主题：Dendritic

¹ Department of Epidemiology and Public Health, Yale University School of Medicine, New Haven, CT 06520
² Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115

¹Graduate School of Public Health and ²School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania; ³Immunex Corporation, Seattle, Washington; ⁴University of Oklahoma Health Sciences Center, Oklahoma City

Received 2 August 2002; revised 3 October 2002; electronically published 19 December 2002.

      Presented in part: Dendritic Cells at the Host-Pathogen Interface Conference, sponsored by the National Institute of Allergy and Infectious Diseases, Warrenton, Virginia, 7 May 2002.
     Informed consent was obtained from the study subjects, and this study was done in accordance with experimentation guidelines of the US Department of Health and Human Services and the Institutional Review Board of the University of Pittsburgh.
     Potential conflict of interest: E.K.T. is an employee of Immunex Corporation, who supplied the CD40 ligand for this study.
     Financial support: National Institutes of Health (grants R01 AI41870, U01 AI37984, U01 AI35041, R01 CA24553, and T32 AI 07487).
      Present affiliation: Department of Immunology, Section of Immunobiology, Yale University School of Medicine, New Haven, Connecticut.
     Reprints or correspondence: Dr. Charles R. Rinaldo, Jr., A427 Crabtree Hall, Graduate School of Public Health, 130 DeSoto St., University of Pittsburgh, Pittsburgh, PA 15261 ().

     Generation of antigen-specific, primary CD8⁺ cytotoxic T lymphocyte (CTL) responses represents a challenge to the development of protein-based vaccines for prevention of human immunodeficiency virus type 1 (HIV-1) infection. The conventional mechanism for antigenic processing of extracellular proteins is through an endocytic pathway for presentation by major histocompatibility complex (MHC) class II molecules to CD4⁺ T cells [1]. One potential strategy to induce vigorous MHC class Irestricted CTL responses with viral proteins involves specific targeting to the cytosol of antigen-presenting cells (APCs), particularly dendritic cells (DCs), with carriers such as liposome for accessing alternative MHC class I antigen-processing pathways (termed "cross-presentation") [2]. We have previously demonstrated that lipofectin, a cationic liposome, provides a means of delivering exogenous proteins in vitro to human DCs for more efficient induction of HIV-1 specific, memory CD8⁺ CTL responses than protein alone [3]. The present study investigated whether DCs loaded with protein-liposome complexes can prime antiHIV-1 CD8⁺ T cell responses.

     Materials and methods.     To obtain immature DCs (iDCs), CD14⁺ monocytes were positive selected from peripheral blood mononuclear cells (PBMC) of healthy donors using anti-CD14 monoclonal antibody (MAb)coated magnetic microbeads (Miltenyi) and cultured in AIM V medium (GIBCO) containing 1000 U/mL of recombinant interleukin (IL)4 and 1000 U/mL recombinant granulocyte-monocyte colony stimulating factor (GM-CSF; Schering-Plough) [4]. The number of viable DCs was determined by typical DC morphology in trypan blue dyestained preparations. The purity of the DCs was 90% on the basis of the expression of HLA-DR molecules and lack of expression of lineage markers by flow cytometry [4]. The maturation state of the DCs was determined by expression of CD80, CD83, and CD86.

     The toxicity of lipofectin, composed of DOTMA (N-[1-{2,3-dioleyloxy} propyl]N, N, N-trimethylammonium chloride)/DOPE (dioleoyl-phosphotidylethanolamine) (GIBCO), was determined by loading iDCs with graded doses at 37°C for 0, 2, 4, and 8 h. The viability of the DCs by trypan blue dye exclusion was >90% when lipofectin was added at 15 g/mL for up to 8 h, and decreased at higher concentrations of liposome. Therefore, 15 g/mL lipofectin was used in this study.

     The vaccinia virus (VV) constructs contained gag p24 or p55 for BH10 and HxB2 strains of HIV-1 (VV-Gag p24 and VV-Gag p55; Therion Biologics). VV-Env was vPE11 containing the env coding region of HIV-1 BH10 minus the signal sequence (B. Moss, National Institutes of Health [NIH], Bethesda, MD). The NYCBH strain of VV (VV-Vac; Therion) was used as control virus. Synthetic peptides representing previously identified HLA class Irestricted Gag and Env epitopes were prepared by the University of Pittsburgh Peptide Synthesis Facility.

     For preparation of APCs, 15 g/mL lipofectin was mixed with 20 g/mL HIV-1 p24 Gag or gp160 Env (IIIB; Protein Sciences) or p55 Gag (SF2; Chiron; AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, NIH) and incubated for 10 min at room temperature [3]. The lipofectin-protein mixture was added to 1 × 10⁶ iDCs on day 7. The cells were incubated for 4 h at 37°C in 5% CO₂ and treated with human CD40L trimer (12.5 g/mL; Immunex) for 2 days to induce phenotypic and functional maturation (defined as mature DCs [mDCs]). Control samples were APCs only and APCs loaded with either liposome or HIV-1 protein. In some experiments, the iDCs were obtained from plastic adherent PBMC and matured with monocyte-conditioned medium (MCM) [4].

