbo P, Cerutti CG, Balbi B, Lusuardi M, Donner CF: Increased MCP-1 and MIP-1ß in bronchoalveolar lavage fluid of chronic bronchitis. Eur Respir J 1999, 14:160-165

Di Stefano A, Capelli A, Lusuardi M, Caramori G, Balbo P, Ioli F, Sacco S, Gnemmi I, Brun P, Adcock IM, Balbi B, Barnes PJ, Chung KF, Donner CF: Decreased T lymphocyte infiltration in bronchial biopsies of subjects with severe chronic obstructive pulmonary disease. Clin Exp Allergy 2001, 31:893-902

Di Stefano A, Donner CF: Severe vs. mild COPD: a different disease? Monaldi Arch Chest Dis 2003, 59:238-239

Clark-Lewis I, Mattioli I, Gong JH, Loetscher P: Structure-function relationship between the human chemokine receptor CXCR3 and its ligands. J Biol Chem 2003, 278:289-295

Kim CH, Kunkel EJ, Boisvert J, Johnston B, Campbell JJ, Genovese MC, Greenberg HB, Butcher EC: Bonzo/CXCR6 expression defines type 1-polarized T-cell subsets with extralymphoid tissue homing potential. J Clin Invest 2001, 107:595-601

Langenkamp A, Nagata K, Murphy K, Wu L, Lanzavecchia A, Sallusto F: Kinetics and expression patterns of chemokine receptors in human CD4+ T lymphocytes primed by myeloid or plasmacytoid dendritic cells. Eur J Immunol 2003, 33:474-482

Sato T, Thorlacius H, Johnston B, Staton TL, Xiang W, Littman DR, Butcher EC: Role for CXCR6 in recruitment of activated CD8+ lymphocytes to inflamed liver. J Immunol 2005, 174:277-283

Shimaoka T, Nakayama T, Fukumoto N, Kume N, Takahashi S, Yamaguchi J, Minami M, Hayashida K, Kita T, Ohsumi J, Yoshie O, Yonehara S: Cell surface-anchored SR-PSOX/CXC chemokine ligand 16 mediates firm adhesion of CXC chemokine receptor 6-expressing cells. J Leukoc Biol 2004, 75:267-274

Unutmaz D, Xiang W, Sunshine MJ, Campbell J, Butcher E, Littman DR: The primate lentiviral receptor Bonzo/STRL33 is coordinately regulated with CCR5 and its expression pattern is conserved between human and mouse. J Immunol 2000, 165:3284-3292

Agace WW, Roberts AI, Wu L, Greineder C, Ebert EC, Parker CM: Human intestinal lamina propria and intraepithelial lymphocytes express receptors specific for chemokines induced by inflammation. Eur J Immunol 2000, 30:819-826

Hislop AD, Annels NE, Gudgeon NH, Leese AM, Rickinson AB: Epitope-specific evolution of human CD8+ T cell responses from primary to persistent phases of Epstein-Barr virus infection. J Exp Med 2002, 195:893-905

Dunne PJ, Faint JM, Gudgeon NH, Fletcher JM, Plunkett FJ, Soares MV, Hislop AD, Annels NE, Rickinson AB, Salmon M, Akbar AN: Epstein-Barr virus-specific CD8+ T cells that re-express CD45RA are apoptosis-resistant memory cells that retain replicative potential. Blood 2002, 100:933-940

Dumortier H, van Mierlo GJ, Egan D, van Ewijk W, Toes RE, Offringa R, Melief CJ: Antigen presentation by an immature myeloid dendritic cell line does not cause CTL deletion in vivo, but generates CD8+ central memory-like T cells that can be rescued for full effector function. J Immunol 2005, 175:855-863

Sallusto F, Lenig D, Forster R, Lipp M, Lanzavecchia A: Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 1999, 401:708-712

Chrysofakis G, Tzanakis N, Kyriakoy D, Tsoumakidou M, Tsiligianni I, Klimathianaki M, Siafakas NM: Perforin expression and cytotoxic activity of sputum CD8+ lymphocytes in patients with COPD. Chest 2004, 125:71-76

Agostini C, Cabrelle A, Calabrese F, Bortoli M, Scquizzato E, Carraro S, Miorin M, Beghe B, Trentin L, Zambello R, Facco M, Semenzato G: Role for CXCR6 and its ligand CXCL16 in the pathogenesis of T-cell alveolitis in sarcoidosis. Am J Respir Crit Care Med 2005, 172:1290-1298

作者单位：From the Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine,* and Department of Pathology, University of Michigan Health System, Ann Arbor; and Pulmonary and Critical Care Medicine Section and Pathology and Laboratory Medicine Service, VA Ann Arbor Healthcare System, A

【摘要】  Isolated lymphoid follicles (ILFs) are organized lymphoid structures that facilitate the efficient interaction of antigen, antigen-presenting cells, and lymphocytes to generate controlled adaptive immune responses within the intestine. Because CC chemokine receptor 6 (CCR6) deficiency affects the generation of mucosal immune responses, we evaluated a potential role for CCR6 in the development of ILFs. We observed that CCR6 and its ligand CCL20 are highly expressed within ILFs and that B lymphocytes are the largest CCR6-expressing population within ILFs. ILF development was profoundly arrested in the absence of CCR6. Concordant with a block in ILF development at a stage corresponding to the influx of B lymphocytes, we observed that CCR6-deficient mice had a diminished population of intestinal B lymphocytes. Bone marrow reconstitution studies demonstrated that ILF development is dependent on CCR6-sufficient B lymphocytes, and adoptive transfers demonstrated that CCR6C/C B lymphocytes were inefficient at localizing to intestinal lymphoid structures. Paralleling these findings, we observed that CCR6-deficient mice had a reduced proportion of Peyer??s patch B lymphocytes and an associated re-duction in the number and size of Peyer??s patch follicular domes. These observations define an essential role for CCR6 expression by B lymphocytes in localizing to intestinal lymphoid structures and in ILF development.
--------------------------------------------------------------------------------
The mucosal immune system is a complex network of lymphoid compartments generating immune responses that both protect the host and mitigate potential damage due to uncontrolled inflammation. In the gastrointestinal tract this system includes diffuse effector sites, such as the intestinal lamina propria, as well as organized lymphoid structures that collectively are referred to as the gastrointestinal-associated lymphoid tissues. Organized lymphoid structures provide sites for the efficient interactions of antigens, antigen-presenting cells, and lymphocytes in a controlled environment. These structures are essential for the initiation of primary immune responses and the regulated environment they provide is felt to be necessary to prevent inappropriate immune responses. Isolated lymphoid follicles (ILFs) have only recently become appreciated as distinct members of the gastrointestinal-associated lymphoid tissues. ILFs resemble Peyer??s patches (PPs) in their architecture, cellular composition, and ability to act as inductive sites for mucosal immune responses.1-5 Our understanding of how these organized structures develop is rapidly evolving. PP formation, like lymph node formation, is developmentally driven, with the early vital events leading to PP formation occurring only during embryogenesis.6 On the other hand, ILF formation occurs after birth and may be induced or augmented by luminal stimuli, including normal intestinal flora.1,2 Further delineation of this process indicates that in the normal animal ILFs are a spectrum of lymphoid aggregates in various stages of formation and that cryptopatches (CPs) are the precursor lymphoid aggregates giving rise to ILFs.2,7-9
Deficiency of CC chemokine receptor 6 (CCR6) has been demonstrated to adversely affect the development of mucosal immune responses,10 but the mechanisms resulting in this defect are still being elucidated.11-14 In contrast to most chemokine receptors, CCR6 pairs monogamously with its ligand, CCL20.15 ß-Defensins have also been identified as potential ligands for CCR6; however, these ligands have a significantly reduced affinity for binding CCR6 and a reduced ability to induce CCR6-dependent chemotaxis when compared with CCL20.16 CCL20 is preferentially produced at mucosal surfaces by a variety of cell types including monocytes, endothelial cells, dendritic cells, fibroblasts, and epithelial cells in the follicle-associated epithelium (FAE).17 Multiple cell types express CCR6, including immature dendritic cells, memory T lymphocytes, and B lymphocytes.15,18,19 Most investigations into the role of CCR6 in mucosal immune responses have revolved around alterations in the function of PP dendritic cells in CCR6-deficient mice.11-14 The contributions of other cell types and the effects of CCR6 deficiency on the development of ILFs have not been previously addressed.
In this study, we evaluated the role for CCR6 in the development of ILFs. We observed elevated expression of CCR6 by ILF B lymphocytes when compared with B lymphocytes from other tissues and elevated expression of CCL20 within ILFs. We found that CCR6C/C mice have significantly reduced numbers of ILFs, but the formation of CPs, as well as CPs containing a large population of dendritic cells, was unaffected. Consistent with this block in ILF formation at a stage before the influx of B lymphocytes, we observed that CCR6C/C mice had a significantly reduced population of non-PP intestinal B lymphocytes, and this defect was most pronounced in the distal small intestine where we have observed the majority of ILFs in wild-type mice. Bone marrow reconstitution demonstrated that CCR6-sufficient B lymphocytes are required for the formation of ILFs, and adoptive transfer studies confirmed that CCR6C/C B lymphocytes have a deficiency in their ability to localize to wild-type PPs and CP/ILFs. Expanding previous reports, and paralleling our observations with ILFs, we observed that CCR6C/C mice have a reduced proportion of B lymphocytes within the PP with an associated decrease in the number of follicles within each PP and a decreased surface area of each PP follicular dome. These findings demonstrate a role for CCR6 expression by B lymphocytes in localizing these cells to organized intestinal lymphoid structures and suggest this requirement is particularly important for ILF development. The profound defect we observed in ILF development in CCR6C/C mice, coupled with the inflammatory nature of CCL20 expression, further highlights the dynamic nature of ILF development and suggests that CCL20 expression facilitates the transition of CPs into ILFs.

【关键词】  chemokine receptor expression lymphocytes essential development isolated lymphoid follicles

Materials and Methods

Mice used for this study were housed in a specific pathogen-free facility and fed routine chow diet. Animal procedures and protocols were performed in accordance with the institutional review board at Washington University School of Medicine. C57BL/6 mice, C57BL/6 congenic mice (B6.Cg-IgHa Thy1a Gpi1a/J), and B-cell-deficient JHC/C mice20 on the C57BL/6 background were purchased from The Jackson Laboratory, Bar Harbor, ME. Lymphotoxin (LT)-deficient mice21 (a gift from Dr. Dave Chaplin, University of Alabama, Birmingham, AL) were bred onto the C57BL/6 background for more than 10 generations before use in experiments. CCR6-deficient mice22 were bred on to the C57BL/6 background for four generations before use in experiments. Timed pregnant C57BL/6 female mice and CCR6C/C mice for use in experiments involving the injection of lymphotoxin ß receptor (LTßR)-Ig fusion protein, were generated by matings with C57BL/6 male mice or CCR6C/C male mice, respectively. Six- to 10-week-old LT C/C mice were used as recipients for bone marrow transfers.

Bone Marrow Transfers

Bone marrow transfers were performed after lethal irradiation as previously described.23 A total of 1 x 107 T-lymphocyte-depleted bone marrow cells from gender-matched donors were injected intravenously into recipients on the second day of irradiation. Mice receiving bone marrow from multiple donors (C57BL/6 and JHC/C or CCR6C/C and JHC/C; see Figure 4 ) received 5 x 106 cells from each donor. Mice were allowed 12 weeks for reconstitution with donor bone marrow before use in experiments. Flow cytometric analysis was performed on splenocytes from recipients at the time of sacrifice to document appropriate B- and T-lymphocyte reconstitution.

Figure 4. CCR6-sufficient B lymphocytes are essential for the development of ILFs. CP and ILF formation can be restored in LTC/C mice following the transfer of LT-sufficient bone marrow. To assess a requirement for CCR6 on B lymphocytes in ILF development, intestines from lethally irradiated LTC/C mice reconstituted with CCR6C/C bone marrow, a combination of CCR6C/C and JHC/C bone marrow, such that the recipients would be selectively deficient in CCR6 expressing B lymphocytes, or a combination of C57BL/6 and JHC/C bone marrow were examined for the presence of iILFs and mILFs. Mice reconstituted with a combination of C57BL/6 and JHC/C bone marrow developed the expected numbers of iILFs and mILFs, whereas mice reconstituted with CCR6C/C bone marrow or a combination of CCR6C/C and JHC/C bone marrow developed few iILFs or mILFs (a). Flow cytometric analysis of splenocytes from recipients was used to confirm appropriate reconstitution of CCR6-sufficient and -deficient lineages (data not shown). Representative photomicrographs of intestinal whole mounts from LTC/C mice receiving CCR6C/C bone marrow (b and e), LTC/C mice receiving CCR6C/C and JHC/C bone marrow (c and f), or LTC/C mice receiving C57BL/6 and JHC/C bone marrow (d and g) stained with UEA-I (bCd) to visualize mature ILFs, or with anti-B220 (eCg) to visualize immature ILFs, demonstrate the requirement for CCR6C/C B lymphocytes in ILF formation. Mature ILFs appear as white domes on the background of nonspecific staining (d); UEA-I-positive M-cells in the FAE cannot be visualized at this magnification. Immature ILFs appear as clusters of brown (B220+) cells (f and g). Recipients of CCR6C/C bone marrow failed to develop iILFs or mILFs (b and e), recipients of a combination of JHC/C and CCR6C/C bone marrow failed to develop mILFs (c), but developed rare iILFs (f, arrow), whereas recipients of a combination of wild-type and JHC/C bone marrow developed mILFs (d, arrowheads) and iILFs (g, arrows). Data are displayed as the mean ?? SD, data for a was generated from five animals in each treatment group. Original magnification for bCg, x20. *P < 0.05 using a one-way analysis of variance.

LTßR-Ig Treatment

LTRß-Ig was purified from supernatants generated by a Chinese hamster ovary cell line producing the LTRß-Ig fusion protein (a gift from Dr. W. Yokoyama, Washington University School of Medicine, St. Louis, MO) as previously described.23 Timed pregnant female mice were injected with 100 µg of LTßR-Ig or 100 µg of human Ig (Bayer Corporation, Elkhart, IN) via tail vein on day 16 postcoitus. Mice receiving LTßR-Ig or human Ig in utero were analyzed for the presence of ILFs at 7 weeks of age (see Figure 2 ).

Figure 2. ILF formation is arrested at a stage corresponding to the influx of B lymphocytes in the absence of CCR6. Intestines from CCR6C/C mice and wild-type mice were evaluated for the presence of CD90+ clusters (a), corresponding to all CP and ILF lymphoid aggregates, CD11c+ clusters (b), corresponding to CPs with a substantial proportion of dendritic cells and ILFs, B220+ clusters (c), corresponding to immature ILFs, and mature ILFs (c) as outlined in Materials and Methods. There were no significant differences in the numbers of CD90+ clusters (a) or CD11c+ clusters (b) between the two groups. There was a significant difference between the groups with respect to the numbers of B220+ clusters or iILFs (c). The defect in the development of ILFs in the CCR6C/C mice could not be overcome by treatment with LTßR-Ig in utero, a manipulation that augments the numbers of ILFs in wild-type mice; treatment with control (human) Ig did not augment ILF numbers (c). CD90+ clusters from wild-type mice (d) and CCR6C/C mice (e) were of equivalent size; however, the CD90+ clusters from CCR6C/C mice had fewer B lymphocytes (d and e). Red, anti-CD90; green, anti-B220; blue, Hoechst dye to visualize nuclei. Original magnification, x400. The defect in the development of ILFs in the CCR6C/C mice correlated with a lack of B lymphocytes in the diffuse lamina propria of the intestine, which contains CPs and ILFs (f). This defect was most pronounced in the distal small intestine, the region in which the majority of ILFs are located in wild-type mice in our colony (f). Data are displayed as the mean ?? SD for a, c, and f. The data from a are generated from two mice from each group. The data from c and f were generated from three or more mice from each treatment group. *P < 0.05 when comparing C57BL/6 and CCR6C/C mice using a standard Student??s t-test for unmanipulated animals and using a one-way analysis of variance to compare LTßR-Ig- and control Ig-treated animals.

Whole Mounts of Small Intestine

Small intestines were removed intact, flushed with ice-cold phosphate-buffered saline (PBS), and opened along the mesenteric border. Intestines were mounted, lumen facing up, and fixed with ice-cold 10% phosphate-buffered formyl saline (Fisher Scientific, Pittsburgh, PA) for 1 hour at 4??C. Intestines were washed three times in ice-cold PBS, incubated in a solution of 20 mmol/L dithiothreitol, 150 mmol/L Tris, and 20% ethanol at room temperature for 45 minutes, washed three times in ice-cold PBS, and incubated in a solution of 1% H2O2 for 15 minutes at room temperature to block endogenous peroxidases. Intestines were washed three times in PBS, followed by incubation in PBS containing 1% bovine serum albumin (BSA) and 0.3% Triton X-100 for 30 minutes. Intestines were incubated with horseradish peroxidase-conjugated lectin from Ulex europaeus (UEA-I; Sigma-Aldrich, St. Louis, MO) in PBS, BSA, and Triton X-100 solution overnight at 4??C to facilitate the identification of PPs and mature ILFs (mILFs). The following day intestines were washed three times in PBS, incubated in DAB metal peroxide substrate (Pierce Chemical Co., Rockford, IL) for 15 minutes, rinsed twice in distilled water, and returned to PBS for further analysis. Investigators unaware of the treatment groups determined the presence of mILF. Under low-power microscopy (25 to 65x) previously established criteria were used to determine the presence of mILF2 : 1) presence of a nodular structure with size equal to or greater than the width of one villus; 2i) nodular structure possessing an overlying dome resembling the FAE of PP; and 3) nodular structures occurring singly or in groups of two.

For the analysis of PPs, whole mounts of intestines were stained with UEA-I as above. The following criteria were used for the purpose of enumerating PPs. PPs were defined as nodular structures more than five villi wide, located along the anti-mesenteric border of the small intestine. These criteria overlap with the criteria used to enumerate mILFs but were necessary because some of the PPs of the CCR6C/C mice contained only one follicle. PPs were identified using a dissecting microscope at 32x magnification and photographs obtained of each PP. Photographs of PPs and a scale were enlarged to 10 x 12 inches, and the diameter of each dome was measured. The surface area of each dome was calculated using the following formula: surface area = (diameter/2).2

For anti-B220 and anti-CD11c staining of whole mounts to determine the numbers of iILFs and CD11c+ clusters, intestines were removed intact, flushed with PBS, opened along the mesenteric border, and mounted as above. Intestines were then incubated three times in Hanks?? balanced salt solution (BioWhittaker, Walkersville, MD) containing 5 mmol/L EDTA at 37??C with shaking for 10 minutes to remove epithelial cells. Intestines were then fixed in 10% phosphate-buffered formyl saline and treated with 1% H2O2 for 15 minutes at room temperature as above. Intestines were incubated in a solution of 50 mmol/L Tris, pH 7.2, 150 mmol/L NaCl, 0.6% Triton X-100, and 0.1% BSA for 1 hour at 4??C to block nonspecific antibody binding and then incubated with rat anti-mouse B220 antibody (PharMingen, San Diego, CA) or biotin-conjugated hamster anti-mouse CD11c (eBiosciences, San Diego, CA) diluted in the above solution overnight at 4??C. Intestines were washed three times in the above solution and incubated with a horseradish peroxidase-conjugated goat anti-rat IgG antibody or streptavidin-conjugated horseradish peroxidase (Jackson Immuno-Research Laboratories, West Grove, PA) diluted in the above solution at room temperature for 1 hour. Intestines were washed three times and incubated in DAB metal peroxide substrate as above. Intestine whole mounts were examined under a dissecting microscope at 25 to 65x. Immature ILFs (iILFs) were counted as clusters of B220+ cells occurring at the base of villi and not containing an overlying dome. Dendritic cell clusters were counted as clusters of CD11c+ cells occurring at the base of villi.

Cell Isolation from Spleen, PPs, and mILFs

Spleens and PPs were removed from unmanipulated C57BL/6 mice and disrupted by mechanical dissociation. Intestines were removed from C57BL/6 mice receiving LTßR-Ig in utero, flushed with ice-cold PBS, opened along the mesenteric border, and mounted with the lumen facing up in cold PBS, as described above. Using the dissecting microscope and a blunt-end 26-gauge needle and syringe, multiple mILFs were aspirated and placed in cold PBS. Red blood cells were lysed from cellular suspensions and then used for flow cytometric analysis as described below. Average yield of viable mononuclear ILF cells ranged from 3 to 7 x 105 cells/intestine.

Flow Cytometric Analysis

Single-cell suspensions were obtained as above and flow cytometric analysis performed as previously described.23 Reagents used for analysis were fluorescein isothiocyanate-conjugated or phycoerythrin-conjugated rat anti-mouse CD19, fluorescein isothiocyanate-conjugated anti-mouse CD45, streptavidin-conjugated phycoerythrin, appropriate isotype control antibodies (all from BD Biosciences, San Diego, CA), and rat anti-mouse CCR6 (R&D Systems, Minneapolis, MN). Dead cells were excluded based on forward and side light scatter. Gates for positive staining were defined such that 1% of the analyzed population stained positive with the appropriate isotype control antibody. Flow cytometric analysis for the expression of CCR6 was performed using directly conjugated anti-CCR6 antibodies in some replicates as well as anti-CCR6 antibodies with an anti-rat IgG secondary antibody (eBiosciences) to augment the fluorescence intensity in some replicates (Figure 1, c and d) ; both methods gave equivalent results.

Figure 1. CCR6 and CCL20 are expressed within ILFs. RNA was isolated from non-PP, non-mILF-bearing small intestine, PPs, and mILFs and used to analyze the expression of CCR6 and CCL20 as described in Materials and Methods (a and b). Cellular populations were isolated from spleen, PPs, and mILFs and used to examine cell surface expression of CCR6 by flow cytometry as described in Materials and Methods (c and d). ILFs demonstrated elevated expression of CCR6 when compared with adjacent non-PP, non-ILF-bearing intestine (a). A large population of ILF cells express cell surface CCR6, and the majority of CCR6+ ILF cells are B lymphocytes (CD19+); gates are set such that less than 1% of the cellular population stains positive with the isotype control antibody (c). In side-by-side comparisons, we observed that CCR6 expression was higher in mILF and PP B lymphocytes when compared with splenic B lymphocytes (d). We observed that CCL20 expression was significantly higher in PPs and mILFs when compared with non-PP, non-mILF bearing small intestine (b). Numeric values in parentheses (d) represent the mean fluorescence intensity for each population; the mean fluorescence intensity for the isotype control staining of the CD19+ cells from the PPs and mILFs in d were 94.6 and 67.3, respectively. Data in a and b represent the mean ?? 1 SD of each of two independent experiments. Data in c and d are representative of one of four independent experiments. *P < 0.05 for each replicate when compared with non-PP non-ILF-bearing intestine in a and b.

Paraffin-embedded sections containing PPs from whole-mount intestines (performed as described above) were deparaffinized, treated with antigen-unmasking solution (Vector Laboratories, Burlingame, CA), treated with avidin/biotin blocking kit (Vector Laboratories), washed three times in PBS, and blocked for 15 minutes at room temperature in PBS containing 1% BSA and 0.1% Triton X-100. Sections were then incubated with biotin-conjugated lectin from Arachis hypogaea (PNA) (Sigma-Aldrich) or biotin-conjugated anti-B220 antibody (BD Biosciences) diluted in PBS containing 1% BSA and 0.1% Triton X-100 overnight at 4??C. Biotinyl-tyramide signal amplification (DuPont/NEN, Boston, MA) followed by incubation with streptavidin-conjugated cyanine 2 dye (Jackson ImmunoResearch) was used for detection of PNA staining. Anti-B220 staining was detected using streptavidin-conjugated cyanine 3 dye (Jackson ImmunoResearch). Sections were counterstained with Hoescht dye (Sigma-Aldrich) to visualize nuclei.

Immunohistochemistry on frozen sections of intestine was used to enumerate CD90+ cellular clusters and to evaluate the localization of adoptively transferred B lymphocytes. Intestines were embedded in OCT compound and serial 8-µm sections were obtained from each block. Slides were fixed with a 1:1 solution of acetone and methanol for 15 minutes, dried, rehydrated, and blocked with PBS containing 1% BSA. Sections were incubated with primary antibodies overnight at 4??C, washed with PBS, and incubated with fluorescently labeled secondary reagents for 1 hour at room temperature. Sections were stained with Hoescht dye (Sigma-Aldrich) to visualize nuclei.

Quantification of CD90+ Cellular Clusters

Segments of small intestine of 1.5 cm were embedded in OCT compound, frozen, and cut at an axis perpendicular to the villi into 8-µm sections. The segments comprised more than one-half of the entire intestine, and identical areas of the intestine were obtained from each animal evaluated. Two serial sections of small intestine were stained with anti-CD90 (eBioscience) and anti-CD3 (eBioscience) as above using two-color immunofluorescence. The serial sections were examined at a magnification of 100x or higher, and the number of CD90+CD3C cellular clusters falling within the crypt area on each section was counted and averaged between the serial sections. Additional sections were stained with anti-CD90 and anti-c-kit to confirm that the CD90 clusters are also c-kit+, stained with anti-B220 to assess the numbers of B lymphocytes associating with the CD90 clusters (Figure 2, d and e) , and stained with anti-CD11c (BD Biosciences) to assess the clustering of dendritic cells associated with the CD90 clusters. Photomicrographs of the sections taken with a 25x objective were assembled, and the crypt area of each section was determined using AxioVision software (Carl Zeiss MicroImaging GmbH, Gottingen, Germany). The average number of CD90+ clusters/mm2 of crypt surface area was then determined.

RNA Isolation and Real-Time Polymerase Chain Reaction

PPs were removed from unmanipulated C57BL/6 mice. Mature ILFs were isolated from C57BL/6 mice receiving LTßR-Ig in utero using a dissection microscope and 26-gauge needle as described above. Non-PP, non-mILF-bearing intestine from the distal small intestine of C57BL/6 mice was identified using a dissecting microscope and removed. The PP and mILF tissue contained the overlying FAE, stromal elements, and mononuclear cells.

RNA was isolated using Trizol (Invitrogen, Carlsbad, CA) and treated with DNase I (Ambion, Austin, TX) to remove contaminating DNA, and cDNA was synthesized from 2 µg of total RNA using Superscript II RNase HC reverse transcriptase (Invitrogen). Expression of targets was detected by real-time polymerase chain reaction using ABI prism 7700 sequence detection system and SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA). The following primers were used for detection of the targets, forward primers are listed first, followed by reverse primers: 18S 5'-CGGCTACCACATCCAAGGAA-3' and 5'-GCTGGAATTACCGCGGCT-3', ß-actin 5'-GCTTCTTTGCAGCTCCTTCGT-3' and 5'-ATATCGTCATCCATGGCGAAC-3', CCL20 5'-TGATGCTTTTTTGGGATGGAA-3' and 5'-AGCCTTCAACCCCAGCTGT-3', and CCR6 5'-TGTTCTGCTATCTGTTCATTATCAAGA-3' and 5'-CACGACTCGGATGGCTCTGT-3'. Samples were measured in triplicate. Relative quantitation of target expression using 18S or ß-actin as a housekeeping gene was determined using the comparative crossing threshold method as described in the ABI Prism 7700 sequence detection system user bulletin.

Adoptive Transfers

To assess the ability of wild-type and CCR6C/C B lymphocytes to localize to the organized intestinal lymphoid tissues, splenocytes isolated from CCR6C/C, C57BL/6 congenic mice (expressing the IgHa allotype) were injected intravenously into JHC/C (B lymphocyte-deficient) recipients. Intestinal B lymphocytes could not be used for these studies because the CCR6C/C mice had few intestinal B lymphocytes (Figure 2f) . Recipients received a total 3 x 107 cells from either CCR6C/C mice, C57BL6 congenic mice, or combined CCR6C/C and C57BL/6 congenic mice. Flow cytometric analysis was performed on the cellular populations at the time of transfer to confirm that equivalent numbers of B lymphocytes from each donor were injected into recipients. Mice were sacrificed 1 week later and frozen sections from PPs and small intestines were evaluated for the presence of transferred cellular populations using immunohistochemistry with anti-IgMb (BioLegend, San Diego, CA), anti-IgMa (BD Biosciences), and anti-B220 antibodies. In co-transfer experiments, serial sections of intestine were stained with either IgMa or IgMb. In experiments involving separately transferred populations, intestinal sections were stained with anti-CD11c and anti-B220. Sections were examined with fluorescence microscopy at a magnification of 200x or higher. The number of IgMa- or IgMb-positive cells in each PP serial section or the number of B220+ cells in each CD11c+ cluster was determined by an individual unaware of the treatment groups.

Statistical Analysis

Data analysis using Student??s t-test and a one-way analysis of variance with a Dunnett??s multiple comparison post-test was performed using GraphPad Prism (GraphPad Software Inc., San Diego, CA).

CCL20 Is Highly Expressed within PPs and ILFs, and CCR6 Is Highly Expressed by ILF and PP B Lymphocytes

To assess the role of CCR6 in ILF formation, we examined the mRNA expression of CCR6 in ILFs using real-time polymerase chain reaction. We noted that CCR6 expression was significantly higher in ILFs when compared with non-PP, non-ILF-bearing intestine (Figure 1a) . To evaluate CCR6 protein expression and to determine the cell types expressing CCR6, we examined ILF cellular populations for CCR6 expression by flow cytometry. We found that a large proportion of the ILF cellular population expresses CCR6; gates for positive staining were set such that less than 1% of cells stain positive for the isotype control (Figure 1c) . The majority of cells falling within the CCR6+ gate are B lymphocytes (CD19+) (Figure 1c) . Directly comparing B-lymphocyte populations from the spleen, PPs, and ILFs, we observed that ILF B lymphocytes displayed greater cell surface expression of CCR6 when compared with splenic B lymphocytes and Peyer??s patch B lymphocytes (Figure 1d) . We observed that T lymphocytes (CD3+) and dendritic cells (CD11c+) displayed staining consistent with CCR6+ and CCR6C populations, which comprised approximately 3% and 2% of the CCR6+ ILF cellular population, respectively; the remaining CCR6+ population of the ILFs was predominantly comprised of lineage c-kit+ cells (data not shown). In attempts to understand further the significance of CCR6 in ILF and PP formation, we examined the expression of CCL20 in the spleen, PPs, and ILFs. We found that both ILFs and PPs displayed CCL20 mRNA expression that was significantly greater than that seen in the non-PP, non-mILF-bearing intestine (Figure 1b) .

ILF Development Is Arrested at a Stage Corresponding to the Influx of B Lymphocytes in the Absence of CCR6

To determine whether CCR6 played an important role in the formation of CPs and ILFs, we examined the intestines of CCR6C/C and C57BL/6 mice for the presence of CPs and ILFs. CPs are clusters of lineage marker-negative c-kit-positive cells randomly distributed throughout the small intestine.24 These CP cells also express other markers such as CD90. CPs can only be identified using immunohistochemistry on frozen sections of intestine cut on an axis perpendicular to the villi. We observed that anti-CD90 is a useful marker for enumerating CPs, as the anti-CD90 staining is robust and CPs can easily be identified using 100x magnification. In contrast, anti-c-kit staining is relatively weak and therefore less useful to enumerate CPs. We observed that CCR6C/C mice had an equivalent number of CD90+ clusters when compared with wild-type mice (Figure 2a) . CPs and ILFs also contain a surrounding population of CD11c+ dendritic cells. In some CPs, this CD11c+ population becomes prominent. We can enumerate these CPs containing a large population of CD11c+ cells as well as CD11c+ clusters within ILFs using whole mounts with anti-CD11c staining. We observed no differences in the numbers of large CD11c+ clusters when comparing CCR6C/C mice to wild-type mice (Figure 2b) .

The transition of CPs to ILFs is thought to occur when a subset of CPs are infiltrated by B lymphocytes. Initially, B lymphocytes cluster in the CP at the base of the villi and form a loosely organized structure that we have termed the iILFs. A subset of these immature structures continue to accumulate B lymphocytes and develop a follicle-associated epithelium and a loosely organized germinal center; we have termed these structures mILFs. Using whole mounts of the intestine to enumerate ILFs, we observed that CCR6C/C have a near absence of both iILFs and mILFs, and the reduced number of iILFs was significantly different from wild-type mice (Figure 2c) . Mature ILFs are rare in C57BL/6 mice in our colony, making it difficult to assess a defect in mILF development by comparing wild-type and CCR6C/C mice. In addition, underdeveloped PPs in the CCR6C/C mice would be counted as mILFs in the current scoring system, further complicating this assessment. However, the numbers of mILFs can be augmented by LTßR-Ig treatment in utero. In utero LTßR-Ig treatment on day 16 of gestation ablates PP development and results in a transient IgA deficiency in the progeny, which is subsequently associated with augmented development of ILFs.25 Therefore, to identify a defect in mILF development, mice were given LTßR-Ig in utero and examined for the presence of ILFs. Treatment of C57BL/6 mice with LTßR-Ig in utero augmented the development of both iILFs and mILFs (Figure 2c) . CCR6C/C mice showed a modest increase in the numbers of iILFs following LTßR-Ig treatment; however, the numbers of iILFs formed were still significantly less than unmanipulated C57BL/6 mice (Figure 2c) . This indicates that in the absence of CCR6 other pathways can facilitate iILF formation, but these pathways are less effective. Treatment of CCR6C/C mice with LTßR-Ig in utero failed to induce the formation of mILFs (Figure 2c) . Consistent with a block in the ability of B lymphocytes to infiltrate the CP in CCR6C/C mice, we observed few B220+ cells within CD90+ clusters in CCR6C/C mice (Figure 2, d and e) . In C57BL/6 mice in our colony, ILFs are located preferentially in the distal small intestine.2 Consistent with the loss of ILFs and the inability of CCR6C/C CP to become infiltrated with B lymphocytes, we observed that CCR6C/C mice have a diminished population of B lymphocytes in their diffuse lamina propria, which contains the CPs and ILFs (Figure 2f) . This defect was most pronounced in the distal small intestine where the majority of ILFs are located in the wild-type mice (Figure 2f) . These findings indicate that in the absence of CCR6 the development of ILFs is blocked at a stage of B-lymphocyte infiltration into CPs, or the formation of iILFs.

PPs Have a Reduced B-Lymphocyte Population and a Reduced FAE in the Absence of CCR6

To determine whether CCR6 deficiency affected the B-lymphocyte population in other intestinal organized lymphoid structures, we examined the PPs of the CCR6C/C mice. The initial observations regarding the PP phenotype in the CCR6C/C mice was unclear, with one group reporting normal PP development and a second group reporting that the CCR6C/C PPs contained fewer domes and had reduced total cellularity.10,13 We analyzed the numbers of PPs, the numbers of domes, and the surface area of each dome in CCR6C/C and wild-type mice. We observed that CCR6C/C mice had an equal number of PPs when compared with wild-type mice (data not shown); however, each PP in the CCR6C/C mice contained significantly fewer domes when compared with wild-type mice (Figure 3, a, e, and f) . Moreover, the average surface area of each PP in the CCR6C/C mice was significantly less than that seen in the wild-type mice (Figure 3, b, e, f, g, and h) . Consistent with these observations, we observed significantly decreased total cellularity of PPs from CCR6C/C mice when compared with wild-type mice (Figure 3c) , and we observed a decrease in the B-lymphocyte population in the PPs of CCR6C/C mice when compared with wild-type mice (Figure 3d) . We did not observe any differences in the proportion of CD4+ or CD8+ T lymphocytes or in the proportion of dendritic cells (MHCII+, CD11c+) within the PP when comparing CCR6C/C and wild-type mice (data not shown). The architecture and organization of the PP from CCR6C/C mice and wild-type mice were similar. Each contained a large population of B220+ cells with a central lucent area with PNA+ staining consistent with the presence of germinal centers (Figure 3, gCl) . These findings confirm and extend earlier findings regarding the PPs of CCR6C/C mice and demonstrate that although CCR6 deficiency affects B-lymphocyte populations in both types of organized intestinal lymphoid structures, this deficiency more dramatically effects ILF development.

Figure 3. CCR6C/C mice have a decreased population of PP B lymphocytes and an associated decrease in the size of the PP FAE. Intestines from CCR6C/C and C57BL/6 mice were analyzed for the numbers of PPs, the numbers of domes per PP, the surface area of each dome, PP cellularity, and PP cellular composition as described in Materials and Methods. Panels e, g, i, and k are representative photomicrographs of PPs from C57BL/6 mice. Panels f, h, j, and l are representative photomicrographs of PPs from CCR6C/C mice. Panels e and f demonstrate the appearance of PPs on UEA-I-stained whole mounts from the small intestine. Panels g and h demonstrate the appearance of PPs on H&E-stained sections. Panels i and j and panels k and l demonstrate the appearance of PPs on anti-B220-stained (red) and PNA-stained (green) sections, respectively. Nuclei are identified by blue staining with Hoescht dye in iCl. CCR6C/C mice had normal numbers of PPs (not shown), but PPs from CCR6C/C mice had a significantly reduced number of domes (a, e, and f). In addition, the surface area of each dome was reduced in PPs from CCR6C/C mice (b, g, and h; arrows in g and h denote the FAE in cross section). Consistent with the reduced number of domes we noted a reduced overall cellularity of PPs from CCR6C/C mice (c). Analysis of cellular composition revealed a reduced population of B lymphocytes in PPs from CCR6C/C mice (d). The size of the PP follicles from CCR6C/C and C57BL/6 mice were comparable (g and h), and both contained a population of B lymphocytes (B220+ cells staining red, i and j) with lucent areas consistent with the presence of germinal centers (PNA+ cells staining green, k and l). Data were generated from nine animals of each genotype. *P < 0.05.

CCR6-Sufficient B Lymphocytes Are Essential for the Formation of ILFs

Given the above findings, we wished to determine whether CCR6-sufficient B lymphocytes were required for the development of ILFs and to examine the stage of ILF development for which a requirement for CCR6-sufficient B lymphocytes might exist. LTC/C mice lack CPs, iILFs, and mILFs, but the formation of ILFs can be reliably induced in LTC/C mice following the transfer of LT-sufficient bone marrow reconstituting the LT/LTßR, as well as the LT/TNFR and LT/HVEM axes.2,25 This provides a useful model to study the requirements of bone marrow-derived cell populations on ILF formation. LTC/C mice reconstituted with CCR6C/C bone marrow, a combination of CCR6C/C and JHC/C bone marrow, such that all B lymphocytes are CCR6-deficient, or a combination of C57BL/6 and JHC/C bone marrow were examined for the presence of iILFs and mILFs. LTC/C recipients of CCR6C/C bone marrow and recipients of a combination of CCR6C/C and JHC/C bone marrow were deficient in the formation of iILFs and mILFs when compared with recipients of a combination of C57BL/6 and JHC/C bone marrow (Figure 4) , thus demonstrating a requirement for CCR6-sufficient B lymphocytes in the development of ILFs.

CCR6C/C B Lymphocytes Have a Diminished Ability to Localize to Organized Gastrointestinal-Associated Lymphoid Tissues

The above findings suggest that CCR6C/C B lymphocytes have a diminished capacity to localize to intestinal lymphoid structures. To confirm this defect we injected equal numbers of CCR6C/C (IgHb allotype) and wild-type (IgHa allotype) splenic B lymphocytes into B-cell-deficient JHC/C mice and looked for their presence in organized intestinal lymphoid structures. When equal numbers of CCR6C/C and wild-type B lymphocytes were injected into the same JHC/C recipient, we observed that wild-type B lymphocytes have a threefold increased ability to localize to PPs as determined by the number of IgMbright cells (Figure 5, aCc) . We also observed a larger number of wild-type IgMdull cells when compared with CCR6C/C IgMdull cells in the recipients?? PPs; however, definitive quantification of these IgMdull cells was not feasible due to the intensity of staining. We were unable to identify many donor B lymphocytes within CD90+ or CD11c+ clusters when using IgM allotype staining, despite being able to identify B220+ CD11cC cells within these clusters. We feel this is due to the relatively lower expression of IgM by the majority of ILF B lymphocytes, making their allotype-specific identification more difficult. To examine the ability of CCR6C/C B lymphocytes to localize to CD11c+ clusters, we transferred equivalent numbers of wild-type or CCR6C/C splenic B lymphocytes to individual recipients and examined the recipients for the presence of B220+ CD11cC cells within the clusters. We observed that wild-type B lymphocytes had approximately a twofold better ability to localize to CD11c+ clusters when compared with CCR6C/C B lymphocytes (Figure 5, dCf) . These findings demonstrate that CCR6C/C B lymphocytes have an impaired ability to localize to organized intestinal lymphoid structures.

Figure 5. CCR6-deficient B lymphocytes have impaired localization to intestinal lymphoid structures. Splenocytes from CCR6C/C (IgHb allotype) mice and wild-type congenic (IgHa allotype) mice were transferred together (aCc) or transferred separately (dCf) into B lymphocyte-deficient (JHC/C) recipients as described in Materials and Methods. Transferred cellular populations contained equivalent numbers of B lymphocytes. Mice were evaluated 7 days later for the presence of wild-type or CCR6C/C B lymphocytes by immunohistochemistry with anti-IgMa (wild type) and anti-IgMb (CCR6C/C) staining when cotransferred (aCc) or anti-B220 staining when transferred separately (dCf). Wild-type B lymphocytes displayed a threefold greater ability to localize to the PP as determined by the numbers of IgMhi cells present in the PP using allotype specific staining (aCc). There were also a significant number of IgMlo IgMa+ (wild type) cells present in the PP, but these could not be accurately quantified due to the intensity of staining (b). CCR6C/C B lymphocytes also displayed a significant, but less pronounced, defect in localizing to CD11c+ clusters as determined by anti-B220 staining (dCf). Data in d are displayed as the mean ?? SD. Data were generated from two independent experiments with a total of four mice in each of the transfer groups. *P < 0.05.

A primary function of the mucosal immune system is to protect the host from invading pathogens while avoiding uncontrolled, damaging immune responses. Organized lymphoid structures within the intestine, including PPs and ILFs, play a key role in this function. They provide an environment allowing for the efficient interaction of antigen, antigen-presenting cells, and lymphocytes to facilitate the development of protective adaptive immune responses. A key distinction between PPs and ILFs are the pathways leading to their formation. The formation of PPs is developmentally driven, with the initial steps in PP formation occurring only during embryogenesis,6 whereas the development of ILFs occurs after birth and can be influenced by luminal stimuli, including normal intestinal flora.1,2,26 Recently, CPs have been suggested to give rise to ILFs.7-9 Therefore, CP and ILF development may be closely linked, and events affecting CP development may subsequently affect ILF development.