     After incubation, the cells were washed and left untreated, or treated with psoralen (10 g/mL; Calbiochem) and UV light (PUV) [5]. Cryopreserved PBMC were primed with antigen-loaded, autologous mDCs for 1 week and then stimulated with antigen-loaded, autologous PBMC as APCs for weeks 25 at an effector cell-to-APC ratio of 10 : 1 in RPMI 1640 medium containing antibiotics, 15% heat inactivated fetal calf serum, and recombinant human interleukin (IL)2 (100 U/mL; Chiron). The effector cell-APC mixtures were seeded into round-bottom microtiter wells, with fresh medium and IL-2 added every 34 days.

     Antigen stimulated cells were harvested and washed after 4 or 5 weeks when there were adequate T cells for CTL assay. In some experiments, CD8⁺ and CD4⁺ T cells were enriched to 98% and 90% purity, respectively, from cultured cells by negative selection using immunomagnetic beads (Dynal) [5]. The targets were autologous B-LCLs infected with the VV vectors or loaded with synthetic HIV-1 peptides (100 g/mL) and labeled with ⁵¹Cr [4]. PBMC were typed for HLA class I by high resolution, reference strand mediated conformation analysis. To block CTL reactivity, target cells were incubated with anti-HLA class I (5 g/mL; Accurate Chemical) or anti-HLA class II MAbs (5 g/mL; Beckman Coulter) for 45 min at 37°C before adding to the effector cells. The CTL lysis assay was done in triplicate at effector/target (E/T) cell ratios of 40, 20, and 10. Specific lysis was calculated as 100 × [(experimental copies per minute {cpm} - spontaneous cpm)/(maximum cpm - spontaneous cpm)]. The percentage of antigen-specific lysis was calculated as (percentage lysis of VV-Gag or VV-Env infected or HIV-1 peptide loaded targets) - (percentage lysis of VV-vac infected or mock treated targets). The mean (±SE) lysis of the uninfected and VV-vac infected targets by the antigen-stimulated effectors at the various E/T ratios was 8% ± 3% (n = 18) and 7% ± 1% (n = 27), respectively. The coefficient of variation for cpm among replicate CTL values was 7% ± 1%. A positive CTL response was defined as 10% antigen-specific lysis.

     Results.     Expression of costimulatory molecules CD80 and CD86, HLA-DR, and the Ig superfamily, DC surface maturation marker, CD83, was up-regulated on CD40L-treated DCs as compared to non-CD40L-treated DCs, indicating that the CD40L treatment induced DC maturation [4, 5]. Loading of iDCs or mDCs with either liposome, p24 Gag, or their combination did not alter expression of these surface molecules (data not shown).

     We next established the parameters for in vitro priming by confirming the work of Zarling et al. [6], which used 1 week of priming of PBMC with HIV-1 Gag peptide-loaded, MCM-matured DCs, followed by weekly, sequential stimulations with Gag peptide-loaded PBMC to induce anti-Gag CD8⁺ CTL responses (data not shown). Priming with peptide-loaded iDCs did not induce CTL reactivity. In subsequent experiments, we used CD40L-matured DCs, because this treatment resulted in a more consistent maturation of the DCs [4].

     Stimulation of PBMC from an HIV-1negative subject with autologous mDCs that had been loaded with HIV-1 Gag p55 protein-liposome induced greater levels of antiHIV-1 CD8⁺ T cell responses than mDCs loaded with HIV-1 Gag p55 protein alone (). Similar priming was elicited by PUV-treated mDCs that had been loaded with Gag p55-liposome (). The lytic reactivity was HLA class I restricted as shown by blocking of lysis by antiHLA class I but not antiHLA class II MAbs (). To date, we have observed that CTLs specific for Gag or Env can be consistently primed by autologous mDCs loaded with these HIV-1 recombinant protein-liposome complexes in multiple PBMC samples obtained from 7 different subjects (data not shown).

fig.ommitted

Figure 1.        Priming of human immunodeficiency virus type 1 (HIV-1)specific CD8⁺ cytotoxic T lymphocyte (CTL) responses by different antigen-presenting cells (APCs). Priming of HIV-1specific CTL in peripheral blood mononuclear cells (PBMC) from a HLA A*0201, B*27 subject by 1 week of stimulation with autologous mature dendritic cells (mDCs; either untreated or psoralen UV [PUV] treated), monocytes, or PBMC loaded with Gag p55-liposome, followed by 4 sequential weekly stimulations with autologous PBMC loaded with the same antigen (A). Results are the mean of triplicate values of antigen-specific lysis. The results show positive CTL responses (10% specific lysis) to autologous B cell lines loaded with known CD8⁺ T cell epitopes for HLA A*0201 p24₁₅₁₁₅₉ (TLNAWVKVV), and B*27 p24₂₆₃₂₇₂ (KRWIILGLNK) and p17₁₉₂₇ (IRLRPGGKK), but not for HLA A*0201 p17₇₇₈₅ (SLYNTVATL). The CTL activity was blocked by anti-HLA class I monoclonal antibody (MAb) but not anti-HLA class II MAb (B). E/T, effector/target cell ratio.