CCL20 has several properties suggesting it is a candidate for participating in ILF development. CCL20 is produced at mucosal surfaces, and specifically produced by the FAE under normal circumstances.17 Similar to ILF formation, CCL20 expression is induced by inflammatory stimuli, and intestinal CCL20 expression is elevated in inflammatory conditions associated with the formation of lymphoid structures.17,27-29 In addition, CCL20 functions to recruit dendritic cells and B lymphocytes; these cell types make up a large proportion of the ILF cellular population.4 CCR6, the only identified receptor for CCL20, plays an important role in mucosal immune responses as evidenced by diminished production of antigen-specific mucosal IgA following oral immunization of CCR6-deficient mice.10 The specific defects resulting in this phenotype are still being elucidated. Initial studies attributed this defect to the absence of CD11b+ dendritic cells in the PP subepithelial dome in CCR6-deficient mice.13 However, subsequent studies demonstrated that this dendritic cell population was present but reduced in CCR6-deficient mice.14 More recent investigations revealed that CCR6 expression by dendritic cells in the subepithelial dome is required to initiate T-cell-dependent responses to an oral pathogen.12 Despite these observations, the role of CCL20 and CCR6 in the development of ILFs is largely uninvestigated with the exception of one report of normal CP development in CCR6C/C mice.30

Here, we demonstrate that CCL20 expression is increased in ILFs and that CCR6 is highly expressed by B lymphocytes within ILFs. We observed that CCR6-deficient mice have a defect in ILF development; however, the formation of CPs and the recruitment of a substantial population of dendritic cells into these CPs are not altered in these animals. This defect in ILF development correlated with a decreased population of B lymphocytes in the diffuse lamina propria, which contains the CPs and ILFs. Accordingly, this defect was most pronounced in the distal small intestine, the region in which we see the majority of ILFs in wild-type mice. These observations indicate that the requirement for CCR6 in ILF development coincides with the influx of B lymphocytes into the CPs and/or ILFs. Using bone marrow chimeric mice, we confirmed the requirement for CCR6 expression by B lymphocytes in ILF development. These findings were further supported by adoptive transfer studies demonstrating that CCR6C/C B lymphocytes had a reduced ability to localize to PPs and CPs. This demonstrates a specific requirement for CCR6 expression by B lymphocytes in localizing to the intestine and is consistent with a block in ILF development at a stage in which B lymphocytes localize to these structures in CCR6-deficient mice. Although not specifically addressed in the studies presented here, our findings do not preclude, but could suggest, that other CCR6-sufficient cell types play a role in ILF development. We observed a small number of iILFs in recipients of a combination of JHC/C and CCR6C/C bone marrow, suggesting that other pathways can partially compensate for the loss of CCR6 expression by B lymphocytes. In combination with this observation, the absence of ILFs in the recipients of CCR6C/C bone marrow suggests that a CCR6-sufficient non-B lymphocyte plays a role in ILF development. In addition to these observations, we noted a trend toward decreased numbers of large CD11c+ clusters in the CCR6C/C mice, suggesting other potential defects in ILF development.

PPs from CCR6C/C mice revealed parallel, but less dramatic, findings. CCR6C/C mice have a normal number of PPs but with diminished size and decreased number of follicles.13 We observed that as the CCR6C/C mice are crossed onto the C57BL/6 background, this phenotype has become more pronounced, such that on average PPs in the CCR6C/C mice have less than two follicles. In our current scoring system, these abnormal PPs are counted as mILFs, thus artificially increasing the number of mILFs in the CCR6C/C mice. Therefore, it is likely that the CCR6C/C mice on the C57BL/6 background are more deficient in mILFs than suggested by the data presented here. We also observed that the PPs in the CCR6C/C mice had small follicular domes, reduced total cellularity, and a reduced proportion of B lymphocytes. The reduced PP total cellular population is consistent with previous reports on the phenotype of the CCR6C/C mice11,13 ; however, the decreased percentage of PP B lymphocytes and the smaller PP follicular dome in the CCR6C/C mice has not been previously noted. The differences we observed in the PP B-lymphocyte populations were small but statistically significant. Evaluation of older animals revealed an increase in the PP B-lymphocyte populations in both wild-type and CCR6C/C mice; however, CCR6C/C mice maintained a statistically significant decrease in the proportion of PP cells that were B lymphocytes when compared with age-matched wild-type mice (data not shown). This indicates that the small, but statistically significant, difference we observed is maintained with aging. The decreased size of the follicle dome associated with a decreased population of PP B lymphocytes in the PPs of the CCR6C/C mice is supported by a prior study documenting a role for B lymphocytes in facilitating the development of the FAE31 as well as a recent study demonstrating a reduced number of M cells in the FAE of CCR6C/C mice.11 The reduced surface area of the FAE and decreased numbers of M cells could provide another explanation for the diminished capacity of the CCR6C/C mice to mount mucosal immune responses to oral antigens.10 We observed no differences in the proportion of the PP cellular population that were dendritic cells (MHCII+, CD11c+) when comparing wild-type and CCR6C/C mice. Our findings are consistent with a recent study using similar methods that found a modest increase in the population of PP cells that are CD11c+ in the CCR6C/C mice.11 In contrast, previous reports demonstrated an absent or diminished CD11b+ dendritic cell population in the PP subepithelial dome of CCR6C/C mice.10,13 Our study did not evaluate dendritic cell subpopulations or the positioning of these subpopulations within the PP. When our findings are evaluated in the context of the significantly decreased total PP cellular population in the CCR6C/C mice, it becomes apparent that PP dendritic cell numbers are decreased in CCR6C/C mice and, therefore, are consistent with a diminished PP CD11b+ DC subpopulation as well as potential defects in positioning of these subpopulations within the PP in the absence of CCR6. Overall, our observations indicate that CCR6 deficiency affects the B-lymphocyte population in both forms of organized intestinal lymphoid structures and do not preclude deficiencies in other cellular populations in either structure.

The findings presented here are consistent with previous observations regarding CCR6 expression and function in B lymphocytes. CCR6 expression is acquired by B-2 B lymphocytes as they mature and exit the bone marrow and is largely restricted to mature, follicular (B-2) B lymphocytes and to a lesser extent a small subset of germinal center and marginal zone B lymphocytes.32 In addition CCR6 expression on follicular B lymphocytes is down-regulated by B-cell receptor ligation.33 Our observation that ILF B lymphocytes are largely CCR6+ is consistent with these findings, as ILF B lymphocytes are exclusively B-2 B lymphocytes and do not have a requirement for antigen activation for their localization to ILFs.1,2,25 We observed a hierarchy of CCR6 expression with ILF B lymphocytes having greater CCR6 expression when compared with PP B lymphocytes, and PP B lymphocytes have greater CCR6 expression than the splenic B lymphocytes. This observation is also consistent with previous studies of CCR6 expression by B lymphocytes, as the PPs and the spleen have more developed germinal centers that contain a population of CCR6-negative B lymphocytes, and the spleen also contains a population of CCR6-negative marginal zone B lymphocytes.32 Although we have consistently observed this hierarchy of CCR6 expression on ILF and PP B lymphocytes, the chemotaxis of freshly isolated ILF or PP B lymphocytes in response to CCL20 has been equivalent to or less than that seen in splenic B lymphocytes (data not shown). Possible explanations for this apparent inconsistency include differences in the local environment of the PPs and ILFs that would desensitize B lymphocytes to CCR6-dependent stimuli, including elevated local expression of CCL20, as we have demonstrated here. In the context of these previous observations regarding the restricted patterns of CCR6 expression on B lymphocytes and our observations documenting that ILFs contain polyclonal follicular B-2 B lymphocytes that can be antigen-naïve,25,34 we interpret the findings of this study to indicate that CCL20 and CCR6 play a role in recruiting antigen naïve (follicular) B-2 B lymphocytes to the sites of ILF formation.

Two LT-dependent steps have been identified in ILF development, differing in the cellular source of LT. Non-B, non-T lymphocytes can deliver the early signals necessary for the formation of iILFs, these events may be mediated by the lineage c-kit+ cells within CPs.8 The transition of immature ILFs into mature ILFs requires LT-sufficient B lymphocytes, and these LT-sufficient B lymphocytes can be antigen-naïve.2,25 Chemokines play a critical role in the development of organized lymphoid structures, with CXCL13 and its receptor CXCR5 playing an essential role in the development of B-cell follicles in the spleen, lymph nodes, and PPs.35 CXCL13 not only recruits CXCR5+ B lymphocytes in this process, bit it also induces the expression of LT by antigen-naïve B lymphocytes, thus inducing the production of additional CXCL13 by LTßR-expressing stromal cells and driving the development of a follicle containing antigen-naïve B lymphocytes.35 Given that CCR6 deficiency affected ILF development to a greater degree than PP development, we questioned whether CCL20 might be playing a similar role to CXCL13 in ILF formation. In support of this role, CCL20 is produced following LTßR ligation in epithelial cells.36 Despite these associations, we did not observe the induction of LT expression in B lymphocytes following stimulation with CCL20 (data not shown). An alternative role for CCR6 in ILF development is suggested by prior studies noting that CCR6+ B lymphocytes are also CXCR5+32 ; therefore these two chemokines may act synergistically, with the recruitment of CCR6+ B lymphocytes facilitating the formation of the CXCL13-driven positive feedback loop driving the development of ILFs in response to CCL20 inducing inflammatory stimuli.

PPs and ILFs are distinct members of gastrointestinal-associated lymphoid tissues. Studies to date indicate that these structures can function in similar manners and initiate noninflammatory adaptive immune responses to luminal antigens.3,4,34 A primary distinction between these structures is the way in which they are formed, with the formation of PPs being developmentally driven and the development of ILFs occurring after birth and being influenced by luminal stimuli.1,2,26 We observed that CCR6 deficiency affects B-lymphocyte populations in intestinal lymphoid structures and resulted in hypoplastic PPs and largely absent ILFs. CCL20 is considered an inflammatory chemokine, and it is reasonable to assume that CCR6 deficiency alters ILF development, which is more affected by inflammatory stimuli, to a greater degree than PP development, which is largely developmentally driven. This assumption is supported by our findings of unchanged numbers of PPs and a more than 10-fold decrease in the numbers of ILFs in the absence of CCR6. Our observations indicate that CCR6 expression by B lymphocytes is essential for the formation of ILFs and suggests that CCL20 production by CPs in response to inflammatory stimuli may be an event facilitating the recruitment of B lymphocytes and the subsequent transition into ILFs.

We thank E. Newberry for assistance and advice with the preparation of this manuscript.

【参考文献】
  Hamada H, Hiroi T, Nishiyama Y, Takahashi H, Masunaga Y, Hachimura S, Kaminogawa S, Takahashi-Iwanaga H, Iwanaga T, Kiyono H, Yamamoto H, Ishikawa H: Identification of multiple isolated lymphoid follicles on the antimesenteric wall of the mouse small intestine. J Immunol 2002, 168:57-64

Lorenz RG, Chaplin DD, McDonald KG, McDonough JS, Newberry RD: Isolated lymphoid follicle formation is inducible and dependent upon lymphotoxin-sufficient B lymphocytes, lymphotoxin beta receptor, and TNF receptor I function. J Immunol 2003, 170:5475-5482

Shikina T, Hiroi T, Iwatani K, Jang MH, Fukuyama S, Tamura M, Kubo T, Ishikawa H, Kiyono H: IgA class switch occurs in the organized nasopharynx- and gut-associated lymphoid tissue, but not in the diffuse lamina propria of airways and gut. J Immunol 2004, 172:6259-6264

Lorenz RG, Newberry RD: isolated lymphoid follicles can function as sites for induction of mucosal immune responses. Ann NY Acad Sci 2004, 1029:44-57

Newberry RD, Lorenz RG: Organizing a mucosal defense. Immunol Rev 2005, 206:6-21

Rennert PD, Browning JL, Mebius R, Mackay F, Hochman PS: Surface lymphotoxin alpha/beta complex is required for the development of peripheral lymphoid organs. J Exp Med 1996, 184:1999-2006

Pabst O, Herbrand H, Worbs T, Friedrichsen M, Yan S, Hoffmann MW, Korner H, Bernhardt G, Pabst R, Forster R: Cryptopatches and isolated lymphoid follicles: dynamic lymphoid tissues dispensable for the generation of intraepithelial lymphocytes. Eur J Immunol 2005, 35:98-107

Eberl G: Inducible lymphoid tissues in the adult gut: recapitulation of a fetal developmental pathway? Nat Rev Immunol 2005, 5:413-420

Eberl G, Littman DR: Thymic origin of intestinal alphabeta T cells revealed by fate mapping of RORgammat+ cells. Science 2004, 305:248-251

Cook DN, Prosser DM, Forster R, Zhang J, Kuklin NA, Abbondanzo SJ, Niu XD, Chen SC, Manfra DJ, Wiekowski MT, Sullivan LM, Smith SR, Greenberg HB, Narula SK, Lipp M, Lira SA: CCR6 mediates dendritic cell localization, lymphocyte homeostasis, and immune responses in mucosal tissue. Immunity 2000, 12:495-503

L?gering A, Floer M, Westphal S, Maaser C, Spahn TW, Schmidt MA, Domschke W, Williams IR, Kucharzik T: Absence of CCR6 inhibits CD4+ regulatory T-cell development and M-cell formation inside Peyer??s patches. Am J Pathol 2005, 166:1647-1654

Salazar-Gonzalez RM, Niess JH, Zammit DJ, Ravindran R, Srinivasan A, Maxwell JR, Stoklasek T, Yadav R, Williams IR, Gu X, McCormick BA, Pazos MA, Vella AT, Lefrancois L, Reinecker HC, McSorley SJ: CCR6-mediated dendritic cell activation of pathogen-specific T cells in Peyer??s patches. Immunity 2006, 24:623-632

Varona R, Villares R, Carramolino L, Goya I, Zaballos A, Gutierrez J, Torres M, Martinez AC, Marquez G: CCR6-deficient mice have impaired leukocyte homeostasis and altered contact hypersensitivity and delayed-type hypersensitivity responses. J Clin Invest 2001, 107:R37-45

Zhao X, Sato A, Dela Cruz CS, Linehan M, Luegering A, Kucharzik T, Shirakawa AK, Marquez G, Farber JM, Williams I, Iwasaki A: CCL9 is secreted by the follicle-associated epithelium and recruits dome region Peyer??s patch CD11b+ dendritic cells. J Immunol 2003, 171:2797-2803

Liao F, Alderson R, Su J, Ullrich SJ, Kreider BL, Farber JM: STRL22 is a receptor for the CC chemokine MIP-3alpha. Biochem Biophys Res Commun 1997, 236:212-217

Yang D, Chertov O, Bykovskaia SN, Chen Q, Buffo MJ, Shogan J, Anderson M, Schroder JM, Wang JM, Howard OM, Oppenheim JJ: Beta-defensins: linking innate and adaptive immunity through dendritic and T cell CCR6. Science 1999, 286:525-528

Tanaka Y, Imai T, Baba M, Ishikawa I, Uehira M, Nomiyama H, Yoshie O: Selective expression of liver and activation-regulated chemokine (LARC) in intestinal epithelium in mice and humans. Eur J Immunol 1999, 29:633-642

Ebert LM, McColl SR: Up-regulation of CCR5 and CCR6 on distinct subpopulations of antigen-activated CD4+ T lymphocytes. J Immunol 2002, 168:65-72

Liao F, Rabin RL, Smith CS, Sharma G, Nutman TB, Farber JM: CC-chemokine receptor 6 is expressed on diverse memory subsets of T cells and determines responsiveness to macrophage inflammatory protein 3 alpha. J Immunol 1999, 162:186-194

Gu H, Zou YR, Rajewsky K: Independent control of immunoglobulin switch recombination at individual switch regions evidenced through Cre-loxP-mediated gene targeting. Cell 1993, 73:1155-1164

De Togni P, Goellner J, Ruddle NH, Streeter PR, Fick A, Mariathasan S, Smith SC, Carlson R, Shornick LP, Strauss-Schoenberger J, Russell JH, Karr R, Chaplin DD: Abnormal development of peripheral lymphoid organs in mice deficient in lymphotoxin. Science 1994, 264:703-707

Kucharzik T, Hudson JT, 3rd, Waikel RL, Martin WD, Williams IR: CCR6 expression distinguishes mouse myeloid and lymphoid dendritic cell subsets: demonstration using a CCR6 EGFP knock-in mouse. Eur J Immunol 2002, 32:104-112

Newberry RD, McDonough JS, McDonald KG, Lorenz RG: Postgestational lymphotoxin/lymphotoxin beta receptor interactions are essential for the presence of intestinal B lymphocytes. J Immunol 2002, 168:4988-4997

Kanamori Y, Ishimaru K, Nanno M, Maki K, Ikuta K, Nariuchi H, Ishikawa H: Identification of novel lymphoid tissues in murine intestinal mucosa where clusters of c-kit+ IL-7R+ Thy1+ lympho-hemopoietic progenitors develop. J Exp Med 1996, 184:1449-1459

McDonald KG, McDonough JS, Newberry RD: Adaptive immune responses are dispensable for isolated lymphoid follicle formation: antigen-naive, lymphotoxin-sufficient B lymphocytes drive the formation of mature isolated lymphoid follicles. J Immunol 2005, 174:5720-5728

Kweon MN, Yamamoto M, Rennert PD, Park EJ, Lee AY, Chang SY, Hiroi T, Nanno M, Kiyono H: Prenatal blockage of lymphotoxin beta receptor and TNF receptor p55 signaling cascade resulted in the acceleration of tissue genesis for isolated lymphoid follicles in the large intestine. J Immunol 2005, 174:4365-4372

Izadpanah A, Dwinell MB, Eckmann L, Varki NM, Kagnoff MF: Regulated MIP-3alpha/CCL20 production by human intestinal epithelium: mechanism for modulating mucosal immunity. Am J Physiol Gastrointest Liver Physiol 2001, 280:G710-G719

Kaser A, Ludwiczek O, Holzmann S, Moschen AR, Weiss G, Enrich B, Graziadei I, Dunzendorfer S, Wiedermann CJ, Murzl E, Grasl E, Jasarevic Z, Romani N, Offner FA, Tilg H: Increased expression of CCL20 in human inflammatory bowel disease. J Clin Immunol 2004, 24:74-85

Kwon JH, Keates S, Bassani L, Mayer LF, Keates AC: Colonic epithelial cells are a major site of macrophage inflammatory protein 3alpha (MIP-3alpha) production in normal colon and inflammatory bowel disease. Gut 2002, 51:818-826

L?gering A, Kucharzik T, Soler D, Picarella D, Hudson JT, 3rd, Williams IR: Lymphoid precursors in intestinal cryptopatches express CCR6 and undergo dysregulated development in the absence of CCR6. J Immunol 2003, 171:2208-2215

Golovkina TV, Shlomchik M, Hannum L, Chervonsky A: Organogenic role of B lymphocytes in mucosal immunity. Science 1999, 286:1965-1968

Bowman EP, Campbell JJ, Soler D, Dong Z, Manlongat N, Picarella D, Hardy RR, Butcher EC: Developmental switches in chemokine response profiles during B cell differentiation and maturation. J Exp Med 2000, 191:1303-1318

Krzysiek R, Lefevre EA, Bernard J, Foussat A, Galanaud P, Louache F, Richard Y: Regulation of CCR6 chemokine receptor expression and responsiveness to macrophage inflammatory protein-3alpha/CCL20 in human B cells. Blood 2000, 96:2338-2345

Wang C, McDonald KG, McDonough JS, Newberry RD: Murine isolated lymphoid follicles contain follicular B lymphocytes with a mucosal phenotype. Am J Physiol 2006, 291:G595-G604

Ansel KM, Ngo VN, Hyman PL, Luther SA, Forster R, Sedgwick JD, Browning JL, Lipp M, Cyster JG: A chemokine-driven positive feedback loop organizes lymphoid follicles. Nature 2000, 406:309-314

Rumbo M, Sierro F, Debard N, Kraehenbuhl JP, Finke D: Lymphotoxin beta receptor signaling induces the chemokine CCL20 in intestinal epithelium. Gastroenterology 2004, 127:213-223

作者单位：From the Department of Internal Medicine,* Washington University School of Medicine, St. Louis, Missouri; the Department of Medicine B, University of Muenster, Muenster, Germany; and the Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia

【摘要】  Idiopathic interstitial pneumonias (IIPs) are a collection of pulmonary fibrotic diseases of unknown etiopathogenesis. CC chemokine receptor 7 (CCR7) is expressed in IIP biopsies and primary fibroblast lines, but its role in pulmonary fibrosis was not previously examined. To study the in vivo role of CCR7 in a novel model of pulmonary fibrosis, 1.0 x 106 primary fibroblasts grown from idiopathic pulmonary fibrosis/usual interstitial pneumonia, nonspecific interstitial pneumonia, or histologically normal biopsies were injected intravenously into C.B-17 severe combined immunodeficiency (SCID)/beige (bg) mice. At days 35 and 63 after idiopathic pulmonary fibrosis/usual interstitial pneumonia fibroblast injection, patchy interstitial fibrosis and increased hydroxyproline were present in the lungs of immunodeficient mice. Adoptively transferred nonspecific interstitial pneumonia fibroblasts caused a more diffuse interstitial fibrosis and increased hydroxyproline levels at both times, but injected normal human fibroblasts did not induce interstitial remodeling changes in C.B-17SCID/bg mice. Systemic therapeutic immunoneutralization of either human CCR7 or CC ligand 21, its ligand, significantly attenuated the pulmonary fibrosis in groups of C.B-17SCID/bg mice that received either type of IIP fibroblasts. Thus, the present study demonstrates that pulmonary fibrosis is initiated by the intravenous introduction of primary human fibroblast lines into immunodeficient mice, and this fibrotic response is dependent on the interaction between CC ligand 21 and CCR7.
--------------------------------------------------------------------------------
A persistently high mortality rate plagues idiopathic pulmonary fibrosis/usual interstitial pneumonia (IPF/UIP), the severest form of idiopathic interstitial pneumonia (IIP).1 This is due to the failure of conventional immunomodulatory therapies, such as corticosteroids, azathioprine, and cyclophosphamide, to halt effectively the aggressive profibrotic and tissue repair processes in this disease.2 Newly diagnosed IPF/UIP patients face respiratory failure and a plethora of complications including cardiovascular disease, lung cancer, and pulmonary embolism; most succumb to this disease within 3 to 5 years of diagnosis.3 With its growing prevalence worldwide,4 the need for novel therapies for IPF/UIP has become a major research focus, but a poor understanding of the etiopathogenesis of this disease has slowed progress in this regard.5 The aberrant parenchymal remodeling in IIP is characterized by the expansion of fibroblasts and myofibroblasts, and previous studies have shown that IPF/UIP fibroblasts have unique proliferation and synthetic properties relative to fibroblasts from other IIPs or normal lung tissues.6-9 These cells may arise from alterations in their microenvironment (due to dysregulated epithelial cell/mesenchymal cell interactions10 ) or be recruited out of the bone marrow (see reviews.11,12 ). Regardless of the source of these cells, controlling their activity in the lung is paramount, and ongoing genomic and proteomic analyses of intact lung biopsies13,14 and biopsy-derived fibroblasts15 have revealed putative targets to achieve this regulation.
Mouse models of pulmonary fibrosis have provided experimental paradigms with which to address abnormal tissue remodeling and scarring in the respiratory system.16 A number of approaches have been used to induce pulmonary fibrosis, and these include transgenic and gene transfer, radiation, inorganic irritants such as silica, and drugs promoting oxidant-induced inflammatory injury such as bleomycin.17 Of these models, the bleomycin model remains the most widely used because of its reproducibility and pathological similarity to human pulmonary fibrosis.17 Accordingly, the bleomycin model has been used to assess a number of targets of interest in IPF/UIP.13,14 Unfortunately, no animal model exists that fully recapitulates the clinico-pathological features of IPF/UIP, and debate still exists pertaining to the relative importance of ongoing inflammatory injury (the primary mode for inducing experimental fibrosis) to end-stage UIP.18,19
Given this dilemma, the present study addressed an alternative strategy for inducing experimental pulmonary fibrosis. Mice that are genetically immunodeficient due to severe combined immunodeficiency (scid) gene mutation or recombinase-activating gene 1 (rag-1) knockout have been extensively used as in vivo hosts of adoptively transferred normal or diseased human cells. Herein, we report that the adoptive intravenous (i.v.) transfer of either IPF/UIP or nonspecific interstitial pneumonia (NSIP; another less severe form of IIP20 ) but not normal fibroblasts into C.B-17 mice with the scid-beige (C.B-17SCID/bg) mutation initiated and maintained pulmonary fibrosis. Histological and biochemical evidence of fibrosis was first evident in both IIP fibroblast groups of C.B-17SCID/bg mice at day 35 and was prominent at day 63 after fibroblast injection. Cytokines and chemokines both seem to have major roles in the pathogenesis of IIP,21 and enzyme-linked immunosorbent assay (ELISA) analysis of whole-lung samples from C.B-17SCID/bg mice that received either IPF/UIP or NSIP fibroblasts revealed significant elevations in murine interleukin (IL)-13, CC ligand (CCL) 6, and CCL21 at day 63 compared with whole-lung levels measured at the earlier time point or in lung tissue from mice that did not receive fibroblasts. IL-1322,23 and CCL624 are mediators of pulmonary fibrosis, but the role of CCL21 in pulmonary remodeling events was unknown. Impetus to examine the role of CCL21 and CC chemokine receptor 7 (CCR7) in the pulmonary remodeling events precipitated by human IIP fibroblasts stemmed from our recent finding that CCR7 expression was increased in IIP biopsies,25 and the migratory, synthetic, and proliferative properties of IIP fibroblasts are significantly enhanced by CCL21 (E.M.P. and C.M.H., unpublished data). In separate immunoneutralization studies, the targeting of either human CCL21 or CCR7 (the receptor for CCL2126 ) from days 35 to 63 after IIP fibroblast injection into C.B-17SCID/bg mice significantly reduced all parameters of pulmonary fibrosis compared with groups of C.B-17SCID/bg mice receiving IIP fibroblasts and IgG. Together, these data highlight the creation of a new murine model of pulmonary fibrosis initiated and maintained by the i.v. introduction of IIP fibroblasts into C.B-17SCID/bg mice and demonstrate a novel role for CCL21 and CCR7 in the maintenance of fibrosis in this model.

【关键词】  therapeutic targeting chemokine receptor abrogates pulmonary fibrosis adoptive transfer pulmonary fibroblasts immunodeficient

Materials and Methods

Female, ICR-scid (ICRSCID), C.B-17-scid (C.B-17SCID), and C.B-17-scid-beige (C.B-17SCID/bg) mice (6 to 8 weeks old) were purchased from Taconic Farms (Germantown, NY), and all SCID mice were housed in a gnotobiotic barrier facility at the University of Michigan Medial School. The first two groups of mice have the scid mutation leading to a lack of both T and B lymphocytes due to a V(D)J recombination defect, whereas C.B-17SCID/bg mice have two mutations: the first is the scid mutation, and the second is a beige mutation leading to a major defect in cytotoxic T-cell and macrophage function and a selective impairment in NK cell function. All mice had access to autoclaved water and pelleted mouse diet ad libitum. All procedures described below were performed in a sterile, laminar environment and were approved by an animal care and use committee at the University of Michigan Medical School.

Human Fibroblast Culture

A mixed cell population was obtained from mechanically dissociated IPF/UIP and NSIP surgical lung biopsies, and pure human fibroblast cultures were derived as previously described in detail.9 Normal lung fibroblasts were purified in the same manner from cell suspensions of normal margins associated with resected lung tumor tissue. In the present study, a total of 10 IPF/UIP, six NSIP, and four normal fibroblast lines were used after the fourth passage in the initial, model characterization, and therapeutic intervention studies described herein. An institutional review board at the University of Michigan Medical School approved this study.

Intravenous Introduction of Human Pulmonary Fibroblasts into SCID Mice

Single-cell preparations of IPF/UIP, NSIP, and normal fibroblasts were obtained after trypsinization of 150-cm2 tissue culture flasks and labeled with PKH26 dye according to the manufacturer??s directions (Sigma Co., St. Louis, MO). Each labeled fibroblast line was diluted to 1 x 106 cells/ml of phosphate-buffered saline (PBS), and 1 ml of this suspension was injected via a tail vain into groups of five SCID mice. Other groups of five SCID mice were injected intravenously with PKH26 and PBS labeling solution alone (ie, control group). Mice were euthanized by anesthesia overdose at days 7, 21, 35, 49, and 63 after the i.v. human pulmonary fibroblast transfer. Whole-lung tissue was dissected at these times for molecular, histological, biochemical, and/or proteomic analysis (see below).

Assessing the Role of CCL21 and CCR7 in C.B-17SCID/bg Mice after the Adoptive Transfer of Human Pulmonary Fibroblasts

To address the role of CCL21 and CCR7, its receptor, in the pulmonary remodeling response after the i.v. adoptive transfer of human fibroblasts, groups of C.B-17SCID/bg mice received IPF/UIP (n = 35 mice), NSIP (n = 20 mice), normal fibroblasts (n = 15 mice), or vehicle (ie, PBS) alone (n = 15 mice). Thirty-five days later, all groups of five C.B-17SCID/bg mice received mouse IgG, mouse anti-human CCL21 monoclonal antibody, or mouse anti-human CCR7 monoclonal antibody (all at 10 µg/ml; R&D Systems, Minneapolis, MN) every other day from days 35 to 63. At day 63, all mice were euthanized by anesthesia overdose, and whole-lung tissue was dissected for molecular, histological, biochemical, and proteomic analysis (see below).

Molecular Analysis

Total RNA was isolated and cDNA generated from whole-lung samples as previously described in detail.27 Changes in gene profiles for human and murine chemokine and chemokine receptors were analyzed in pooled samples (n = 5) using nonradioactive GEArray gene array membranes according to the manufacturer??s instructions (SuperArray, Inc., Bethesda, MD), and signal intensities were determined as previously described in detail.28 Individual whole-lung cDNA samples (n 5) were analyzed for human CCR7, collagen I, cathepsin E, matrix metalloproteinase (MMP)-2, MMP-9, MMP-19, fibronectin, tissue inhibitors of metalloproteinases (TIMP)-1, and glyceraldehyde-3-phosphate dehydrogenase expression by real-time quantitative RT-PCR procedure using an 7500 Real Time PCR System (Applied Biosystems, Foster City, CA) as previously described.27

Histological Analysis

After anesthesia-induced euthanasia, the right lobes from each mouse were dissected, fully inflated with 10% formalin solution, and placed in fresh formalin for 24 hours. Standard histological techniques were used to paraffin-embed each lobe, and 5-µm sections were stained with hematoxylin and eosin and Mason trichrome for histological analysis. Additional unstained whole-lung tissue sections were analyzed via fluorescent microscopy.

Biochemical Analysis

Whole-left lung samples were homogenized in 1x PBS and pelleted by centrifugation. The cell-free supernatants were removed for ELISA analysis, and the pellets were vacuum-dried and resuspended in 0.5 mol/L glacial acetic acid. The tissue was then processed for hydroxyproline concentration as previously described.29

Proteomic Analysis

Murine IL-13, CCL6, CCL21, interferon-, IL-12, IL-4, CCL2, CCL7, CCL17, CCL3, CXC chemokine ligand (CXCL)13, tumor necrosis factor-, CXCL10, CXCL9, and CXCL2 proteins were analyzed in 50 µl of cell-free supernatants from homogenized whole-lung samples using a standardized sandwich ELISA technique (R&D Systems) as previously described in detail.27

Statistical Analysis

All results are expressed as mean ?? SEM. One-way analysis of variance analysis and Tukey-Kramer or Dunnett??s multiple comparisons tests were used to detect statistical differences between UIP, NSIP, normal, and control SCID mouse groups. Significance was set at P < 0.05.

The Adoptive Intravenous Transfer of Either IPF/UIP or NSIP, but Not Normal, Pulmonary Fibroblasts Promoted Lung Histopathology and Remodeling in C.B-17SCID/bg Mice

Initial studies were undertaken to assess the impact of adoptively transferred normal and IIP fibroblasts on the lung architecture in various strains of SCID mice including ICRSCID, C.B-17SCID, and C.B-17SCID/bg (n = 5 per time point). These initial studies were undertaken using three IPF/UIP and two normal human fibroblast lines. All human fibroblast lines were labeled with PKH26 before injection into the SCID mouse groups thereby allowing for the detection of labeled cells in histological sections. At days 7 (Figure 1, B and C) and 21 (not shown) after injection, collections of human fibroblasts were detected in pulmonary blood vessels in each SCID group. However, the intensity of this marker diminishes after 21 days (information from the provided Sigma data sheet), and hence, we failed to detect PKH26 fluorescence in histological sections from each at day 35 after fibroblast injection (not shown). Other organs (ie, liver, spleen, and kidney) presumably contained human pulmonary fibroblasts, but we observed no evidence of gross macroscopic alterations to these organs. Because IIP is a lung-specific disease,30 which does not exhibit fibrogenesis in any other organ, a detailed histological analysis of other organs was not undertaken in the present study.

Figure 1. Representative whole-lung tissues sections from C.B-17SCID/bg mice that received PBS + PKH26 (A) or IPF/UIP fibroblasts + PBS + PKH26 (B and C). The presence of fluorescently labeled human fibroblasts in the lung was observed in C.B-17SCID/bg mice that received human IPF/UIP fibroblasts 7 days previously (B and C). The clumped human fibroblasts were typically detected in the walls of pulmonary blood vessels (B and C). Original magnifications: x100 (A), x200 (B), and x400 (C).

Examination of whole-lung sections in groups of SCID mice at later time points revealed that marked lung remodeling was only present in C.B-17SCID/bg mice. Specifically, no fibroproliferation was observed at day 49 after the introduction of IPF/UIP fibroblasts into ICRSCID mice (n = 5 mice; not shown). Likewise, the i.v. adoptive transfer of IPF/UIP fibroblasts into C.B-17SCID mice failed to elicit histologically evident pulmonary remodeling at days 7 (n = 5 mice), 21 (n = 10 mice), 35 (n = 5 mice), or 49 (n = 5 mice) after the i.v. injection of IPF/UIP fibroblasts.

Given the presence of marked pulmonary histopathology in the C.B-17SCID/bg group, all subsequent studies described below involved the adoptive transfer of normal and IIP fibroblasts into C.B-17SCID/bg mice. In the model characterization study, four IPF/UIP, four NSIP, and one normal fibroblast lines were adoptively transferred into separate groups of C.B17-SCID/bg mice, and pulmonary histopathological, genomic, and proteomic alterations were analyzed at days 35 and 63 after fibroblast transfer.

Little or no pulmonary histopathology was observed in C.B-17SCID/bg mice that received normal pulmonary fibroblasts (Figure 2A) . However, C.B-17SCID/bg mice exhibited significant pulmonary histopathology, which was evident at day 35 after the i.v. injection of either NSIP (Figure 2B) or IPF/UIP (Figure 2C) fibroblast lines. The pulmonary histopathology in C.B-17SCID/bg mice following NSIP or IPF/UIP pulmonary fibroblasts was characterized by disruption of the alveolar space, apparent fibroproliferation, and the presence of eosinophilic granulocytes (Figure 2, B and C) . Mason trichrome staining of histological tissue sections from the lungs of C.B-17SCID/bg mice at day 35 after the adoptive transfer of normal (Figure 2D) , NSIP (Figure 2E) , or IPF/UIP (Figure 2F) human pulmonary fibroblasts revealed the presence of extracellular matrix (stained light blue) in remodeled areas in C.B-17SCID/bg groups that received the IIP but not normal fibroblasts.

Figure 2. Representative hematoxylin and eosin-stained histological sections from C.B-17SCID/bg mice that received normal (A), NSIP (B), or IPF/UIP (C) fibroblasts. Representative Mason trichrome-stained histological sections from C.B-17SCID/bg mice that received normal (D), NSIP (E), or IPF/UIP (F) fibroblasts are shown. Lung samples were removed at day 35 after the adoptive i.v. transfer of human pulmonary fibroblasts into C.B-17SCID/bg mice. Original magnification, x200 (A, CCF) or x400 (B).

Later analysis of histological sections from C.B-17SCID/bg groups revealed major differences in the extent and appearance of the pulmonary remodeling precipitated by the introduction of IPF/UIP or NSIP fibroblasts. The lungs of C.B-17SCID/bg mice that received IPF/UIP fibroblasts 63 days previously exhibited a heterogeneous appearance with areas of relatively normal-appearing lung tissue (Figure 3A) adjacent to areas of severe interstitial disruption and remodeling (Figure 3B) . In addition, foci of human fibroblasts were apparent in the lungs of C.B-17SCID/bg mice that received IPF/UIP fibroblasts (Figure 3B , inset), but these foci were detected in blood vessels and not in interstitial areas as in clinical UIP.1 The histological pattern in whole-lung tissue sections from C.B-17SCID/bg mice that received NSIP fibroblasts 63 days previously was characterized by interstitial thickening and overtly fibrotic areas, and the remodeling in these mice appeared to involve most of the lung (Figure 3, C and D) . Together, these data showed that the introduction of human IIP fibroblasts into C.B-17SCID/bg mice caused fibrotic lesions in these mice.

Figure 3. Representative Mason trichrome-stained histological sections from C.B-17SCID/bg mice that received IPF/UIP (A and B) or NSIP (C and D) fibroblasts 35 or 63 days previously. Normal-appearing areas (A), intensely fibrotic areas (B), and foci of human fibroblasts (observed at day 35 after fibroblast injection; inset to B) were apparent in whole-lung histological sections from C.B-17SCID/bg mice that received IPF/UIP fibroblasts. In contrast, whole-lung histological sections from C.B-17SCID/bg mice that received NSIP showed a more generalized involvement of the lung tissue examined with varying degrees of fibrosis and remodeling (C and D). Lung samples were removed at days 35 or 63 after the adoptive i.v. transfer of human fibroblasts into C.B-17SCID/bg mice. Original magnification, x200.

Hydroxyproline Levels Were Significantly Altered in a Temporally Dependent Manner after the Introduction of IIP Fibroblasts into C.B-17SCID/bg Mice

Hydroxyproline is a commonly used marker of de novo collagen synthesis in experimental models involving pulmonary remodeling.31 In the present study, hydroxyproline levels were measured in whole-lung samples from C.B-17SCID/bg mice that had received no fibroblasts or normal, NSIP, or IPF/UIP human pulmonary fibroblasts either 35 or 63 days previously. As shown in Figure 4 , hydroxyproline levels were unchanged at these time points after the introduction of normal fibroblasts. The levels of hydroxyproline in these C.B-17SCID/bg groups were 4.7 ?? 0.3 and 5.6 ?? 0.5 µg/mg protein at days 35 and 63, respectively, and these hydroxyproline levels were similar to those detected in C.B-17SCID/bg mouse groups that did not receive human fibroblasts (4.5 ?? 1.3 µg/mg protein). However, the lungs of mice contained greater amounts of hydroxyproline at day 35 after intravenously transferred NSIP (11.4 ?? 3.7 µg/mg protein) or IPF/UIP (8.2 ?? 2 µg/mg protein) fibroblasts compared with the normal fibroblast group. In addition, hydroxyproline levels were further increased 3- and 2.5-fold in the NSIP (31 ?? 11 µg/mg protein) and IPF/UIP (22 ?? 4 µg/mg protein) fibroblast groups, respectively, at day 63 after i.v. adoptive transfer (Figure 4) . At the day-63 time point, the increase in hydroxyproline levels in the C.B-17SCID/bg groups that received either NSIP or IPF/UIP human fibroblasts reached statistical significance compared with the C.B-17SCID/bg group that received normal fibroblasts. Thus, the intravenous injection of human IIP fibroblasts enhanced hydroxyproline levels at day 35 and, most demonstrably, at day 63 after adoptive transfer.

Figure 4. Whole-lung hydroxyproline levels in C.B-17SCID/bg mice that received normal, NSIP, or IPF/UIP fibroblasts. Lung samples were removed at days 35 and 63 after the adoptive i.v. transfer of human fibroblasts into C.B-17SCID/bg mice. Data shown are mean ?? SEM. *P 0.05 compared with hydroxyproline levels measured at day 63 after adoptive transfer of normal fibroblasts into C.B-17SCID/bg mice.

Whole-Lung Cytokine Analysis Showed That Murine IL-13, CCL6, and CCL21 Were Significantly Elevated in the Lungs of C.B-17SCID/bg Mice That Received IIP Fibroblasts

Whole-lung ELISA analysis of several cytokines, CC ligand, and CXC ligand chemokines at days 35 and 63 after the i.v. adoptive transfer of human normal or IIP fibroblasts revealed a number of statistically significant changes in murine IL-13, CCL6, and CCL21 (Figure 5) . Although the whole-lung levels of IL-13 in the C.B-17SCID/bg group that received normal fibroblasts were below the level of ELISA detection, whole-lung IL-13 levels were significantly greater in the C.B-17SCID/bg group that received either NSIP or IPF/UIP fibroblasts compared with the C.B-17SCID/bg group that received normal fibroblasts 35 and/or 63 days previously (Figure 5 , top). In addition, significantly more IL-13 was detected in the lungs of C.B-17SCID/bg mice that received IPF/UIP fibroblasts at day 63 versus 35 after adoptive transfer of fibroblasts. At days 63 after fibroblast injection, whole-lung CCL6 levels were significantly greater in the C.B-17SCID/bg groups that received either IPF/UIP or NSIP fibroblasts compared with the C.B-17SCID/bg group that received normal fibroblasts (Figure 5 , middle). Significantly greater CCL6 was detected in the lungs of C.B-17SCID/bg mice that received IPF/UIP or NSIP fibroblasts at day 63 versus 35 after adoptive transfer of fibroblasts (Figure 5 , middle). The only other murine CC ligand that was altered by the introduction of human fibroblasts into C.B-17SCID/bg mice was CCL21. This chemokine was significantly greater in the NSIP and IPF/UIP fibroblast C.B-17SCID/bg groups at day 63 compared with the normal fibroblast C.B-17SCID/bg groups at day 63. In addition, whole-lung murine CCL21 levels were significantly elevated in IPF/UIP fibroblast groups at day 63 after fibroblast transfer compared with the NSIP fibroblast group at the same time (Figure 5 , bottom). Finally, significantly higher levels of CCL21 were present in whole-lung samples from the IIP fibroblasts C.B-17SCID/bg group at day 63 after adoptive transfer compared with the IIP fibroblasts C.B-17SCID/bg groups at day 35 after adoptive transfer. Thus, taken together, these data suggested that the presence of IIP fibroblasts, but not normal fibroblasts, in C.B-17SCID/bg mice significantly altered the whole-lung levels of murine cytokines and chemokines with established (ie, IL-1322,23 and CCL624 ) and putative (ie, CCL21) profibrotic roles in the lung.

Figure 5. Whole-lung murine IL-13, CCL6, and CCL21 levels in C.B-17SCID/bg mice that received normal, NSIP, or IPF/UIP fibroblasts. Lung samples were removed at days 35 and 63 after the adoptive i.v. transfer of human fibroblasts into C.B-17SCID/bg mice, and all soluble proteins were measured by specific ELISA. Data shown are mean ?? SEM. *P 0.05, **P 0.01, ***P 0.001 compared with appropriate the C.B-17SCID/bg group that received normal fibroblasts. P 0.01, P 0.001 compared with whole-lung cytokine and chemokine levels at the day 35 time point in the C.B-17SCID/bg groups with either IPF/UIP or NSIP fibroblasts The whole-lung cytokine and chemokine levels in control C.B-17SCID/bg group that did not receive fibroblasts were as follows: IL-13, 0.18 ?? 0.014 ng/mg protein; CCL6, 0.37 ?? 0.05 ng/mg protein; and CCL21, 2.0 ?? 0.1 ng/mg protein.