     The HLA class I specificity and breadth of this primary CD8⁺ T cell response was further demonstrated by killing of autologous targets expressing 3 of 4 HLA A*0201 and HLA B*27 HIV-1 peptides (). We confirmed the specificity of CD8⁺ T cells primed by mDC loaded with either Gag p24 or p55 and liposome for p24_151-159 (TLNAWVKVV) in 5 of 6 HLA A*0201 subjects. In further experiments, priming of CTLs from another HLA A*0201 person with mDCs loaded with gp160-liposome induced CTLs specific for HLA A*0201 epitopes gp120₃₁₁₃₂₀ (RGPGRAFVTI) and gp41₇₄₇₇₅₅ (RLVNGSLAL), but not gp120₁₉₂₁₉₉ (KLTSCNTSN) or gp41_818-827 (SLLNATDIAV) (data not shown). Similarly, CTLs from an HLA A*03 B*07 person that were primed by mDCs loaded with Gag p24- and Env gp160-liposome complexes were specific for HLA B*07 p24₁₇₉₁₈₇ (ATPQDLNTM) and HLA A*03 gp41₇₇₅₇₈₅ (RLRDLLLIVTR) epitopes, but not for the HLA B*07 p24₁₄₈₁₅₆ (SPRTNAWV) epitope (data not shown).

     We showed that Gag p55-liposome loaded mDCs more efficiently primed HIV-1specific CD8⁺ CTLs than did monocytes or PBMC for recognition of targets expressing 4 different Gag epitopes (). Furthermore, mDCs loaded with Gag p55-liposome were superior to iDCs for priming of CTL specific for targets expressing naturally processed Gag proteins (i.e., VV-p24 and VV-p55), as well as targets loaded with a known HLA A*0201 p24 epitope (p24_151-159) ().

fig.ommitted

Figure 2.        Priming of human immunodeficiency virus (HIV)-1specific cytotoxic T lymphocyte (CTL) responses by immature dendritic cells (iDCs) and mature (m) DCs loaded with HIV-1 protein-liposome (lipo) complexes. Peripheral blood mononuclear cells (PBMC) from an HLA A*0201 subject were primed for HIV-1specific CTL by 1 week of stimulation with autologous iDCs and mDCs loaded with Gag p24-lipo complexes, followed by 4 sequential weekly stimulations with autologous PBMC loaded with the same antigen. Targets were autologous B cell ines infected with vaccinia virus expressing Gag p55 or p24, or loaded with an HIV-1 peptide (p24₁₅₁₁₅₉ [TLNAWVKVV]) representing a known CTL epitope for HLA A*0201. Results are the mean of triplicate values of antigen-specific lysis. E/T, effector/target cell ratio.

     Discussion.     We were consistently able to prime antiHIV-1 CD8⁺ T cell responses with mDCs loaded with HIV-1 protein-liposome complexes but not with mDCs loaded with HIV-1 proteins alone. In vitro priming of CD8⁺ T cells to HIV-1 was first demonstrated by Engleman et al. [7, 8] who showed that multiple, sequential stimulations of naive CD8⁺ T cells with autologous or allogeneic, HLA class Imatched DCs freshly isolated from blood samples and loaded with HIV-1 Gag and Env peptides, could activate HIV-1specific CTL. More recently, monocyte-derived iDCs transduced with an env- and nef-deleted HIV-1 vector pseudotyped by VSV G protein [9], HIV gag-encoded mRNA-transfected DCs [10], or iDCs that were matured with MCM or CD40L and loaded with HIV-1 peptides representing HLA class I epitopes [6, 11], have been used to prime antiHIV-1 CTL in vitro. There is only one report of in vitro priming of CD8⁺ T cells with whole HIV-1 protein, where Nef-specific CD8⁺ T cells were primed by repetitive stimulation of PBMC with -irradiated iDCs loaded with HIV-1 Nef protein [12]. In this case, optimal T cell priming required addition of interferon (IFN).

     We found that priming of antiHIV-1 CD8⁺ T cell responses in vitro required liposome as a delivery vehicle. We believe that this was due, in part, to the unique fusogenic property of the DOPE portion of lipofectin that enhances access of exogenous protein from the endosomes/lysosomes into the cytosol of DCs by destabilizing the endosomes and allowing the release of HIV-1 protein into the cytosol [13]. Moreover, a single stimulation of PBMC for one week with HIV-1 protein-liposome loaded mDCs, followed by multiple, weekly stimulations with protein-liposome loaded PBMC, was sufficient to induce a primary, antiHIV-1 CD8⁺ T cell response. Initial stimulation with antigen-loaded mDCs was essential for priming, as iDCs, monocytes or PBMC loaded with liposome-complexed HIV-1 protein were poor primers of HIV-1 specific T cells. The mDCs expressed enhanced levels of costimulatory and HLA class II molecules, and these were not altered by addition of liposome-protein complexes. Maturation of DCs with CD40L increases surface expression of HLA and costimulatory molecules and release of cytokines including IL-12 p70 (when combined with IFN- stimulation) and IL-15 from mDCs that enhance antigen-specific CD8⁺ T cell responses [14]. Such costimulatory CD40/CD40L interactions lower the threshold of antigen required to activate primary T cell responses, and may augment survival of antigen-activated T cells.

     Our data, that PUV-treated mDCs could prime antiHIV-1 CD8⁺ T cell responses, are concordant with our previous findings among HIV-1infected persons [4]. The PUV-treated DCs are likely undergoing apoptosis, which suggest that viable DCs are not essential for triggering of T cell priming. This may relate to the high levels of costimulatory molecule expression that is retained on PUV-treated DCs (X.-L.H., Z.F., and C.R.R., unpublished data).

     Priming of CD8⁺ T cells with mDCs loaded with HIV-1 proteins and liposome elicited CTL reactivity against multiple HIV-1 HLA class I epitopes. The levels of lysis were within the range expected for CTL primed by a multi-epitope protein immunogen, using single epitope, individual peptides as targets. Lack of recognition of some HIV-1 peptides by the HIV-1 protein-primed T cells could reflect immunodominance among the peptides for their HLA class I alleles, differences in proteasomal cleavage and heterogeneity of peptide binding due to polymorphism of HLA class I alleles [2].

     We reported elsewhere activation of HIV-1 specific, CD8⁺ T cells from HIV-1infected persons in vitro by cross-presentation of liposome-complexed HIV-1 proteins by DCs [3]. These studies suggest that complexing of HIV-1 protein with liposome promotes processing of the protein through an alternative, cytosolic pathway for cross-presentation of peptides by HLA class I molecules to both naive and memory CD8⁺ T cells. Our studies, together with a recent report showing in vitro priming of Gag-specific CD4⁺ T cells by mDCs loaded with uncomplexed HIV-1 p24 protein [15], suggest that a protein-based antigen approach could be used to prime or boost both antiHIV-1 CD8⁺ and CD4⁺ T cell activity in prophylactic and therapeutic vaccine protocols.

Acknowledgments

     We thank K. Picha (Immunex; Seattle, WA) for assistance in providing the CD40L, S. Narula (Schering-Plough; Kenilworth, NJ) for providing hIL-4 and hGM-CSF, and J. Malenka for administrative assistance.

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Yewdell JW, Norbury CC, Bennink JR. Mechanisms of exogenous antigen presentation by MHC class I molecules in vitro and in vivo: implications for generating CD8⁺ T cell responses to infectious agents, tumors, transplants, and vaccines. Adv Immunol 1999; 73:177.

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Schlienger K, Craighead N, Lee KP, Levine BL, June CH. Efficient priming of protein antigen-specific human CD4⁺ T cells by monocyte-derived dendritic cells. Blood 2000; 96:34908.

    Departments of Microbiology, Medicine, Biostatistics
    Computer Science and Engineering, University of Minnesota, Minneapolis, AIDS Vaccine Program, SAIC Frederick, Inc., National Cancer Institute, Frederick, Maryland

    Osteopontin is a multifunctional protein with known roles in bone remodeling, wound healing, and normal and pathological immune responses. We showed in microarray studies that osteopontin gene expression is increased in human immunodeficiency virus type 1 (HIV-1)infected lymphatic tissues after treatment, and we undertook mapping experiments to study osteopontin's possible functions in this context. We discovered species-specific colocalization of osteopontin with the follicular dendritic cell (FDC) network in lymphatic tissues in HIV-1 and simian immunodeficiency virus infections, and we found that changes in FDC-associated osteopontin covary with changes in lymphoid follicles during acute and late stages of infection and in response to treatment. We propose that this localization normally facilitates antibody production and plays a role in B cell abnormalities in infection and in the reconstitution of lymphoid follicles with treatment and that mapping genes identified in microarray studies is a useful experimental approach to gaining a better understanding of function in the context of a particular tissue and disease.

    Osteopontin is a secreted, phosphorylated, acidic glycoprotein that was first identified in studies of bone repair but was subsequently shown to have multiple functions in the repair of other tissues, immune responses, and inflammation [1, 2]. In mineralized tissues, osteopontin binds to the extracellular matrix through interactions between its asparagine-glycine-aspartate sequence and integrins, and it regulates calcium deposition during the stress-induced remodeling of bone. In other tissues, osteopontin promotes wound healing and inhibits ectopic calcification [35]. Osteopontin also functions in the induction of cellular and humoral immune responses [68] and participates in both physiological inflammation during wound healing and immune responses and pathological inflammatory conditions that include arthritides [9, 10], multiple sclerosis [11], and autoimmune diseases [8, 12, 13].