Human CCR7 and CCL21 Gene Transcripts Were Present in the Lungs of C.B-17SCID/bg Mice That Received Human Pulmonary Fibroblasts 35 Days Previously

The changes in whole-lung levels of murine CCL21 in C.B-17SCID/bg mice that received either NSIP or IPF/UIP fibroblasts were intriguing in light of previous studies demonstrating the important remodeling role of this CC ligand in the kidney32 and liver.33 This prompted further analysis of this CC ligand and its receptor, CCR7, during the pulmonary remodeling responses elicited by human IIP fibroblasts. Human transcripts for CCR7 and CCL21 were detected by SuperArray gene analysis, and among the three C.B-17SCID/bg groups, the greatest transcript expression for CCR7 and CCL21 was present in lung samples from the IPF/UIP fibroblast group (Table 1) . Furthermore, the presence of CCR7 was confirmed by TaqMan analysis, and again the IPF/UIP group exhibited the highest CCR7 transcript expression among the C.B-17SCID/bg groups (Table 1) . SuperArray and TaqMan analysis of murine CCR7 and CCL21 also confirmed the presence of these transcript products in all three C.B-17SCID/bg groups that received human fibroblasts, and the highest levels of both transcripts were present in the C.B-17SCID/bg groups that received IPF/UIP fibroblasts (not shown). Thus, the i.v. adoptive transfer of normal and IIP fibroblasts resulted in the presence of human gene transcripts for CCR7 and CCL21.

Table 1. SuperArray Analysis of Human CCR7 and CCL21 and TaqMan Analysis of Human CCR7 in Whole-Lung Samples from C.B-17SCID/bg Mice at Day 35 after i.v. Human Fibroblast Injection

Quantitative TaqMan PCR Analysis of Murine Extracellular Matrix-Associated Genes after the Adoptive Transfer of Human Fibroblasts into C.B-17SCID/bg Mice

Lung alterations in murine extracellular matrix-associated genes were analyzed using quantitative PCR analysis. As shown in Figure 6 , top, the expression of collagen 1, cathepsin E, MMP-19, and TIMP-1 was present in C.B-17SCID/bg mice that received normal, NSIP, and IPF/UIP human fibroblasts 63 days previously. Most importantly, when transcript levels for these genes in fibroblast-challenged mice were compared with transcript levels in control C.B-17SCID/bg mice, the increases in collagen 1 and cathepsin transcript expression in C.B-17SCID/bg mice that received either type of IIP fibroblast and MMP-19 and TIMP-1 transcript expression in mice that received IPF/UIP fibroblasts reached statistical significance when compared with the transcript increases for these genes in C.B-17SCID/bg mice that received normal human fibroblasts (Figure 6 , top). Other extracellular matrix genes were analyzed by TaqMan, and the presence of MMP-2, MMP-9, and fibronectin was confirmed. No significant differences in levels of these transcripts were observed among the groups of C.B-17SCID/bg mice, but the greatest increase in MMP-2 and fibronectin was observed in the IPF/UIP C.B-17SCID/bg group, whereas the smallest increase in MMP-9 was observed in this same C.B-17SCID/bg group (Figure 6 , bottom). Thus, these data showed that transcript levels for murine extracellular matrix-associated genes were altered by the presence of human fibroblasts in C.B-17SCID/bg mice.

Figure 6. Quantitative TaqMan PCR analysis of extracellular matrix-associated genes, collagen 1, cathepsin E, MMP-19, TIMP-1, MMP-2, MMP-9, and fibronectin in C.B-17SCID/bg mice that received normal, NSIP, or IPF/UIP fibroblasts. Changes in gene expression are expressed as mean ?? SEM of the fold increase in transcript expression above a group of C.B-17SCID/bg mice that received PBS and PKH26 alone. *P 0.05, **P 0.01 compared with the C.B-17SCID/bg group that received normal fibroblasts.

Immunoneutralization of Human CCR7 or Human CCL21 Abrogated Pulmonary Remodeling in C.B-17SCID/bg Mice That Received Human IIP Fibroblasts

In the next series of experiments, the roles of human CCR7 and CCL21 were assessed in C.B-17SCID/bg mice that received human normal (n = 1 line) or IIP fibroblasts (n = 3 IPF/UIP and n = 2 NSIP lines). Although attempts to measure human CCL21 in whole-lung samples were unsuccessful, presumably because of the presence of this CC ligand at levels below the level of ELISA detection (not shown), previous studies have shown that both mouse and human CCL21 can promote cellular calcium flux via human CCR7.34 In addition, the immunoneutralization of mouse CCL21 using a polyclonal antibody has been shown to abrogate the migration of human dendritic cells and the priming of human T cells in a humanized model of house dust mite-induced allergic airway disease.35 Given the reported cross-reactivity of mouse and human CCL21, a therapeutic protocol was implemented in which either human CCL21 or human CCR7 was targeted by monoclonal antibody administration from days 35 to 63 after the adoptive transfer of fibroblasts. A representative histological survey of whole-lung tissues at day 63 in the three C.B-17SCID/bg groups with the three treatment modalities tested is shown in Figure 7 . In the normal fibroblast C.B-17SCID/bg group, no evidence of interstitial pulmonary remodeling was evident in any of the treatment groups (IgG treatment, Figure 7A ; anti-CCL21 antibody treatment, Figure 7B ; and anti-CCR7 antibody treatment, Figure 7C ). However, the vascular accumulation of normal fibroblasts was histologically apparent in these C.B-17SCID/bg groups, and an example is shown in Figure 7B . None of the treatments altered this feature in C.B-17SCID/bg mice that received normal fibroblasts. Interstitial remodeling was apparent in the NSIP fibroblast C.B-17SCID/bg group that received IgG (Figure 7D) , but this remodeling response was abrogated by either anti-CCL21 antibody (Figure 7E) or anti-CCR7 antibody (Figure 7F) administration from days 35 to 63 after adoptive transfer. Likewise, the interstitial remodeling apparent in the IgG C.B-17SCID/bg group that received IPF/UIP fibroblasts (Figure 7G) was absent in groups of C.B-17SCID/bg mice that received anti-CCL21 antibody (Figure 7H) or anti-CCR7 antibody (Figure 7I) administration from days 35 to 63 after adoptive transfer IPF/UIP fibroblasts. Thus, therapeutic targeting of either CCL21 or CCR7 abrogated histological evidence of interstitial remodeling in C.B-17SCID/bg mice that received IIP fibroblasts.

Figure 7. Representative Mason trichrome-stained histological sections from C.B-17SCID/bg mice that received normal (ACC), NSIP (DCF), or IPF/UIP (GCI) fibroblasts. No interstitial remodeling was apparent in C.B-17SCID/bg mice that received normal fibroblasts, but vascular anomalies were observed in this group (B), and the IgG (A), anti-CCL21 monoclonal antibody (B), and anti-CCR7 monoclonal antibody (C) therapies did not alter the lung histological appearance in this group. Pulmonary remodeling was apparent in C.B-17SCID/bg mice that received NSIP fibroblasts, and this pattern was not altered by IgG (D), whereas the anti-CCL21 antibody (E) or anti-CCR7 antibody (F) therapies markedly reduced the interstitial remodeling in whole-lung samples. Interstitial pulmonary fibrosis was apparent in C.B-17SCID/bg mice that received IPF/UIP fibroblasts, and this pattern was not altered by IgG (G), whereas the anti-CCL21 antibody (H) or anti-CCR7 antibody (I) therapies markedly reduced the interstitial remodeling in whole-lung samples. Monoclonal antibody therapies began at day 35 and continued to day 63, and lung samples were removed at day 63 after the adoptive i.v. transfer of human fibroblasts into C.B-17SCID/bg mice. Original magnification, x400.

Quantitative TaqMan PCR Analysis of Murine Extracellular Matrix-Associated Genes after the Therapeutic Targeting of Either Human CCL21 or CCR7 in C.B-17SCID/bg Mice That Received Human Normal and IIP Fibroblasts

Quantitative TaqMan analysis of the antibody treatment groups also confirmed that the anti-CCL21 and anti-CCR7 treatments also significantly altered the transcript levels of MMP-2 and MMP-19. Transcript levels for these MMPs in antibody-treated, control C.B-17SCID/bg mice were compared with the levels of these transcripts in antibody-treated, fibroblast-challenged C.B-17SCID/bg mice. Anti-CCL21 antibody treatment significantly reduced the fold increase in MMP-2 transcript levels in C.B-17SCID/bg groups that received normal fibroblasts compared with the fold increase in the transcript levels in C.B-17SCID/bg groups that received IgG (Figure 8 , top). The anti-CCR7 antibody treatment significantly reduced the fold increase in MMP-2 transcript levels compared with the appropriate IgG group. Quantitative TaqMan analysis of MMP-19 revealed that anti-CCR7 significantly increased the fold change in the C.B-17SCID/bg group that received normal fibroblasts, whereas both antibody treatments significantly increased the fold change in this transcript compared with the appropriate IgG group (Figure 8 , bottom). Thus, the antibody treatments used in C.B-17SCID/bg mice challenged with human fibroblasts markedly altered transcript expression for murine extracellular matrix-associated genes.

Figure 8. Quantitative TaqMan PCR analysis of extracellular matrix-associated genes MMP-2 (top) and MMP-19 (bottom) in C.B-17SCID/bg mice that received normal, NSIP, or IPF/UIP fibroblasts. Changes in gene expression are expressed as mean ?? SEM of the fold increase in transcript expression above a group of C.B-17SCID/bg mice that received PBS, PKH26, and one of IgG, anti-CCL21 antibody, and anti-CCR7 antibody. *P 0.05, ***P 0.001 compared with the appropriate C.B-17SCID/bg group that received human fibroblasts and IgG treatment.

Immunoneutralization of Human CCR7 or Human CCL21 Significantly Reduced Whole-Lung Hydroxyproline Levels in C.B-17SCID/bg Mice That Received Human IIP Fibroblasts

Hydroxyproline levels in whole-lung samples from IgG-, anti-CCL21-, or anti-CCR7-treated control C.B-17SCID/bg mice (ie, mice that did not receive human fibroblasts) and groups of treated mice that received normal, NSIP, or IPF/UIP fibroblasts are shown in Figure 9 . Hydroxyproline levels were increased in whole-lung samples from C.B-17SCID/bg mice that received normal fibroblasts and IgG, but neither antibody treatment altered these levels (Figure 9) . In the NSIP fibroblast C.B-17SCID/bg groups, hydroxyproline levels were significantly increased above those levels measured in the control C.B-17SCID/bg group, and the anti-CCL21 antibody and anti-CCR7 antibody therapies significantly reduced hydroxyproline levels by 52 ?? 6.7 and 51 ?? 5.6%, respectively, compared with the IgG treatment group (Figure 9) . In the IPF/UIP fibroblast C.B-17SCID/bg group that received IgG, hydroxyproline levels were again significantly increased above those levels measured in the control C.B-17SCID/bg group. In addition, hydroxyproline levels in IPF/UIP fibroblast challenged mice were reduced by 66 ?? 7.2 and 59 ?? 7.1% in the anti-CCL21 and anti-CCR7 antibody treatment groups, respectively, compared with the IgG-treated C.B-17SCID/bg IPF/UIP fibroblast group (Figure 9) . Thus, these data confirmed that the targeting of either CCL21 or CCR7 markedly and significantly reduced pulmonary remodeling precipitated by the adoptive transfer of NSIP or IPF/UIP fibroblasts in C.B-17SCID/bg mice.

Figure 9. Whole-lung hydroxyproline levels in C.B-17SCID/bg mice that received no fibroblasts (ie, control) or received normal, NSIP, or IPF/UIP fibroblasts. All groups of mice received either IgG or monoclonal antibody therapy. IgG, anti-CCL21, and anti-CCR7 monoclonal antibody therapies began in separate groups of C.B-17SCID/bg mice at day 35 and continued to day 63. Lung samples were removed at day 63 after the adoptive i.v. transfer of human fibroblasts into C.B-17SCID/bg mice. Data shown are mean ?? SEM. *P 0.05 compared with the control C.B-17SCID/bg group, which did not receive any human fibroblasts; P 0.001 compared with the appropriate C.B-17SCID/bg group that received human fibroblasts and IgG treatment; P 0.05 compared with the appropriate C.B-17SCID/bg group that received human fibroblasts and IgC treatment.

Whole-Lung Cytokine Analysis Showed That Murine CCL21 Levels Were Significantly Reduced in the Lungs of C.B-17SCID/bg Mice That Received IPF/UIP Fibroblasts and Anti-CCR7 Antibody Therapy

Because whole-lung levels of murine IL-13, CCL6, and CCL21 were altered and/or increased by the presence of NSIP and IPF/UIP fibroblasts in C.B-17SCID/bg mice, the effects of the IgG and monoclonal antibody treatments on these mediators at day 63 were assessed. As shown in Figure 10 , the anti-CCR7 and anti-CCL21 antibody therapies did not affect whole-lung levels of IL-13 or CCL6 in any of the C.B-17SCID/bg fibroblast groups. However, the anti-CCR7 antibody therapy significantly reduced whole-lung levels of murine CCL21 in the C.B-17SCID/bg group that received IPF/UIP fibroblasts compared with the C.B-17SCID/bg group that received IPF/UIP fibroblasts + IgG (Figure 10) . Thus, the therapeutic targeting of CCR7 reduced murine CCL21 levels but did not affect the levels of the other two mouse mediators found to be elevated in the IIP fibroblast models.

Figure 10. Whole-lung murine IL-13, CCL6, and CCL21 levels in C.B-17SCID/bg mice that received normal, NSIP, or IPF/UIP fibroblasts and IgG or monoclonal antibody therapies. Anti-CCL21 and anti-CCR7 monoclonal antibody therapies began at day 35 and continued to day 63. Lung samples were removed at day 63 after the adoptive i.v. transfer of human fibroblasts into C.B-17SCID/bg mice. Specific ELISA was used to measure all soluble proteins. Data shown are mean ?? SEM. *P 0.05, **P 0.01 compared with indicated protein levels measured in whole-lung samples from C.B-17SCID/bg mice that received human fibroblasts.

The present study addressed the following two questions: Do adoptively transferred human pulmonary fibroblasts remodel the lung architecture in SCID mice, and what role do CCL21 and CCR7 exert in the remodeling response precipitated by the adoptive transfer of human fibroblasts? The response to the first question was affirmative because the i.v. adoptive transfer of either 1 x 106 IPF/UIP or NSIP fibroblasts into mice C.B-17SCID/bg mice lacking both adaptive and innate immune features caused fibrosis, which was confirmed using histological, molecular, and biochemical analyses. This fibrosis was abrogated by the therapeutic administration of monoclonal antibodies directed against either human CCL21 or CCR7, thereby demonstrating a major role for this ligand and its receptor in pulmonary fibrosis similar to their roles in renal32 and liver33 fibrosis. Although murine equivalents of known and putative profibrotic mediators were increased in C.B-17SCID/bg mice that had received either IPF/UIP or NSIP fibroblasts, the role of these mediators in the fibrotic process is questionable because their levels were unchanged in many groups of C.B-17SCID/bg mice, which showed a significant decrease in lung remodeling. Thus, the present study provides evidence that the adoptive transfer of IIP fibroblasts promotes fibrosis in C.B-17SCID/bg mice and identifies a novel therapeutic target in IIP.

The modeling of clinical pulmonary fibrosis remains a major challenge in the laboratory.17 Although bleomycin sulfate is the agent of choice in the induction of experimental fibrosis, bleomycin-induced pulmonary fibrosis has been criticized as a less than ideal model of IPF/UIP.17 Recognizing that newer models of pulmonary fibrosis should incorporate as many of the features of clinical IIP as possible, the present study capitalized on the observation that the fibroblast is primarily responsible for the profound and often lethal remodeling in these diseases.4 The adoptive transfer of either IPF/UIP or NSIP fibroblasts initiated interstitial remodeling and histologically evident fibrosis was observed at day 35, but not earlier, after the i.v. adoptive transfer of these lines. The delay in appearance of fibrosis is not readily explainable, but these findings are consistent with previous studies in which the adoptive transfer of human endothelial cells into C.B-17SCID/bg mice required approximately 30 to 40 days before distinct blood vessels were apparent.36 Thus, the present study highlights that a clinically relevant model of pulmonary fibrosis can be initiated in immunodeficient mice with the adoptive transfer of human pulmonary fibroblasts.

The obvious benefit of the IPF/UIP and NSIP fibroblast C.B-17SCID/bg models described herein is their utility in the testing of novel therapeutics for these diseases. The present study addressed the roles of human CCL21 and CCR7 because we have observed that both are prominently expressed in IIP biopsies25 and cultured IIP fibroblasts (E.M.P. and C.M.H., unpublished observations). Our present hypothesis is that the therapeutic effect of anti-human CCL21 and anti-human CCR7 antibody treatments relates to their negations of the pro-proliferative effect of CCL21 on IPF/UIP and NSIP fibroblasts (E.M.P. and C.M.H., unpublished observations). Although it is unlikely that the monoclonal antibodies used herein affected the migration of i.v.-injected fibroblasts because the antibody treatments were delayed until a time point when fibrosis was histologically apparent in C.B-17SCID/bg mice, it is conceivable that this therapeutic approach, if used in other models of pulmonary fibrosis, may have an effect on the recruitment of fibrocytes into the lung. CCR7 is prominently expressed on fibrocytes,37,38 and CCL21 promotes their recruitment into various tissue sites.37 Studies are presently underway to address the role of CCR7-positive fibrocytes in bleomycin-induced pulmonary fibrosis using CCR7 wild-type and gene-deficient mice.

The precise contribution of the adoptively transferred human IIP fibroblasts and the mouse-associated fibrotic components (ie, cells and mediators) to the overall pulmonary remodeling response observed in C.B-17SCID/bg mice remains to be determined. In the present study, it would seem that there was an interaction between the transferred human fibroblasts and mouse components as evidenced by the dynamic changes in murine extracellular matrix-associated transcripts and murine-soluble profibrotic proteins in the lungs of C.B-17SCID/bg mice that most notably received either of the two IIP fibroblast types. Although the relative importance of the soluble murine proteins detected in the C.B-17SCID/bg fibrotic response is questionable at this point, the alterations in cathepsin E, MMPs, extracellular matrix components (ie, collagen and fibronectin), and the inhibitors of MMPs in the IIP fibroblast C.B-17SCID/bg groups relative to the normal fibroblast C.B-17SCID/bg group may indicate that the mouse-associated fibrotic components were actively involved in the remodeling response. Quantitative TaqMan PCR confirmed that collagen 1, cathepsin E, MMP-19, and TIMP-1 transcript levels were significantly increased in the IIP fibroblast C.B-17SCID/bg groups relative to changes in transcript levels for these genes in C.B-17SCID/bg mice that received normal fibroblasts. These transcript changes are relevant to clinical pulmonary fibrosis because cathepsins,39,40 MMPs and TIMPs (reviewed in Ref. 41 ), and extracellular matrix42,43 are increased in the more severe forms of IIP such as IPF/UIP and NSIP. MMP-19 was increased most notably in C.B-17SCID/bg mice that received IPF/UIP fibroblasts, and although this proteinase seems to have a major role in dermal wound healing responses (commentary by Mauch44 ), its role in pulmonary fibrosis is unknown. Almost without exception, the anti-CCL21 and anti-CCR7 antibody therapies reduced all of the abovementioned murine extracellular matrix-associated gene products analyzed using quantitative TaqMan PCR. The one exception was MMP-19, and further study of the relative importance of MMP-19 in the pulmonary fibrotic response evoked after the adoptive transfer of human IIP fibroblasts is warranted in light of this and our other findings.

In summary, the present study confirms that pulmonary fibrosis can be transferred to C.B-17SCID/bg mice after the i.v. adoptive transfer of either IPF/UIP or NSIP primary fibroblast lines. The utility of this model in the testing of novel therapeutic strategies was demonstrated, and our findings raise the possibility that the CCL21-CCR7 interaction may be an important target in clinical NSIP and IPF/UIP.