    We recently found that osteopontin gene expression increased in HIV-1infected lymphatic tissues after the institution of highly active antiretroviral therapy (HAART) [14]. We provisionally interpreted this result as possible evidence of the wound-healing functions of osteopontin (described above) in a previously unexamined tissue, but we sought further insight into osteopontin's functions in the reparative response to HAART in lymphatic tissues by mapping the localization of osteopontin. During the course of these studies, we discovered a previously unrecognized association of osteopontin with the follicular dendritic-cell (FDC) network of humans and primates but not that of mice, and we propose that this species-specific association reflects the evolution of osteopontin to facilitate antibody production. In support of this hypothesis, we document (1) changes in FDC-associated osteopontin that parallel the well-known hypergammaglobulinemia and B cell abnormalities observed during the early stages of HIV-1 and simian immunodeficiency virus (SIV) infections, (2) decreases in FDC-associated osteopontin that parallel the destruction of lymphoid follicles during late stages of infection, and (3) during HAART, a coordinate normalization of follicular architecture and FDC-associated osteopontin.

    MATERIAL AND METHODS

    Human lymph-node biopsies.

    After we obtained signed, informed consent, inguinal lymph-node biopsies were performed, by use of standard surgical techniques, on individuals under local anesthesia who were participating in University of Minnesota institutional review boardapproved protocols [15]. A portion of the tissue was placed in 4% paraformaldehyde for 34 h and was then transferred to 70% ethanol and embedded in paraffin for in situ hybridization, immunohistochemical staining, and histological examination.

    Lymph-node biopsies from SIV-infected rhesus macaques.

    Axillary lymph-node biopsies were performed on adult female SIV- and simian retrovirusnegative rhesus macaques under anesthesia before and 1 and 4 weeks after intravenous (iv) inoculation with 2 × 104 ID50 of SIVmac239 (provided by R. Desrosiers, New England National Primate Research Center, Southborough, MA). All animal housing, care, and research were performed in accordance with the Guide for the Care and Use of Laboratory Animals [16] and with protocols approved by the Institutional Animal Care and Use Committee of the National Cancer Institute.

    Immunohistochemical staining.

    For immunohistochemical staining, after blocking to reduce nonspecific binding, sections were reacted sequentially with primary antibody, biotinylated secondary antibody, and ABC reagent, and then were stained with diaminobenzidine and counterstained with hematoxylin-eosin. For osteopontin staining, we used monoclonal antibodies to human osteopontin peptides at the C terminus 3 aa from the thrombin cleavage site (Immuno-Biological Laboratories), goat polyclonal antibodies to full-length recombinant mouse osteopontin (AF808; R&D Systems) and proteolytically derived N- and C-terminal fragments (provided by L. Fisher, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD) [17, 18], rabbit polyclonal antibodies to full-length and N- and C-terminal fragments of recombinant mouse osteopontin (provided by L. Liaw, Maine Medical Center Research Institute, Scarborough, ME), and rabbit polyclonal antibodies that recognize full-length and C-terminal fragments of osteopontin and cross-react with human, pig, and dog osteopontin (abcam 8448; Novus Biologicals).

    In situ hybridization.

    Tissue sections of 8 m were cut and adhered to slides, deparaffinized, and subsequently pretreated for the detection of HIV RNA by in situ hybridization, as described elsewhere [19]. In brief, sections were pretreated with HCl, digitonin, and proteinase K, to enhance the diffusion of probes, and then acetylated, to reduce the nonspecific binding of probes. After the hybridization of a collection of 35S-labeled HIV-1specific riboprobes, sections were washed, treated with ribonuclease, dehydrated, coated with NTB2 (Kodak), and developed and stained after radioautographic exposure. For osteopontin, cDNA from the American Type Culture Collection (ATCC number 61052) was subcloned into the pBluescript II SK(-) plasmid, and an 35S-labeled riboprobe was prepared by incorporation of the label with T7 RNA polymerase.

    Statistical analysis.

    A random-effects model was used to determine the statistical significance of changes in the percentage of FDC-associated osteopontin/lymphatic tissue area attributable to HAART. This analysis was necessary for combining information from 2 small, slightly different studies. The model assumed that the changes in each individual were normally distributed, given some study-specific mean effect, and that these study-specific mean effects were drawn from another normal distribution. The mean of the latter distribution was the estimate of the effect due to HAART. A Bayesian approach was used, and computation was done by first integrating the variance parameters out of the posterior distribution, then directly sampling the other 3 parameters from a histogram approximation of their posterior distribution. Calculations were conducted by use of S-plus software (version 3.4; MathSoft).

    RESULTS

    FDC-associated osteopontin in HIV-1infected and uninfected lymphatic tissues as revealed by mapping studies.

    With the rationale that knowing where osteopontin was expressed in lymphatic tissues would provide additional clues to its function, we determined sites of osteopontin expression by immunohistochemical staining. We had expected from the results of other studies [1, 20] that we would detect osteopontin in cells, but we had not seen a report of an association of osteopontin with lymphoid follicles. We were thus surprised to find that (1) before treatment, during the late stage of HIV-1 infection, osteopontin colocalized with the FDC network in small residual secondary follicles; and (2) 2 months after the initiation of HAART, there was increased osteopontin staining in a pattern characteristic of the FDC network in the large follicles that had reformed by that time (figure 1A and 1B). We confirmed the colocalization of osteopontin protein with the FDC network by staining for the FDC marker CD35 and by revealing, through in situ hybridization, the virions bound to the FDC network [19] before treatment (figure 1C and 1D). We then investigated and documented osteopontin colocalization with the FDC network in follicles in other HIV-1infected and uninfected individuals (figure 1E).