【参考文献】
  American Thoracic Society/European Respiratory Society (ATS/ERS) international multidisciplinary consensus classification of idiopathic interstitial pneumonias. Am J Respir Crit Care Med 2002, 165:277-304

Antoniou KM, Pataka A, Bouros D, Siafakas NM: Pathogenetic pathways and novel pharmacotherapeutic targets in idiopathic pulmonary fibrosis. Pulm Pharmacol Ther 2006,

Strieter RM: Pathogenesis and natural history of usual interstitial pneumonia: the whole story or the last chapter of a long novel. Chest 2005, 128:526S-532S

Zisman DA, Keane MP, Belperio JA, Strieter RM, Lynch JP, III: Pulmonary fibrosis. Methods Mol Med 2005, 117:3-44

Swigris JJ, Kuschner WG, Kelsey JL, Gould MK: Idiopathic pulmonary fibrosis: challenges and opportunities for the clinician and investigator. Chest 2005, 127:275-283

Keane MP, Arenberg DA, Lynch JP, III, Whyte RI, Iannettoni MD, Burdick MD, Wilke CA, Morris SB, Glass MC, DiGiovine B, Kunkel SL, Strieter RM: The CXC chemokines, IL-8 and IP-10, regulate angiogenic activity in idiopathic pulmonary fibrosis. J Immunol 1997, 159:1437-1443

Ramos C, Montano M, Garcia-Alvarez J, Ruiz V, Uhal BD, Selman M, Pardo A: Fibroblasts from idiopathic pulmonary fibrosis and normal lungs differ in growth rate, apoptosis, and tissue inhibitor of metalloproteinases expression. Am J Respir Cell Mol Biol 2001, 24:591-598

White ES, Atrasz RG, Hu B, Phan SH, Stambolic V, Mak TW, Hogaboam CM, Flaherty KR, Martinez FJ, Kontos CD, Toews GB: Negative regulation of myofibroblast differentiation by PTEN (phosphatase and tensin homolog deleted on chromosome 10). Am J Respir Crit Care Med 2006, 173:112-121

Jakubzick C, Choi ES, Carpenter KJ, Kunkel SL, Evanoff H, Martinez FJ, Flaherty KR, Toews GB, Colby TV, Travis WD, Joshi BH, Puri RK, Hogaboam CM: Human pulmonary fibroblasts exhibit altered interleukin-4 and interleukin-13 receptor subunit expression in idiopathic interstitial pneumonia. Am J Pathol 2004, 164:1989-2001

Selman M, Thannickal VJ, Pardo A, Zisman DA, Martinez FJ, Lynch JP, III: Idiopathic pulmonary fibrosis: pathogenesis and therapeutic approaches. Drugs 2004, 64:405-430

Quan TE, Cowper SE, Bucala R: The role of circulating fibrocytes in fibrosis. Curr Rheumatol Rep 2006, 8:145-150

Lama VN, Phan SH: The extrapulmonary origin of fibroblasts: stem/progenitor cells and beyond. Proc Am Thorac Soc 2006, 3:373-376

Kaminski N, Allard JD, Pittet JF, Zuo F, Griffiths MJ, Morris D, Huang X, Sheppard D, Heller RA: Global analysis of gene expression in pulmonary fibrosis reveals distinct programs regulating lung inflammation and fibrosis. Proc Natl Acad Sci USA 2000, 97:1778-1783

Pardo A, Gibson K, Cisneros J, Richards TJ, Yang Y, Becerril C, Yousem S, Herrera I, Ruiz V, Selman M, Kaminski N: Up-regulation and profibrotic role of osteopontin in human idiopathic pulmonary fibrosis. PLoS Med 2005, 2:e251

Choi ES, Jakubzick C, Carpenter KJ, Kunkel SL, Evanoff H, Martinez FJ, Flaherty KR, Toews GB, Colby TV, Kazerooni EA, Gross BH, Travis WD, Hogaboam CM: Enhanced monocyte chemoattractant protein-3/CC chemokine ligand-7 in usual interstitial pneumonia. Am J Respir Crit Care Med 2004, 170:508-515

Gharaee-Kermani M, Ullenbruch M, Phan SH: Animal models of pulmonary fibrosis. Methods Mol Med 2005, 117:251-259

Chua F, Gauldie J, Laurent GJ: Pulmonary fibrosis: searching for model answers. Am J Respir Cell Mol Biol 2005, 33:9-13

Gauldie J: Pro: inflammatory mechanisms are a minor component of the pathogenesis of idiopathic pulmonary fibrosis. Am J Respir Crit Care Med 2002, 165:1205-1206

Strieter RM: Con: Inflammatory mechanisms are not a minor component of the pathogenesis of idiopathic pulmonary fibrosis. Am J Respir Crit Care Med 2002, 165:1206-1207discussion 1207C1208

Flaherty KR, Thwaite EL, Kazerooni EA, Gross BH, Toews GB, Colby TV, Travis WD, Mumford JA, Murray S, Flint A, Lynch JP, III, Martinez FJ: Radiological versus histological diagnosis in UIP and NSIP: survival implications. Thorax 2003, 58:143-148

Agostini C, Gurrieri C: Chemokine/cytokine cocktail in idiopathic pulmonary fibrosis. Proc Am Thorac Soc 2006, 3:357-363

Zhu Z, Homer RJ, Wang Z, Chen Q, Geba GP, Wang J, Zhang Y, Elias JA: Pulmonary expression of interleukin-13 causes inflammation, mucus hypersecretion, subepithelial fibrosis, physiologic abnormalities, and eotaxin production. J Clin Invest 1999, 103:779-788

Jakubzick C, Kunkel SL, Puri RK, Hogaboam CM: Therapeutic targeting of IL-4- and IL-13-responsive cells in pulmonary fibrosis. Immunol Res 2004, 30:339-350

Belperio JA, Dy M, Burdick MD, Xue YY, Li K, Elias JA, Keane MP: Interaction of IL-13 and C10 in the pathogenesis of bleomycin-induced pulmonary fibrosis. Am J Respir Cell Mol Biol 2002, 27:419-427

Choi ES, Pierce EM, Jakubzick C, Carpenter KJ, Kunkel SL, Evanoff H, Martinez FJ, Flaherty KR, Moore BB, Toews GB, Colby TV, Kazerooni EA, Gross BH, Travis WD, Hogaboam CM: Focal interstitial CC chemokine receptor 7 (CCR7) expression in idiopathic interstitial pneumonia. J Clin Pathol 2006, 59:28-39

Kellermann SA, Hudak S, Oldham ER, Liu YJ, McEvoy LM: The CC chemokine receptor-7 ligands 6Ckine and macrophage inflammatory protein-3 beta are potent chemoattractants for in vitro- and in vivo-derived dendritic cells. J Immunol 1999, 162:3859-3864

Jakubzick C, Choi ES, Joshi BH, Keane MP, Kunkel SL, Puri RK, Hogaboam CM: Therapeutic attenuation of pulmonary fibrosis via targeting of IL-4- and IL-13-responsive cells. J Immunol 2003, 171:2684-2693

Jakubzick C, Choi ES, Kunkel SL, Joshi BH, Puri RK, Hogaboam CM: Impact of interleukin-13 responsiveness on the synthetic and proliferative properties of Th1- and Th2-type pulmonary granuloma fibroblasts. Am J Pathol 2003, 162:1475-1486

Zhang K, Gharaee-Kermani M, Jones ML, Warren JS, Phan SH: Lung monocyte chemoattractant protein-1 gene expression in bleomycin-induced pulmonary fibrosis. J Immunol 1994, 153:4733-4741

Ryu JH, Colby TV, Hartman TE: Idiopathic pulmonary fibrosis: current concepts. Mayo Clin Proc 1998, 73:1085-1101

Jakubzick C, Wen H, Matsukawa A, Keller M, Kunkel SL, Hogaboam CM: Role of CCR4 ligands, CCL17 and CCL22, during Schistosoma mansoni egg-induced pulmonary granuloma formation in mice. Am J Pathol 2004, 165:1211-1221

Banas B, Wornle M, Berger T, Nelson PJ, Cohen CD, Kretzler M, Pfirstinger J, Mack M, Lipp M, Grone HJ, Schlondorff D: Roles of SLC/CCL21 and CCR7 in human kidney for mesangial proliferation, migration, apoptosis, and tissue homeostasis. J Immunol 2002, 168:4301-4307

Bonacchi A, Petrai I, Defranco RM, Lazzeri E, Annunziato F, Efsen E, Cosmi L, Romagnani P, Milani S, Failli P, Batignani G, Liotta F, Laffi G, Pinzani M, Gentilini P, Marra F: The chemokine CCL21 modulates lymphocyte recruitment and fibrosis in chronic hepatitis C. Gastroenterology 2003, 125:1060-1076

Jenh CH, Cox MA, Kaminski H, Zhang M, Byrnes H, Fine J, Lundell D, Chou CC, Narula SK, Zavodny PJ: Cutting edge: species specificity of the CC chemokine 6Ckine signaling through the CXC chemokine receptor CXCR3: human 6Ckine is not a ligand for the human or mouse CXCR3 receptors. J Immunol 1999, 162:3765-3769

Hammad H, Lambrecht BN, Pochard P, Gosset P, Marquillies P, Tonnel AB, Pestel J: Monocyte-derived dendritic cells induce a house dust mite-specific Th2 allergic inflammation in the lung of humanized SCID mice: involvement of CCR7. J Immunol 2002, 169:1524-1534

Skovseth DK, Yamanaka T, Brandtzaeg P, Butcher EC, Haraldsen G: Vascular morphogenesis and differentiation after adoptive transfer of human endothelial cells to immunodeficient mice. Am J Pathol 2002, 160:1629-1637

Abe R, Donnelly SC, Peng T, Bucala R, Metz CN: Peripheral blood fibrocytes: differentiation pathway and migration to wound sites. J Immunol 2001, 166:7556-7562

Hashimoto N, Jin H, Liu T, Chensue SW, Phan SH: Bone marrow-derived progenitor cells in pulmonary fibrosis. J Clin Invest 2004, 113:243-252

Kimura M, Tani K, Miyata J, Sato K, Hayashi A, Otsuka S, Urata T, Sone S: The significance of cathepsins, thrombin and aminopeptidase in diffuse interstitial lung diseases. J Med Invest 2005, 52:93-100

Wattiez R, Hermans C, Cruyt C, Bernard A, Falmagne P: Human bronchoalveolar lavage fluid protein two-dimensional database: study of interstitial lung diseases. Electrophoresis 2000, 21:2703-2712

Pardo A, Selman M: Matrix metalloproteases in aberrant fibrotic tissue remodeling. Proc Am Thorac Soc 2006, 3:383-388

Kuhn C, Mason RJ: Immunolocalization of SPARC, tenascin, and thrombospondin in pulmonary fibrosis. Am J Pathol 1995, 147:1759-1769

Adachi K, Yamauchi K, Bernaudin JF, Fouret P, Ferrans VJ, Crystal RG: Evaluation of fibronectin gene expression by in situ hybridization: differential expression of the fibronectin gene among populations of human alveolar macrophages. Am J Pathol 1988, 133:193-203

Mauch C: Matrix metalloproteinase-19: what role does this enzyme play in wound healing? J Invest Dermatol 2003, 121:xix-xx

作者单位：From the Department of Pathology,* Division of Pulmonary Medicine, and the Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan

【摘要】  We investigated the pathogenic roles of CC chemokine ligand (CCL)3 and its receptors, CC chemokine receptor (CCR)1 and CCR5, in bleomycin (BLM)-induced pulmonary fibrosis (PF). An intratracheal injection of BLM into wild-type (WT) mice caused a massive infiltration of granulocytes and macrophages, followed by the development of diffuse PF with fibrocyte accumulation. Intrapulmonary CCL3 expression was enhanced rapidly and remained at elevated levels until PF developed. Moreover, CCL3 protein was detected mainly in infiltrating granulocytes and macrophages, whereas transforming growth factor-ß1 protein was detected in macrophages and myofibroblasts. Compared with WT mice, collagen accumulation was reduced in CCL3C/C and CCR5C/C but not CCR1C/C mice. Moreover, the BLM-induced increases in intrapulmonary macrophage and fibrocyte numbers were attenuated in CCL3C/C and CCR5C/C but not CCR1C/C mice, although BLM increased bone marrow (BM) fibrocyte number to a similar extent in these strains. BM transplantation from CCR5C/C to WT, but not that from WT to CCR5C/C mice, recapitulated the phenotypes in CCR5C/C mice. Furthermore, CCR5+/C mice exhibited a significant reduction in BLM-induced fibrotic changes. These results demonstrated that locally produced CCL3 was involved in BLM-induced recruitment of BM-derived macrophages and fibrocytes, main producers of transforming growth factor-ß1, and subsequent development of PF by interacting mainly with CCR5.
--------------------------------------------------------------------------------
Pulmonary fibrosis (PF) is a chronic lung disease characterized histologically by diffuse interstitial inflammation and fibrosis.1,2 PF frequently develops because of well-defined entities, such as irradiation injury, oxygen toxicity pneumonitis, and scleroderma, but approximately half of the cases are idiopathic. Idiopathic pulmonary fibrosis (IPF) is a progressive and irreversible chronic lung disease, and most patients experience severe hypoxemia and cyanosis in the advanced stages. Given the absence of proven therapies for IPF, current clinical management is primarily supportive, and lung transplantation is sometimes tried to cure the situation.1,2
Bleomycin (BLM), a copper-chelating peptide that can cleave DNA, is widely used as an anti-tumor agent for various types of malignancies, including squamous carcinomas and lymphomas, because BLM administration seldom causes myelosuppression, a common adverse effect of other various types of anti-tumor agents.3 However, BLM treatment frequently causes pulmonary injury, and irreversible PF sometimes ensues as its severe form.3 Because pathological changes in BLM-induced PF resemble those in IPF, a BLM-induced PF model is used to elucidate the molecular and cellular pathogenesis of IPF. On BLM administration, mice develop acute alveolitis and interstitial inflammation, characterized by the sequential infiltration of neutrophils, macrophages, and lymphocytes. The infiltration of inflammatory cells is postulated to lead to myofibroblast hyperplasia and disordered collagen deposition.4 Recently, in addition to these inflammatory cells, several independent groups proposed that fibrocytes expressing both leukocyte (CD45, CD13) and mesenchymal antigens (collagen I, fibronectin) have crucial roles in fibrosis, including BLM-induced PF and renal fibrosis.5-12
Chemokines can recruit and activate a selected type(s) of inflammatory cells, and some can mobilize even fibrocytes.7-10,12 BLM treatment can augment the intrapulmonary expression of various chemokines including macrophage inflammatory protein-1/CC chemokine ligand (CCL)3, a member of CC chemokines.13-20 CCL3 exhibits chemotactic and activating effects on neutrophils, macrophages, lymphocytes, and immature dendritic cells.21-23 Moreover, CC chemokine receptor (CCR)1 and CCR5, a specific receptor for CCL3,24,25 are expressed by fibrocytes.7,9 Indeed, passive immunization of BLM-challenged mice with anti-CCL3 antibodies attenuated fibrotic changes,13 implying CCL3 as a mediator responsible for BLM-induced fibrosis. However, its precise roles have not been determined yet, particularly with reference to fibrocytes. Moreover, it remains to be investigated how CCR1 and CCR5 are used by a single ligand, CCL3, in BLM-induced PF.
Here, we investigated the pathophysiological roles of the CCL3 axis in BLM-induced PF by using mice deficient in each gene. We demonstrated that the genetic ablation of CCL3 markedly attenuated PF induced by BLM administration. Moreover, homozygous and to a lesser degree, heterozygous deficiency in CCR5, ameliorated significantly fibrotic changes in the lungs, whereas CCR1 deficiency has minimal effects on BLM-induced PF. Furthermore, the recruitment of fibrocytes in both CCL3C/C and CCR5C/C mice was significantly reduced compared with WT and CCR1C/C mice. Finally, bone marrow (BM) transplantation from WT to CCR5C/C mice restored the susceptibility to BLM, whereas WT mice receiving CCR5C/C BM were protected from BLM-induced PF. Thus, CCL3 used CCR5 but not CCR1 to recruit and activate BM-derived cells including fibrocytes in BLM-induced PF.

【关键词】  essential chemokine chemokine receptor bleomycin-induced pulmonary fibrosis regulation macrophage fibrocyte infiltration

Materials and Methods

Reagents and Antibodies (Abs)

BLM was purchased from Sigma Chemical Co. (St. Louis, MO). Rabbit anti-mouse CCR5 polyclonal antibodies (pAbs) were prepared as described previously.26 The following monoclonal antibodies (mAbs) and pAbs were commercially obtained: rat anti-mouse F4/80 mAb (Dainippon Pharmaceutical Co., Osaka, Japan); rat anti-mouse Ly-6G mAb, fluorescein isothiocyanate (FITC)-labeled rat anti-mouse CXC chemokines receptor (CXCR)4 mAb, peridinin chlorophyll-a protein (PerCP)-labeled rat anti-mouse CD45 mAb, and FITC-labeled rat anti-mouse CD45 mAb (BD Pharmingen, San Jose, CA); goat anti-mouse CCL3 pAbs (R&D Systems, Inc., Minneapolis, MN); rabbit anti-transforming growth factor (TGF)-ß1 pAbs and goat anti-CCR5 pAbs (Santa Cruz Biotechnology, Santa Cruz, CA); mouse anti--smooth muscle actin (-SMA) mAb (Boehringer Mannheim GmbH, Mannheim, Germany); Alexa Fluor 488-labeled hamster anti-CCR5 (Biolegend Inc., San Diego, CA); Cy3-conjugated donkey anti-rat IgG pAbs, FITC-conjugated donkey anti-goat IgG pAbs, and FITC-conjugated donkey anti-rabbit IgG pAbs (Jackson Immunoresearch Laboratories, West Grove, PA); and rabbit anti-type I collagen (Col I) pAbs (Chemicon International, Temecula, CA).8,11,12

Pathogen-free 8-week-old male C57BL/6 mice were obtained from Sankyo Laboratories (Tokyo, Japan) and designated as wild-type (WT) mice. Homozygous CCL3-deficient (CCL3C/C) mice were obtained from Jackson Laboratories (Bar Harbor, ME). CCR1-deficient (CCR1C/C) mice were a generous gift from Drs. P.M. Murphy and J.-L. Gao (National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD).27 CCR5-deficient (CCR5C/C) mice were generated as previously described.28 All these mice were backcrossed to C57BL/6 mice for 8 to 10 generations. In some experiments, CCR5C/C mice were mated with C57BL/6 mice to generate heterozygous CCR5-deficient (CCR5+/C) mice. All mice were kept under the specific pathogen-free conditions at the Animal Research Center of Kanazawa University (Kanazawa, Japan). Age- and sex-matched mice were used for the experiments. All animal experiments in this study complied with the Guidelines for the Care and Use of Laboratory Animals on the Takara-Machi Campus of Kanazawa University.

BLM-Induced Lung Injury

After mice were anesthetized with an intraperitoneal injection of pentobarbital (50 µg/g weight), a cervical midline incision was made, and the trachea was exposed. Thereafter, BLM (0.075 U) in 50 µl of sterile saline was intratracheally administered by the use of a syringe with a 26-guage needle. At the indicated time intervals after BLM administration, the mice were sacrificed by an overdose of diethylether, and both lungs were removed for subsequent analyses.

Generation of BM Chimera Mice

The following BM chimeric mice were prepared: male CCR5C/C BMfemale WT mice, male WT BMfemale CCR5C/C mice, male WT BMfemale WT mice, and male CCR5C/C BMfemale CCR5C/C mice. BM cells were collected from the femurs of donor mice by aspiration and flushing. Recipient mice were irradiated to 15 Gy using an RX-650 irradiator (Faxitron X-ray Inc., Wheeling, IL). Then, the animals received intravenously 5 x 106 BM cells from donor mice in a volume of 200-µl sterile phosphate-buffered saline (PBS)(C) under the anesthesia. Thereafter, the mice were housed in sterilized microisolator cages and were fed normal chow and autoclaved hyperchlorinated water for 60 days. To verify successful engraftment and reconstitution of the BM in the transplanted mice, genomic DNA was isolated from peripheral blood and tail tissues of each chimeric mouse 30 days after BMT with a NucleoSpin tissue kit (Macherey-Nagel, Duren, Germany). Then, we performed polymerase chain reaction (PCR) to detect the Sry gene contained in the Y chromosome (forward primer, 5'-TTGCCTCAACAAAA-3'; reverse primer, 5'-AAACTGCTGCTTCTGCTGGT-3'). The amplified PCR products were fractionated on a 2% agarose gel and visualized by ethidium bromide staining. After durable BM engraftment was confirmed, mice were treated with BLM as described above.

Histopathological Analyses

The lung tissues were fixed in 10% formalin buffered with PBS (pH 7.2) and embedded in paraffin. Six-µm-thick sections were made and stained with hematoxylin and eosin or Masson??s trichrome for the detection of collagen deposition. Histopathological changes were evaluated by an examiner without prior knowledge on the experimental procedures. Immunohistochemical analyses were also performed using anti-Ly-6G, anti-F4/80, anti-CCL3, or TGF-ß1 Abs, as described previously.29 The numbers of infiltrating granulocytes or macrophages were enumerated on 10 randomly chosen visual fields at x200 magnification, and the average of the 10 selected microscopic fields was calculated. All measurements were performed by an examiner without previous knowledge on the experimental procedures. The incubation of isotype-matched control Ab did not give rise to any positive reaction, indicating the specificities of each primary Ab (data not shown). As described previously,29,30 preadsorption of anti-CCL3 or anti-TGF-ß1 Abs with an excess amount of each recombinant protein abolished the positive signals, further indicating the specificity of the reaction (data not shown).

Double-Color or Triple-Color Immunofluorescence Analysis

A double-color immunofluorescence analysis was conducted to identify the types of CCL3- and TGF-ß1-expressing cells in the lung, as described previously.30 A triple-color immunofluorescence analysis was also performed in the combination of anti-CD45, anti-Col I, and anti-CCR5 Abs. Thereafter, the sections were observed under a fluorescence microscopy. In triple-color analysis, we titrated the concentrations of anti-Col I to determine the concentration that did not give rise to a positive staining in extracellular matrix.

Determination of Hydroxyproline (Hyp) Contents

At 21 days after BLM administration, lung tissues were removed, and the contents of Hyp, a major component of collagen, were determined as previously described.29 The data were expressed as the amount (µg) per lung.

Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)

Five µg of total RNA extracted from lung samples was reverse-transcribed as described previously. The resultant cDNAs were amplified together with Taq polymerase (Takara Shuzo, Kyoto, Japan) using the specific sets of primers with the optimal cycles consisting of 94??C for 30 seconds, the optimal annealing temperature for 30 seconds, and 72??C for 1 minute, followed by incubation at 72??C for 5 minutes (Table 1) . The amplified PCR products were fractionated on a 2% agarose gel along with a constant amount of a standard DNA and visualized by ethidium bromide staining.29,30 The band intensities were measured using NIH Image Analysis software (version 1.63), and the ratios to ß-actin were calculated on the assumption that the ratios of untreated animals are 1.0.

Table 1. Sequences of the Primers Used for RT-PCR

Flow Cytometric Analysis

Mononuclear cells were isolated from lungs, BM, and peripheral blood, as described previously.8 Nonspecific binding was blocked by incubating with 25 µg/ml of Fc block (BD Biosciences Pharmingen, Piscataway, NJ) for 15 minutes at 4??C. The cells were stained with FITC-labeled anti-CXCR4 or Alexa Fluor 488-labeled anti-CCR5 and PerCP-labeled anti-CD45. After an extensive washing with PBS (C), the cells were permeabilized using cytofix/cytoperm (BD Biosciences Pharmingen) and stained with anti-Col I pAbs followed by the incubation with PE-conjugated goat anti-rabbit Ig (Molecular Probes Inc., Eugene, OR) as previously described.8,11,12 After being washed twice with PBS (C), the cells were fixed in 2% paraformaldehyde. Control staining was performed by using isotype-matched mAbs for CD45, CXCR4, or CCR5. For immunostaining with anti-collagen I pAb, normal rabbit IgG was used as a negative control. A flow cytometric analysis with FITC-labeled anti-CD45 and Via-Probe7-AAD (BD Pharmingen) indicated that dead cells were consistently less than 0.5% in the cells gated with anti-CD45 mAb. Moreover, the cells were stained with anti-Col I, anti-CXCR4, and CCR5 for the evaluation of CCR5-positive fibrocytes co-expressing CXCR4. The stained cells were analyzed on a FACS Calibur flow cytometer (Becton-Dickinson, Mountain View, CA), and the obtained data were analyzed using CellQuest Pro software (BD Biosciences, San Jose, CA).