    Binding but not production of osteopontin by FDCs.

    Although the FDC network clearly binds osteopontin, we did not detect osteopontin mRNA in FDCs by in situ hybridization, although we did detect osteopontin mRNA (figure 2A) in cells in the paracortical T cell zone identified by double-label staining as T cells and macrophages (figure 2B and 2C). We conclude that these cells are the local source of osteopontin that binds to the FDC network.

    Increase in FDC-associated osteopontin during HAART.

    We found that the size of the follicles and FDC-associated osteopontin gene expression (figure 1A and 1B) increased in parallel, which suggests that follicle formation and osteopontin synthesis and binding are concurrently regulated. Because the FDC network is slowly reconstituted during HAART [21], we would therefore expect corresponding increases in FDC-associated osteopontin gene expression. We quantified FDC-associated osteopontin as a percentage of the area of lymphatic tissues stained by antibody in a total of 12 individuals in 2 separate studies over the course of 1.5 years of HAART; indeed, we found, in a meta-analysis of the separate studies, statistically significant increases (P = .02) in FDC-associated osteopontin gene expression that closely matched the increases previously documented in the FDC network (data not shown) [21].

    Increase in FDC-associated osteopontin gene expression during acute SIV infection.

    The increases in both osteopontin gene expression and the number and size of lymphoid follicles imply that FDC-bound osteopontin gene expression should also increase with the well-documented and particularly dramatic increases in the numbers and sizes of the follicles during the early stage of HIV-1 infection [22]. For obvious practical and ethical reasons, lymph-node biopsies cannot be obtained during the relevant time frame (before and shortly after HIV-1 infection) for testing this hypothesis. However, we were able to study lymph nodes obtained before and during acute SIV infection of rhesus macaques, a nonhuman primate model in which the changes in lymphatic tissues correspond closely to those in HIV-1 infection [23, 24]. Osteopontin colocalized to the rhesus macaque FDC network (figure 3A) and to cells in the paracortex (figure 3A, inset); in accordance with the hypothesis, FDC-bound osteopontin gene expression increased substantially and proportionately with the increased numbers and sizes of the follicles accompanying immune activation in response to infection at 1 and 4 weeks after iv infection with SIV (figure 3).

    No detection of FDC-associated osteopontin gene expression in the mouse.

    In addition to the follicular hyperplasia described above, polyclonal B cell activation and hypergammaglobulinemia are also characteristic of early HIV-1 infection [2427]. Because the increased FDC-associated osteopontin gene expression observed in acute SIV infection pointed to a potential role of osteopontin in these B cell abnormalities that we could not directly test in fixed tissues, we designed experiments with model antigens to examine the relationship between osteopontin gene expression and antibody production in mice. Because we had not seen descriptions of osteopontin colocalizing with the FDC network in mice, and because of the many examples of differences in mouse and human immune systems [28], we first undertook some preliminary experiments to determine whether osteopontin was, in fact, associated with murine FDCs. Although the osteopontin antibodies readily detected osteopontin-positive cells in the paracortex and follicles (figure 4) of mouse (B6 and other strains) lymph nodes, there was no detectable FDC-associated osteopontin in polyclonal and monoclonal antibodies to full-length recombinant mouse osteopontin and proteolytically derived N- and C-terminal fragments. Thus, osteopontin's association with the FDC network is species specific.

    DISCUSSION

    In the present studies, we found that (1) there is a species-specific association of osteopontin with the FDC network and (2) changes in the levels of FDC-associated osteopontin gene expression are correlated with pathological abnormalities in lymphoid follicles in HIV-1 and SIV infections and the reparative response to HAART. We now discuss what these findings might imply about FDC-associated osteopontin's roles, both normally in antibody production and in immunodeficiency virus infections.

    The association of osteopontin with the FDC network locates a powerful antibody-stimulating cytokine in the lymphoid follicle structure, which is organized to bring together antigen bound to the FDC network, B cells, and germinal-center CD4+ cells to stimulate B cell proliferation and differentiation for the production of high-affinity antibodies. The FDC-associated osteopontin could be contributing to antibody production at this site by facilitating FDCCD4+ cell germinal-center interactions that augment Th1 cytokine and CD40L expression. Because osteopontin is known to augment CD40L expression in T cells [32], FDC-associated osteopontin is ideally sited anatomically to increase the expression of CD40L on CD4+ cells in close proximity to B cells, thereby inducing B cell proliferation and antibody production.

    Osteopontin in mice, similarly, has long been known to stimulate antibody production and B cell proliferation through CD40-CD40L and other interactions with CD4+ cells. T cells and macrophages also produce osteopontin in mice, just as we have shown in humans and nonhuman primates. However, osteopontin was not detectably bound by FDCs in mice, in contrast to what was seen in primate species. Although the murine and human osteopontin sequences are closely related [3335], we conclude from the present studies that the additional sequences that osteopontin has acquired through evolution enable it to bind to FDCs for the facilitation of antibody production.