In Situ Hybridization Combined with Immunofluorescence Analysis on CD45+ Cells from the Whole Lungs and BM

At 14 days after BLM administration, mononuclear cells were isolated from lungs and BMs and cultured for 10 to 14 days, as described previously.9 The trypsinized cells were stained with anti-CD45 mAb coupled to magnetic beads (Miltenyi Biotech, Auburn, CA). The cells were then sorted by LS-positive selection columns using a SuperMACS apparatus (Miltenyi Biotech) according to the manufacturer??s instructions. After being washed extensively, CD45+ cells, which were retained on the column, were removed from the magnetic field and were used for in situ hybridization combined with immunofluorescence analysis.31 Nested RT-PCR products of collagen I were obtained using the pair of primers with the addition of T7- and Sp6-RNA polymerase promoter to the 5' end of each sense and anti-sense primer of collagen I, respectively (Table 1) . Digoxigenin-labeled sense and anti-sense probes were obtained by using DIG RNA labeling kit (Boehringer Mannheim Biochemica, Mannheim, Germany) according to the manufacturer??s instructions. The sense probe was used as a negative control. After the resultant CD45+ cells were incubated with anti-sense probe of collagen I, the bound probe was detected using rhodamine-conjugated anti-digoxigenin Abs. Thereafter, anti-CXCR4 Abs were further applied to the cells, followed by the incubation of FITC-labeled secondary Abs. Images were observed under a fluorescent microscopy and digitally merged.

Enzyme-Linked Immunosorbent Assay (ELISA)

Lung samples were homogenized with PBS containing Complete protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) and centrifuged at 5000 x g for 10 minutes. Supernatants were used to determine CCL3, TGF-ß, and CXC chemokine ligand (CXCL)12 levels with commercial ELISA kits (R&D Systems), according to the manufacturer??s instructions. The detection limits in each method were as follows: CCL3 > 1.5 pg/ml, TGF-ß1 > 7 pg/ml, and CXCL12 > 40 pg/ml. Total protein in the supernatant was measured with a commercial kit (bicinchoninic acid protein assay kit; Pierce, Rockford, IL). The data were expressed as the target molecule (pg) per total protein (mg) for each sample.

Statistical Analysis

The means and SEMs were calculated for all parameters determined in this study. Statistical significance was evaluated by using one-way analysis of variance with posthoc testing with the Scheff???s F multiple comparisons test or Mann-Whitney??s U-test. P < 0.05 was accepted as statistically significant.

Chemokine Expression in Lung after BLM Treatment

CCL3 mRNA expression was increased nearly threefold as early as 1 day and remained elevated until 14 days after BLM injection (Figure 1, A and B) . In contrast, CCL4 mRNA expression was marginally increased and returned to a basal level at 14 days after the injection and CCL5 mRNA expression was enhanced only later than 14 days after the injection (Figure 1, A and D) . These observations suggest that CCL3 was a major chemokine expressed in lungs after BLM treatment among these chemokines. Moreover, intrapulmonary CCL3 protein contents started to be increased 1 day after BLM treatment and reached a peak at 14 days (Figure 2A) . Furthermore, CCL3 protein was immunohistochemically detected in polymorphonuclear or mononuclear leukocytes recruited into the alveolar spaces after BLM treatment (Figure 2B) . A double-color immunofluorescence analysis detected CCL3 protein in Ly-6G- and F4/80-positive cells (Figure 2C) but not CD3-positive cells (data not shown) after BLM treatment, suggesting that granulocytes and macrophages were main producers of CCL3.

Figure 1. CC chemokine expression in the lungs of WT mice after BLM treatment. ACD: RT-PCR was performed on total RNAs extracted from the lungs at the indicated time intervals after BLM treatment as described in Materials and Methods. Representative results from six independent experiments are shown in A. The ratios of CCL3 (B), CCL4 (C), and CCL5 (D) to ß-actin were calculated. Each value represents mean ?? SEM (n = 6). *P < 0.05; **P < 0.01, versus untreated WT lungs.

Figure 2. Intrapulmonary CCL3 protein expression after BLM treatment. A: Intrapulmonary CCL3 contents were determined by ELISA and are shown here. Each value represents mean ?? SEM (n = 6). **P < 0.01, versus untreated WT lungs. B: Immunohistochemical detection of CCL3 protein in the lungs of WT mice at 7 days after BLM treatment. A representative result from six independent experiments is shown here. C: A double-color immunofluorescence analysis of CCL3-expressing cells in the lungs of WT mice at 7 days after BLM treatment. Representative results from six independent experiments are shown here. The fluorescent images were digitally merged (right column). Original magnifications, x200 (B).

Attenuated PF in CCR5C/C and CCL3C/C Mice after BLM Treatment

To evaluate the contribution of CCL3 and its receptors to BLM-induced lung injury, the mice deficient in CCL3, CCR1, and CCR5 were injected with BLM, along with WT mice. Before BLM challenge, no apparent histological differences were observed in the lungs among these mice (data not shown). By 3 days after administration, the lungs of WT mice exhibited marked hemorrhages and congestion with infiltration of inflammatory cells predominantly consisting of Ly-6G-positive polymorphonuclear cells (Figures 3 and 4) . Later than 7 days, F4/80-positive mononuclear inflammatory cells predominated in infiltrating cells, with a marked thickening of the alveolar septa (Figures 3 and 4) . Concomitantly, fibrotic changes and fibroblast proliferation become apparent, starting from the subpleural areas and extending to the central areas. At 21 days after BLM administration, Masson??s trichrome staining revealed a massive blue coloration in the lungs from WT mice (Figure 2) , indicating a dense collagen deposition. CCR1C/C mice also exhibited similar histopathological changes as WT mice after BLM treatment (Figure 3) . In contrast, these pathological changes were remarkably attenuated in CCL3C/C and CCR5C/C mice with a marked reduction in Ly-6G-positive granulocyte and F4/80-positive macrophage infiltration, compared with WT mice (Figures 3 and 4) . Moreover, we evaluated intrapulmonary Hyp contents, an index of collagen deposition. Before BLM challenge, there was no significant difference in intrapulmonary Hyp contents among these strains (data not shown). Consistent with histopathological analysis, intrapulmonary Hyp contents, an index of collagen deposition, were increased approximately threefold in WT and CCR1C/C mice 21 days after BLM administration, whereas the increases in Hyp contents were negligible in CCL3C/C and CCR5C/C mice (Figure 5) . These observations would indicate that the CCL3-CCR5 but not the CCL3-CCR1 axis was involved in BLM-induced lung injury. Moreover, the intrapulmonary Hyp contents in CCR5+/C mice were intermediate between BLM-treated WT and CCR5C/C mice 21 days after BLM treatment (data not shown), suggesting that CCR5 gene has dose effects on BLM-induced PF.

Figure 3. Histopathological observations on lungs from WT, CCR5C/C, CCR1C/C, and CCL3C/C mice after BLM treatment. Representative results from six animals at each time point are shown. Original magnifications: x100 (H&E stain); x40 (Masson??s stain).

Figure 5. Hyp contents in the lung at 21 days after BLM treatment. Hyp contents were determined as an indicator of collagen contents. Each value represents mean ?? SEM (n = 6). **P < 0.01, versus untreated WT lungs. ##P < 0.01, versus BLM-treated WT lungs.

Contribution of BM Cells to BLM-Induced PF

Evidence is accumulating to indicate the crucial contribution of BM cells to PF.8-10 To evaluate the roles of BM-derived cells to BLM-induced PF, we made several BM chimeric mice. Although radiation can induce pneumonitis including fibrosis, the untreated BM chimeric mice did not exhibit a significant increase in intrapulmonary Hyp (Figure 6) , indicating that radiation alone cannot induce PF under the present conditions. BLM injection increased Hyp contents markedly and to a similar level in WT BMCCR5C/C mice and WT BMWT mice as WT mice (Figure 6) . Although the intrapulmonary Hyp contents were slightly increased in CCR5C/C BMWT mice and CCR5C/C BMCCR5C/C mice, the extent of fibrotic changes was significantly reduced, compared with mice transplanted with WT BM. Thus, BM-derived cells were mainly responsible for BLM-induced PF.

Figure 6. The effects of BM transplantation on BLM-induced PF. Recipient female mice were transplanted with BM cells from CCR5C/C or WT male donors as described in Materials and Methods. BM chimera mice were injected with BLM 60 days after BM transplantation. Intrapulmonary Hyp contents were determined as an indicator of collagen contents 21 days after BLM treatment as described in Materials and Methods. There was no difference in Hyp contents among all four BM chimera mice before BLM challenge. Each value represents mean ?? SEM (n = 6). *P < 0.05; **P < 0.01, versus untreated WT lungs. ##P < 0.01, versus BLM-treated WT mice.

CCR5 Deficiency Impaired Fibrocyte Migration into the BLM-Injected Lungs

The potential contribution of BM-derived cells to BLM-induced fibrosis prompted us to examine the roles of BM-derived fibrocytes, which are presumed to be involved in PF.8,9 In line with previous reports,8 we detected specific collagen I mRNA in some of CXCR4+ cells among CD45+ cells isolated from whole lungs (Figure 7A) and BMs (data not shown). Sense probes showed no positive signals, indicating the specificity of the reaction (data not shown). Before BLM treatment, there was no difference in the number of the intrapulmonary fibrocytes, defined as CD45+ColI+CXCR4+ cells, among untreated WT mice and mice deficient in CCR5, CCR1, and CCL3. BLM increased the number of the intrapulmonary fibrocytes to similar extents in WT and CCR1C/C mice 14 days after the injection (Figure 7, B and C) , but the increase was marginal in CCR5C/C or CCL3C/C mice, and the increment was intermediate in CCR5+/C mice (Figure 7C) . Thus, the intrapulmonary fibrocyte numbers were well correlated with BLM-induced increases in intrapulmonary Hyp contents among these strains. Moreover, similar numbers of fibrocytes were present in BM and, to a lesser degree, peripheral blood among these four untreated strains (Figure 7D) . An intratracheal injection of BLM increased the fibrocyte numbers in BM but not peripheral blood, to similar extents among these four strains 14 days after the injection (Figure 7D) , indicating that BM is a source of fibrocytes. Moreover, CCR5 was detected on CD45+Col I+ cells (Figure 7E) , consistent with the previous reports.7,9 Moreover, CXCR4 was coexpressed by 90% of CCR5+ fibrocytes, which were recruited into the lungs of WT mice after BLM treatment (Figure 7F) . In the next series, we analyzed the number of CD45+ColI+CCR5+ fibrocytes in WT and CCL3C/C mice. Although there was no difference in intrapulmonary CCR5+ fibrocyte numbers between untreated WT and CCL3C/C mice (data not shown), BLM induced less CCR5+ fibrocyte recruitment into the lungs in CCL3C/C mice, compared with WT mice (Figure 8, ACC) . Thus, the CCL3-CCR5 axis can regulate the migration of fibrocytes into the lungs from the BM, and eventually PF was induced by BLM treatment.

Figure 7. A: Detection of collagen I mRNA in CXCR4+ CD45+ cells. CD45+ cells, which were isolated from the whole lung were processed to in situ hybridization and immunofluorescence analysis, to detect collagen I mRNA and CXCR4 expression, respectively, as described in Materials and Methods. Representative results from six independent experiments are shown here. Similar results were obtained when BM-derived CD45+ cells were used. Original magnifications, x400. B: Flow cytometric analysis on CD45+CXCR4+Col I+ fibrocytes in the lungs, BM, and peripheral blood at 14 days after BLM challenge. Single-cell suspensions were isolated from the BM, peripheral blood, and lungs, and stained with the combination of anti-CD45, anti-CXCR4, and anti-Col I Abs, followed by a flow cytometric analysis, as described in Materials and Methods. Representative results on lung single cell suspensions from six individual WT mice are shown here with a contour plotting. Normal rabbit IgG was used as a negative control to gate collagen-positive signals (B, top left). C: The numbers of fibrocytes were calculated on lungs. Each value represents mean ?? SEM (n = 6). *P < 0.05; **P < 0.01, versus BLM-treated WT mice. D: The numbers of fibrocytes were calculated on BM and peripheral blood. Each value represents mean ?? SEM (n = 6). *P < 0.05, versus untreated mice. E: A triple-color immunofluorescence analysis of CCR5-expressing cells in the lungs of WT mice at 14 days after BLM challenge. CD45+Col I+ fibrocytes expressed CCR5 (arrows). Representative results from six independent experiments are shown here. Original magnifications, x400. F: Flow cytometric analysis on the portion of CCR5-positive fibrocytes co-expressing CXCR4. Representative results are shown here.

Figure 8. A and B: Flow cytometric analysis on CCR5+ fibrocytes in the lungs of WT and CCL3C/C mice at 14 days after BLM treatment. Single-cell suspensions were isolated from lungs and stained with the combination of anti-CD45, anti-CCR5, and anti-Col I Abs, followed by a flow cytometric analysis. Representative results from six independent experiments are shown in A (WT) and B (CCL3C/C). C: The numbers of intrapulmonary CCR5+ fibrocytes were calculated, and are shown here. Each value represents mean ?? SEM (n = 6). *P < 0.05, WT versus CCL3C/C mice.

Attenuated Expression of Intrapulmonary CXCL12 Expression in CCR5C/C and CCL3C/C Mice

CXCL12, a single ligand for CXCR4, is also presumed to be involved in the migration of fibrocytes from the BM.8 In WT mice, intrapulmonary CXCL12 protein contents started to be increased 1 day and reached a peak 7 days after BLM challenge. Moreover, CXCL12 protein contents remained at fourfold higher levels than untreated ones, even at 21 days after BLM treatment (Figure 9) . In contrast, both CCR5C/C and CCL3C/C mice exhibited a remarkable attenuation in intrapulmonary CXCL12 protein levels, compared with WT mice. Thus, the lack of the CCL3-CCR5 axis might impair fibrocyte recruitment directly and indirectly by reducing CXCL12 expression.

Figure 9. Intrapulmonary CXCL12 protein contents after BLM treatment. Intrapulmonary CXCL12 contents in WT, CCR5C/C, and CCL3C/C mice were determined by ELISA and are shown here. Each value represents mean ?? SEM (n = 6). **P < 0.01 versus WT mice at the same time points.

TGF-ß1 Expression after BLM Treatment

We finally examined the intrapulmonary expression of TGF-ß1, a potent stimulating factor for fibroblast proliferation and collagen production.32,33 Among untreated WT, CCR5C/C, and CCL3C/C mice, there were no significant differences in TGF-ß1 expression at mRNA and protein levels (Figure 10, A and B) . BLM treatment markedly enhanced TGF-ß1 expression at mRNA and protein levels in WT mice, whereas the enhanced TGF-ß1 expression was significantly reduced in both CCR5C/C and CCL3C/C mice, compared with WT mice (Figure 10, A and B) . Moreover, immunohistochemical and double-color immunofluorescence analyses demonstrated that F4/80- or -SMA-positive cells were cellular sources of TGF-ß1 in WT mice after BLM treatment (Figure 10, C and D) . Thus, the absence of CCL3 or CCR5 may reduce the intrapulmonary migration of fibrocytes, a precursor of fibroblasts, and F4/80-positive macrophages, cell populations that produced TGF-ß1, a factor responsible for fibrotic changes.

Figure 10. TGF-ß1 expression in lungs after BLM treatment. A: RT-PCR analysis for TGF-ß1 was performed. The ratios of TGF-ß1 to ß-actin of WT, CCR5C/C, and CCL3C/C mice were determined and are shown here. Each value represents mean ?? SEM (n = 6). *P < 0.05; **P < 0.01 versus WT mice at the same time points. B: Intrapulmonary TGF-ß1 protein contents were determined by ELISA as described in Materials and Methods. Each value represents mean ?? SEM (n = 6). **P < 0.01 versus WT mice at the same time points. C: Immunohistochemical analysis for TGF-ß protein was performed. A representative result at 14 days after BLM treatment is shown here. D: A double-color immunofluorescence analysis of TGF-ß1-expressing cells. Lungs were obtained from WT mice at 14 days after BLM treatment. The sections were stained with the combination of anti--SMA (Cy3) and anti-TGF-ß1 (FITC) and observed under fluorescent microscopy. The fluorescent images were digitally merged (right column). Representative results from six independent experiments are shown here. Original magnifications: x200 (C); x400 (D).

Some chemokines show redundancy in terms of utilization of receptors. CCL3 utilizes two distinct receptors, CCR1 and CCR5, as its specific receptors. Simultaneously, these two receptors are used by CC chemokines other than CCL3.24,25,34 In vitro studies have revealed that there are slight differences in expression patterns between CCR1 and CCR5. CCR1 is expressed by macrophages, granulocytes, NK cells, T cells, and immature dendritic cells,35-37 whereas CCR5 is expressed by T cells, B cells, immature and mature dendritic cells, and macrophages.38-41 In the present study, the deficiency of CCL3 and CCR5 but not CCR1 gene remarkably attenuated BLM-induced fibrotic changes in lungs, implying that the CCL3-CCR5 axis was more essential in the development of BLM-induced PF than the CCL3-CCR1 axis. Moreover, the observations on BM-chimeric mice have revealed the essential roles of BM-derived cells.

Our observations on CCL3C/C mice were consistent with the previous observation that the passive immunization of BLM-challenged mice with anti-CCL3 Ab attenuated markedly fibrotic changes.13 In contrast, our observations on CCR1-deficient mice are seemingly contradictory to the previous report demonstrating that anti-CCR1 Ab improved BLM-induced intrapulmonary fibrotic changes.18 In that study, a much higher dose of BLM was used and caused subacute massive lung injury, resulting in 45% mortality 14 days after BLM challenge, whereas we did not see any mortality with our present dose. Moreover, granulocyte infiltration persisted longer in their model than ours. Because granulocytes express CCR1 but not CCR5, anti-CCR1 Ab might act on the infiltrating granulocytes, thereby reducing BLM-induced PF in their model. On the other hand, because some CCR1-positive cells in lungs also expressed CCR5 simultaneously (our unpublished data), anti-CCR1 Ab treatment may impair the functions of these double-positive cells as genetic CCR5 deficiency may do, thereby reducing lung fibrotic changes.

Moore et al19 reported that CCR5C/C mice on a C57BL/6 x 129 F2 genetic background developed lung fibrosis to a similar extent as WT mice when administered with 0.025 U of BLM. However, we observed the same dose did cause little pathological changes in the lungs of WT mice on a C57BL/6 background (our unpublished data). Similar differences were also found in the overall magnitude of the fibrotic response to FITC between C57BL/6 and C57BL/6 x 129 F2 mice.9 Thus, the genetic background may account for this seeming discrepancy.

Fibrotic changes are a hallmark of the BLM-induced lung injury and are characterized histologically by fibroblast hyperplasia and increased collagen deposition. Several lines of evidence have revealed that BLM can augment the intrapulmonary expression of TGF-ß1, one of the most potent fibrogenic factors32,33 and that BLM-induced PF was ameliorated by counteracting the signal pathways of TGF-ß1.42,43 These observations imply TGF-ß1 as one of the main mediators responsible for BLM-induced PF. We also demonstrated that BLM-induced enhancement of TGF-ß1 expression was significantly attenuated in the lungs of CCR5C/C and CCL3C/C mice, compared with WT mice. We observed that CCR5-positive cells started to infiltrate to the lungs 3 days after BLM injection and that most CCR5-positive cells were F4/80-positive macrophages (our unpublished data). Given that F4/80-positive macrophages were one of the main producers of TGF-ß1, the CCL3-CCR5 axis can regulate the trafficking of macrophages, thereby contributing to development of BLM-induced PF.

In addition to F4/80-positive macrophages, -SMA-positive myofibroblasts were positive for TGF-ß1 in our model. Because TGF-ß1 can induce fibroblasts to produce collagen,32,33 TGF-ß1 may act in an autocrine manner. Several lines of evidence suggest that some but not all fibroblasts are derived from a circulating population of cells, termed fibrocytes, which express both leukocyte (CD45, CD34, CD13) and mesenchymal markers (type I collagen, fibronectin).5-12,44-46 Of interest is that fibrocytes can exhibit a chemotactic response to several chemokines including CXCL12, CCL2, and CCL21.7-9,12 Indeed, the neutralization of CXCL12 or the lack of CCR2 (a specific receptor for CCL2) reduced the recruitment of fibrocytes into the lungs and eventually reduced intrapulmonary collagen deposition after BLM or FITC challenge.8,9 These observations would indicate that chemokine-mediated trafficking of fibrocytes to the lung may essentially be involved in the pathogenesis of PF.

Two independent groups detected CCR5 mRNA and protein and CCR1 mRNA in fibrocytes,7,9 but the pathophysiological relevance has not been determined yet. Hence, we examined the fibrocyte numbers in BM, peripheral blood, and lungs among CCL3C/C, CCR1C/C, and CCR5C/C mice before and after BLM injection. BLM treatment increased fibrocyte numbers in BM to similar extents among these strains, but the increments in peripheral blood were negligible. However, the intrapulmonary fibrocyte numbers were increased to a similar extent in WT and CCR1C/C but not CCL3C/C and CCR5C/C mice. Moreover, CCL3C/C mice exhibited impaired CCR5+ fibrocyte recruitment compared with WT mice. These observations suggest the involvement of the CCL3-CCR5 axis in fibrocyte migration from BM but not fibrocyte expansion in BM. Moreover, fibrocyte numbers in lungs were correlated with intrapulmonary collagen deposition after BLM treatment, among these strains. Given that CCL3 was abundantly expressed in lungs from the early time points after BLM treatment, locally produced CCL3 may interact with CCR5 on fibrocytes to induce their migration from BM to lungs. Migrated fibrocytes may eventually differentiate to myofibroblasts, one of main sources of fibrogenic factor, TGF-ß1, thereby causing fibrotic changes.9

Intrapulmonary CXCL12 protein was also significantly reduced in CCL3C/C and CCR5C/C mice, compared with WT mice. A double-color immunofluorescence analysis detected CXC12 protein mainly in F4/80-positive macrophages (our unpublished data). Given a potent capacity of CXCL12 to induce fibrocyte migration, the absence of CCL3 and CCR5 attenuated BLM-induced intrapulmonary fibrocyte accumulation directly and/or indirectly by reducing the migration of macrophages, a main source of CXCL12.