    In all 3 species, elevated levels of osteopontin were implicated in B cell abnormalities. In mice, high levels of osteopontin were associated with polyclonal B cell activation, autoimmune disease, and murine AIDS, respectively, in MRL/lpr mice, osteopontin-transgenic mice, and mice infected with the retrovirus LP-BM5 [8, 12, 13, 20]. In human and nonhuman primate lymphatic tissues, changes in the levels of FDC-associated osteopontin that could be contributing to the B cell abnormalities observed in HIV-1 and SIV infections and to the pathological changes in lymphoid follicles in these infections that cover a spectrum from follicular hyperplasia, to involution, to the development of lymphomas [22, 29]. During the early stages of infection, the deposition of viral immune complexes in the FDC network is associated with increases in the number and size of the follicles and with the concomitant generalized polyclonal activation of B cells that results in hypergammaglobulinemia [24, 26, 27, 30]. Recent evidence has suggested that hyperactivated naive B cells are responsible for increased immunoglobulin production [31] and that the abnormal differentiation of naive B cells is driven by CD40-CD40L interactions that would be enhanced by FDC-associated osteopontin gene expression.

    By the later stages of infection, HIV-1 and SIV have destroyed lymphoid follicles, with accompanying defects in the humoral immune response to recall antigens and vaccination that reflect structural damage and reductions in the number of memory B cells [31]. We had shown previously that this destructive process is reversible by HAART. During HAART, follicles reformed over a period from a few months to 1.5 years of treatment; by the latter time point, levels were similar in proportion to numbers of follicles in HIV-1uninfected individuals [36]. Here, we have provided encouraging evidence that the capacity to regenerate FDC-associated osteopontin is retained as another component of the immune reconstitution that takes place during HAART.

    Finally, the present studies illustrate how mapping the cellular and anatomic location of genes discovered in microarray studies can help define function in a particular tissue and disease. In particular, in the immunodeficiency virus infection of lymphatic tissues, there would have been no a priori reason or literature on osteopontin to suspect binding to FDCs and, therefore, a potentially new role in normal and abnormal B cell states. Thus, mapping studies can complement gene profiling by pointing the way forward from hypothesis generation to hypothesis testing.

    Acknowledgments

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    Baylor Institute for Immunology Research
    Division of Pediatric Infectious Diseases, Department of Pediatrics, University of Texas Southwestern Medical Center at Dallas and Children's Medical Center Dallas, Dallas

    Background.

    Respiratory syncytial virus (RSV) is the principal etiologic agent of bronchiolitis and viral pneumonia in infants and young children. Yet, many aspects of its immunopathogenesis are not well understood.

    Methods.

    We analyzed the immune cells that are mobilized by RSV and other respiratory viruses, by studying nasal wash samples from children hospitalized with acute viral respiratory infections.

    Results.

    RSV mobilizes virtually all blood immune cells, including myeloid dendritic cells (DCs) and plasmacytoid DCs (pDCs), to the nasal mucosa. DCs were also mobilized to the nasal mucosa of children with other viral respiratory infections. The increased number of pDCs in the nasal compartment significantly correlates with RSV load (P = .022), and it is associated with a significant decrease in the number of pDCs in the blood (P = .007). The influx of DCs in the nasal mucosa is not transient, as even higher numbers of both DC subsets were found in respiratory secretions weeks after the acute symptoms of RSV infection had resolved. Immunochemistry analysis of respiratory samples has demonstrated the presence of the RSV fusion protein within HLA-DRpositive cells.

    Conclusion.

    Infection with RSV and other respiratory viruses mobilizes DCs to the site of viral entry.

    Control of viral infections represents a formidable challenge for the immune system. Mucosal viral infections are controlled by (1) preformed local and circulating antibodies [1, 2], (2) innate immunity mechanisms (such as local production of interferon [IFN] [35]), and (3) adaptive immunity mechanisms (such as cytotoxic responses [1] and antibody-secreting plasma cells [6, 7]).

    Respiratory syncytial virus (RSV) is the leading viral respiratory pathogen in young children [811]. RSV infection does not lead to the development of protective immunity, and repeated infections are common. Additionally, there is a strong association between RSV bronchiolitis in infancy and recurrent wheezing [1216].

    The inflammatory response initiated by RSV infection likely contributes to the manifestations of RSV disease [1721]. Little is known about the mobilization of the immune effectors and regulators at the site of viral entry (the nasal mucosa) in humans. Neutrophils were the predominant leukocyte found in both bronchoalveolar lavage and nasopharyngeal specimens from infants with RSV bronchiolitis, although lymphocytes and monocytes were also identified [2224].

    Despite considerable efforts, no RSV vaccine is currently available [25]. A better understanding of the interplay of RSV with immune cells may facilitate the development of an effective vaccine. Given the critical role of dendritic cells (DCs) in the initiation of immune responses, it is important to understand the relationship between RSV infection and DCs.

    DCs constitute a complex system of cells that induce and control immune responses [2630]. Two subsets of DC precursors circulate in the blood: lineage-negative CD11c+ myeloid DCs (mDCs) and CD11c- interleukin (IL)3R+ plasmacytoid DCs (pDCs) [29, 31], which produce large amounts of type I IFN in response to viruses [32, 33]. pDCs have been found in lymphoid organs [3436] and in increased numbers in allergen-challenged nasal mucosa [37] and in the skin of patients with systemic lupus erythematosus [38].