We have demonstrated that both homozygous and heterozygous CCR5-deficient mice were less prone to develop a major adverse effect of BLM lung fibrosis. In humans, 32-bp deletion allele in ccr5 gene is prevalent among Caucasians, with a frequency of 0.092.47-49 This deletion results in a frame-shift, with a premature stop codon. Thus, homozygous persons do not express functional CCR5 protein at all, similarly to CCR5C/C mice. Both homozygous and hemizygous individuals do not exhibit any apparent health problems but are resistant to human immunodeficiency virus (HIV)-1 infection because CCR5 is a co-receptor for T-tropic HIV.50-53 Thus, it is probable that the polymorphisms of CCR5 among individuals may determine the sensitivities to BLM. If so, typing of human CCR5 polymorphisms may help to individualize BLM treatment for malignancies.

Figure 4. The numbers of granulocytes and macrophages in the lung tissue after BLM treatment. Lung tissues were obtained from WT, CCL3C/C, and CCR5C/C mice at the indicated time intervals after BLM treatment and were immunostained with anti-Ly6G and anti-F4/80 antibodies to determine the numbers of granulocytes (A) and macrophages (B), respectively. The cell numbers were counted per microscopic field at x200 magnification. Each value represents mean ?? SEM (n = 6). *P < 0.05; **P < 0.01, versus WT mice at the same time points.

We thank Drs. Philip M. Murphy and Ji-Liang Gao (National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD) for providing us with CCR1C/C mice, and Ms. Tomoko Miyashita for her excellent immunohistochemical technique.

【参考文献】
  Gross TJ, Hunninghake GW: Idiopathic pulmonary fibrosis. N Engl J Med 2001, 345:517-525

: American Thoracic Society: Idiopathic pulmonary fibrosis: diagnosis and treatment. International consensus statement. American Thoracic Society (ATS), and the European Respiratory Society (ERS). Am J Respir Crit Care Med 2000, 161:646-664

Chabner BA, Ryan DP, Paz-Ares L, Garcia-Carbonero R, Calabresi P: Antineoplastic agents. Hardman JG Limbird LE eds. The Pharmacological Basis of Therapeutics, ed 10 2003:pp 1429-1430 McGraw-Hill, New York

Bowden DH: Unraveling pulmonary fibrosis: the bleomycin model. Lab Invest 1984, 50:487-488

Chesney J, Metz C, Stavitsky AB, Bacher M, Bucala R: Regulated production of type I collagen and inflammatory cytokines by peripheral blood fibrocytes. J Immunol 1998, 160:419-425

Bucala R, Spiegel LA, Chesney J, Hogan M, Cerami A: Circulating fibrocytes define a new leukocyte subpopulation that mediates tissue repair. Mol Med 1994, 1:71-81

Abe R, Donnelly SC, Peng T, Bucala R, Metz CN: Peripheral blood fibrocytes: differentiation pathway and migration to wound sites. J Immunol 2001, 166:7556-7562

Phillips RJ, Burdick MD, Hong K, Lutz MA, Murray LA, Xue YY, Belperio JA, Keane MP, Strieter RM: Circulating fibrocytes traffic to the lungs in response to CXCL12 and mediate fibrosis. J Clin Invest 2004, 114:438-446

Moore BB, Kolodsick JE, Thannickal VJ, Cooke K, Moore TA, Hogaboam C, Wilke CA, Toews GB: CCR2-mediated recruitment of fibrocytes to the alveolar space after fibrotic injury. Am J Pathol 2005, 166:675-684

Hashimoto N, Jin H, Liu T, Chensue SW, Phan SH: Bone marrow-derived progenitor cells in pulmonary fibrosis. J Clin Invest 2004, 113:243-252

Mori L, Bellini A, Stacey MA, Schmidt M, Mattoli S: Fibrocytes contribute to the myofibroblast population in wounded skin and originate from the bone marrow. Exp Cell Res 2005, 304:81-90

Sakai N, Wada T, Yokoyama H, Lipp M, Ueha S, Matsushima K, Kaneko S: Secondary lymphoid tissue chemokine (SLC/CCL21)/CCR7 signaling regulates fibrocytes in renal fibrosis. Proc Natl Acad Sci USA 2006, 103:14098-14103

Smith RE, Strieter RM, Phan SH, Lukacs NW, Huffnagle GB, Wilke CA, Burdick MD, Lincoln P, Evanoff H, Kunkel SL: Production and function of murine macrophage inflammatory protein-1 alpha in bleomycin-induced lung injury. J Immunol 1994, 153:4704-4712

Smith RE, Strieter RM, Zhang K, Phan SH, Standiford TJ, Lukacs NW, Kunkel SL: A role for C-C chemokines in fibrotic lung disease. J Leukoc Biol 1995, 57:782-787

Smith RE, Strieter RM, Phan SH, Kunkel SL: C-C chemokines: novel mediators of the profibrotic inflammatory response to bleomycin challenge. Am J Respir Cell Mol Biol 1996, 15:693-702

Keane MP, Belperio JA, Moore TA, Moore BB, Arenberg DA, Smith RE, Burdick MD, Kunkel SL, Strieter RM: Neutralization of the CXC chemokine, macrophage inflammatory protein-2, attenuates bleomycin-induced pulmonary fibrosis. J Immunol 1999, 162:5511-5518

Keane MP, Belperio JA, Arenberg DA, Burdick MD, Xu ZJ, Xue YY, Strieter RM: IFN-gamma-inducible protein-10 attenuates bleomycin-induced pulmonary fibrosis via inhibition of angiogenesis. J Immunol 1999, 163:5686-5692

Tokuda A, Itakura M, Onai N, Kimura H, Kuriyama T, Matsushima K: Pivotal role of CCR1-positive leukocytes in bleomycin-induced lung fibrosis in mice. J Immunol 2000, 164:2745-2751

Moore BB, III, Paine R, Christensen PJ, Moore TA, Sitterding S, Ngan R, Wilke CA, Kuziel WA, Toews GB: Protection from pulmonary fibrosis in the absence of CCR2 signaling. J Immunol 2001, 167:4368-4377

Jiang D, Liang J, Hodge J, Lu B, Zhu Z, Yu S, Fan J, Gao Y, Yin Z, Homer R, Gerard C, Noble PW: Regulation of pulmonary fibrosis by chemokine receptor CXCR3. J Clin Invest 2004, 114:291-299

Wolpe SD, Davatelis G, Sherry B, Beutler B, Hesse DG, Nguyen HT, Moldawer LL, Nathan CF, Lowry SF, Cerami A: Macrophages secrete a novel heparin-binding protein with inflammatory and neutrophil chemokinetic properties. J Exp Med 1988, 167:570-581

Davatelis G, Tekamp-Olson P, Wolpe SD, Hermsen K, Luedke C, Gallegos C, Coit D, Merryweather J, Cerami A: Cloning and characterization of a cDNA for murine macrophage inflammatory protein (MIP), a novel monokine with inflammatory and chemokinetic properties. J Exp Med 1988, 167:1939-1944

Taub DD, Conlon K, Lloyd AR, Oppenheim JJ, Kelvin DJ: Preferential migration of activated CD4+ and CD8+ T cells in response to MIP-1 alpha and MIP-1 beta. Science 1993, 260:355-358

Combadiere C, Ahuja SK, Tiffany HL, Murphy PM: Cloning and functional expression of CC CKR5, a human monocyte CC chemokine receptor selective for MIP-1, MIP-1ß, and RANTES. J Leukoc Biol 1996, 60:147-152

Boring L, Gosling J, Monteclaro FS, Lusis AJ, Tsou CL, Charo IF: Molecular cloning and functional expression of murine JE (monocyte chemoattractant protein 1) and murine macrophage inflammatory protein 1alpha receptors: evidence for two closely linked C-C chemokine receptors on chromosome 9. J Biol Chem 1996, 271:7551-7558

Murai M, Yoneyama H, Harada A, Yi Z, Vestergaard C, Guo B, Suzuki K, Asakura H, Matsushima K: Active participation of CCR5(+)CD8(+) T lymphocytes in the pathogenesis of liver injury in graft-versus-host disease. J Clin Invest 1999, 104:49-57

Gao JL, Wynn TA, Chang Y, Lee EJ, Broxmeyer HE, Cooper S, Tiffany HL, Westphal H, Kwon-Chung J, Murphy PM: Impaired host defense, hematopoiesis, granulomatous inflammation and type 1-type 2 cytokine balance in mice lacking CC chemokine receptor 1. J Exp Med 1997, 185:1959-1968

Murai M, Yoneyama H, Ezaki T, Suematsu M, Terashima Y, Harada A, Hamada H, Asakura H, Ishikawa H, Matsushima K: Peyer??s patch is the essential site in initiating murine acute and lethal graft-versus-host reaction. Nat Immunol 2003, 4:154-160

Ishida Y, Kondo T, Takayasu T, Iwakura Y, Mukaida N: The essential involvement of cross-talk between IFN- and TGF-ß in the skin wound-healing process. J Immunol 2004, 172:1848-1855

Ishida Y, Maegawa T, Kondo T, Kimura A, Iwakura Y, Nakamura S, Mukaida N: Essential involvement of IFN- in Clostridium difficile toxin A-induced enteritis. J Immunol 2004, 172:3018-3025

Kimura A, Ishida Y, Hayashi T, Wada T, Yokoyama H, Sugaya T, Mukaida N, Kondo T: Interferon- plays protective roles in sodium arsenite-induced renal injury by up-regulating intrarenal multidrug resistance-associated protein 1 expression. Am J Pathol 2006, 169:1118-1128

Giri SN, Hyde DM, Hollinger MA: Effect of antibody to transforming growth factor beta on bleomycin induced accumulation of lung collagen in mice. Thorax 1993, 48:959-966

Coker RK, Laurent GJ, Shahzeidi S, Lympany PA, du Bois RM, Jeffery PK, McAnulty RJ: Transforming growth factors-beta 1, -beta 2, and -beta 3 stimulate fibroblast procollagen production in vitro but are differentially expressed during bleomycin-induced lung fibrosis. Am J Pathol 1997, 150:981-991

Raport CJ, Gosling J, Schweickart VL, Gray PW, Charo IF: Molecular cloning and functional characterization of a novel human CC chemokine receptor (CCR5) for RANTES, MIP-1ß, and MIP-1. J Biol Chem 1996, 271:17161-17166

Ogata M, Zhang Y, Wang Y, Itakura M, Zhang YY, Harada A, Hashimoto S, Matsushima K: Chemotactic response toward chemokines and its regulation by transforming growth factor-beta1 of murine bone marrow hematopoietic progenitor cell-derived different subset of dendritic cells. Blood 1999, 93:3225-3232

Sallusto F, Lanzavecchia A: Mobilizing dendritic cells for tolerance, priming, and chronic inflammation. J Exp Med 1999, 189:611-614

Su SB, Mukaida N, Wang J, Nomura H, Matsushima K: Preparation of specific polyclonal antibodies to a C-C chemokine receptor, CCR1, and determination of CCR1 expression on various types of leukocytes. J Leukoc Biol 1996, 60:658-666

Qin S, Rottman JB, Myers P, Kassam N, Weinblatt M, Loetscher M, Koch AE, Moser B, Mackay CR: The chemokine receptors CXCR3 and CCR5 mark subsets of T cells associated with certain inflammatory reactions. J Clin Invest 1998, 101:746-754

Loetscher P, Uguccioni M, Bordoli L, Baggiolini M, Moser B, Chizzolini C, Dayer JM: CCR5 is characteristic of Th1 lymphocytes. Nature 1998, 391:344-345

Bonecchi R, Bianchi G, Bordignon PP, D??Ambrosio D, Lang R, Borsatti A, Sozzani S, Allavena P, Gray PA, Mantovani A, Sinigaglia F: Differential expression of chemokine receptors and chemotactic responsiveness of type 1 T helper cells (Th1s) and Th2s. J Exp Med 1998, 187:129-134

Rottman JB, Ganley KP, Williams K, Wu L, Mackay CR, Ringler DJ: Cellular localization of the chemokine receptor CCR5. Correlation to cellular targets of HIV-1 infection. Am J Pathol 1997, 151:1341-1351

Zhao J, Shi W, Wang YL, Chen H, Bringas P, Jr, Datto MB, Frederick JP, Wang XF, Warburton D: Smad3 deficiency attenuates bleomycin-induced pulmonary fibrosis in mice. Am J Physiol 2002, 282:L585-L593

Nakao A, Fujii M, Matsumura R, Kumano K, Saito Y, Miyazono K, Iwamoto I: Transient gene transfer and expression of Smad7 prevents bleomycin-induced lung fibrosis in mice. J Clin Invest 1999, 104:5-11

Schmidt M, Sun G, Stacey MA, Mori L, Mattoli S: Identification of circulating fibrocytes as precursors of bronchial myofibroblasts in asthma. J Immunol 2003, 171:380-389

Aiba S, Tagami H: Inverse correlation between CD34 expression and proline-4-hydroxylase immunoreactivity on spindle cells noted in hypertrophic scars and keloids. J Cutan Pathol 1997, 24:65-69

Yang L, Scott PG, Dodd C, Medina A, Jiao H, Shankowsky HA, Ghahary A, Tredget EE: Identification of fibrocytes in postburn hypertrophic scar. Wound Repair Regen 2005, 13:398-404

Bamshad MJ, Mummidi S, Gonzalez E, Ahuja SS, Dunn DM, Watkins WS, Wooding S, Stone AC, Jorde LB, Weiss RB, Ahuja SK: A strong signature of balancing selection in the 5' cis-regulatory region of CCR5. Proc Natl Acad Sci USA 2002, 99:10539-10544

Blanpain C, Lee B, Tackoen M, Puffer B, Boom A, Libert F, Sharron M, Wittamer V, Vassart G, Doms RW, Parmentier M: Multiple nonfunctional alleles of CCR5 are frequent in various human populations. Blood 2000, 96:1638-1645

Mummidi S, Bamshad M, Ahuja SS, Gonzalez E, Feuillet PM, Begum K, Galvis MC, Kostecki V, Valente AJ, Murthy KK, Haro L, Dolan MJ, Allan JS, Ahuja SK: Evolution of human and non-human primate CC chemokine receptor 5 gene and mRNA. Potential roles for haplotype and mRNA diversity, differential haplotype-specific transcriptional activity, and altered transcription factor binding to polymorphic nucleotides in the pathogenesis of HIV-1 and simian immunodeficiency virus. J Biol Chem 2000, 275:18946-18961

Dean M, Carrington M, Winkler C, Huttley GA, Smith MW, Allikmets R, Goedert JJ, Buchbinder SP, Vittinghoff E, Gomperts E, Donfield S, Vlahov D, Kaslow R, Saah A, Rinaldo C, Detels R, O??Brien SJ: Genetic restriction of HIV-1 infection and progression to AIDS by a deletion allele of the CKR5 structural gene. Science 1996, 273:1856-1862

Zimmerman PA, Buckler-White A, Alkhatib G, Spalding T, Kubofcik J, Combadiere C, Weissman D, Cohen O, Rubbert A, Lam G, Vaccarezza M, Kennedy PE, Kumaraswami V, Giorgi JV, Detels R, Hunter J, Chopek M, Berger EA, Fauci AS, Nutman TB, Murphy PM: Inherited resistance to HIV-1 conferred by an inactivating mutation in CC chemokine receptor 5: studies in populations with contrasting clinical phenotypes, defined racial background, and quantified risk. Mol Med 1997, 3:23-36

Samson M, Libert F, Doranz BJ, Rucker J, Liesnard C, Farber CM, Saragosti S, Lapoumeroulie C, Cognaux J, Forceille C, Muyldermans G, Verhofstede C, Burtonboy G, Georges M, Imai T, Rana S, Yi Y, Smyth RJ, Collman RG, Doms RW, Vassart G, Parmentier M: Resistance to HIV-1 infection in Caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene. Nature 1996, 382:722-725

Liu R, Paxton WA, Choe S, Ceradini D, Martin SR, Horuk R, MacDonald ME, Stuhlmann H, Koup RA, Landau NR: Homozygous defect in HIV-1 coreceptor accounts for resistance of some multiply exposed individuals to HIV-1 infection. Cell 1996, 86:367-377

作者单位：From the Department of Forensic Medicine,* Wakayama Medical University, Wakayama; the Divisions of Molecular Bioregulation and Stem Cell Biology, Kanazawa University, Cancer Research Institute, Kanazawa; and the Department of Molecular Preventive Medicine, Graduate School of Medicine, The University

　克罗米酚与HCG促排卵作用的临床观察 (pdf)　

    克罗米酚（clomiphene citrate，CC）是具有弱雌激素和抗雌激素双重作用的三苯乙烯消旋混合物，由于CC对内源性排卵机制的调节作用更符合生理状态，故被广泛用于调节月经、诱导排卵治疗不孕症，已成为临床上首选的促排卵药物。又因其价格便宜，来源容易，使用方便、安全。由于大多数单纯使用CC的患者只在家中服药，对这一人群缺少详细的临床资料。为此，我们对这一人群详细观察了排卵率、子宫内膜厚度及回声类型、监测雌孕激素水平的变化，有望对临床工作给予帮助。

    1  资料与方法

    1.1  一般资料  2002年12月~2006年7月于我院就诊的生育年龄妇女，有正常月经，6个月内未使用过激素类药物，B超及盆腔检查无异常发现。将观察对象95例随机分为3组：A组39例为对照组，B组30例为单独服用CC组，C组26例为CC/HCG组。

    1.2  方法  （1）用药方法：B组于月经周期第5~9天口服CC 50 mg/d；C组于月经周期第5~9天口服CC 50mg/d，B超监测优势卵泡≥18 mm时，肌肉注射HCG 5000 IU。（2）B超监测排卵：从自然周期第10天左右或服CC后，卵泡直径≥10 mm开始每日或隔日监测卵泡发育及排卵。

    1.3  排卵指征  优势卵泡消失或缩小、形态改变、壁皱缩或卵泡内出现不规则回声，有时伴有后陷凹积液。

    1.4  子宫内膜回声分型  A型：三线征，子宫内膜呈现3条线性强回声。B型：子宫内膜回声与肌壁回声相似，中线回声可见到但并不强。C型：子宫内膜回声强于肌壁回声。

    A组黄素化不破裂卵泡综合征2例（5.13%），B组LUFS 11例（36.67%），两组比较P<0.001。C组中LUFS 5例（19.23%），与B组比较P>0.05。将应用CC的患者（B组与C组合并）56例按有无LUFS分为2组，将LUFS组（n=40）：LH+0 d卵泡最大直径（21.15±2.76） mm，内膜厚度（8.80±2.08）mm；有LUFS组（n=16）：卵泡最大直径（22.34±LH+0d）mm。

    2  结果

    2.1  卵泡与子宫内膜发育  阴道B超监测卵泡大小、内膜厚度及内膜回声类型。LH+0 d A组卵泡最大直径（18.30±1.47）mm，B组（21.73±3.09）mm，两组比较P=0.0001。LH+0d A组A型内膜33例（84.62%）、B型内膜6例（15.38%）、C型内膜0例；B组A型内膜21例（70.00%）、B型内膜4例（13.33%）、C型内膜5例（16.67%），两组比较：P<0.05。内膜厚度（8.56±2.591）mm，差异均无显著性（P>0.05）。

    2.2  血清E2、P水平  （1）CC对血清E2、P水平的影响，排除3组中LUFS 18例进行比较，应用CC后排卵前及种植窗期血清E2、P水平均高于对照组，差异有极显著性。种植窗期P/E2对照组为128∶1，CC组为99∶1，两组比较，P<0.05。（2）将应用CC的患者56例按有无LUFS分为2组，比较血清E2、P水平。

    3  讨论

    B超监测下CC对排卵前子宫内膜的影响：文献报道排卵前子宫内膜厚度<6 mm时妊娠不易发生；内膜厚度6~9 mm时，妊娠流产率为40%；而当内膜厚度≥9 mm时，妊娠流产率只有6.3%。Check等对促排卵治疗的妇女研究发现，94%的妊娠者排卵前子宫内膜厚度≥10 mm，且回声类型为A、B型，而C型内膜无一例妊娠。多数学者认为A型内膜且厚度≥9 mm为适当型，此时种植有较高的妊娠率。但内膜过厚也对种植造成不利影响，Weissman等发现注射HCG当日内膜厚度>14 mm着床率和妊娠率均明显下降，流产率增加。应用CC是否会对内膜厚度和回声类型造成影响？Dickey等提出，子宫内膜厚度与CC的使用剂量呈负相关。我们的结果显示，对照组子宫内膜厚度集中于9~14 mm，主要内膜类型为A型。应用CC后子宫内膜厚度变薄，A型内膜比例减少，C型内膜比例增加，似乎不利于种植的发生。目前的文献报道多关注于子宫内膜回声类型与妊娠结局的关系，而不同回声类型的形成机制尚不清楚。有学者认为，应用CC后，子宫内膜腺体结构、细胞结构、腺上皮纤毛数量均受到影响，内膜变薄及回声类型的改变是否是上述变化的直观反映，值得临床进一步探讨。

   作者单位： 266753 山东平度，平度市第三人民医院

　 （编辑：乔  雨）