    Viruses employ many different strategies to escape the immune system, including targeting DCs and subverting their function [39, 40]. The measles virus, another paramyxovirus, modulates release of cytokines by DCs [41, 42] and renders DCs cytotoxic through the up-regulation of both FasL/CD95L and tumor necrosis factorrelated apoptosis-inducing ligand [43]. There is little information concerning the interplay of RSV with DCs. Therefore, we have analyzed the mDC and pDC subsets in children hospitalized with RSV infection and other viral respiratory infections. The present study demonstrates that both mDCs and pDCs are recruited to the site of viral replication in the respiratory tract.

    PATIENTS, MATERIALS, AND METHODS

    Patient population.

    Inclusion criteria for enrollment into the RSV group included the following: age <15 months, illness requiring hospitalization, and diagnosis of acute RSV infection by direct fluorescent antibody (DFA; Diagnostic Hybrids) test and/or viral culture (in HEp-2 or A549 cells). Our viral control group included patients with acute infections caused by respiratory viruses other than RSV, isolated from nasal wash samples by culture and/or identified by DFA test. DFA tests and viral cultures, which are routinely performed by the clinical virology laboratory, identified RSV; influenza A and B; parainfluenza 1, 2, and 3; adenovirus; and rhinovirus. Our control population consisted of healthy infants with no history of upper respiratory illness within the preceding 4 weeks. These children were typically identified in the operating room while undergoing elective surgical procedures that did not involve the respiratory tract. Despite the lack of respiratory symptoms, both DFA tests and viral culture of nasal wash samples were performed at enrollment, for all the healthy control children and for all patients with RSV infection who had undergone follow-up evaluations. Exclusion criteria for all groups included the following: age >15 months, receipt of immunosuppressive medications, and history of chronic lung disease. Informed consent was obtained from the parents of all patients at enrollment into the study, and the institutional review board at the University of Texas Southwestern Medical Center approved the study.

    Collection of nasal wash samples and cell isolation.

    Nasal wash samples were collected from patients by nasal suctioning, in accordance with a validated protocol [44]. A standardized volume of 3 mL of viral transport media (Bartels) at 4°C was then added, and the sample was maintained on ice. The sample was then centrifuged for 10 min at 300 g at 4°C. The cell/mucus pellet was resuspended in PBS/2% fetal calf serum at 4°C (Sigma) and centrifuged again, as above. This was repeated until no visible mucus clumps remained in the cell/mucus solution. The sample was filtered over a Filcon 100-m filter (Dako). Viable cells were visualized by trypan blue exclusion and counted by use of a Reichert Bright Line hemocytometer.

    Cell staining for flow-cytometric analysis.

    For nasal wash cell staining, 200,000 cells/tube were incubated with antibodies for 30 min, rinsed with PBS, centrifuged at 300 g for 10 min, and resuspended in 1% paraformaldehyde. For blood studies, whole blood was collected in tubes containing acid citrate dextrose. For staining, 100 L of blood and 310 L of each antibody were incubated for 30 min. The blood was then lysed with FACS Lysing Solution (BD Biosciences), rinsed with PBS, centrifuged at 300 g for 10 min, and resuspended in 1% paraformaldehyde. Samples were then acquired on a FACSCalibur flow cytometer and analyzed with CellQuest software (BD Biosciences).

    The following fluorochrome-conjugated antihuman antibodies were used for both nasal wash and whole-blood stainings: LINEAGEfluorescein isothiocyanate (FITC) cocktail (containing CD3, CD14, CD16, CD19, CD20, and CD56), CD123phycoerythrin (PE), HLA-DRperidin chlorophyll protein (PerCP), CD11callophycocyanin (APC), CD4-FITC, CD8-PE, CD3-PerCP, and CD14-APC (BD Biosciences). In selected experiments, nasal wash cells were also stained with monoclonal antibody (MAb) directed against the fusion protein of RSV (Fitzgerald Industries International).

    Quantification of RSV in nasal wash samples.

    Quantitative RSV cultures were performed using the standard plaque assay technique, as described elsewhere [23, 44].

    Quantification of cytokines and chemokines in nasal wash samples.

    Concentrations of cytokines in nasal wash samples were measured by Luminex XMAP technology (Luminex), by use of commercially available kits (Upstate USA), following the manufacturers' instructions.

    Confocal microscopy.

    In several patients with RSV infection, cells from respiratory secretions were mounted onto slides and stained with MAbs directed against HLA-DRFITC and RSV fusion protein (conjugated to Texas Red). Cells were then visualized by confocal microscopy (Leica TCS SP confocal microscope with a Planapo 63/1.32X objective).

    Statistical analysis.

    The nonparametric Mann-Whitney U test or the parametric unpaired t test was used, as appropriate, to compare numbers of DCs and concentrations of cytokines between groups. The Wilcoxon signed rank test was used to compare numbers of DCs in patients with acute versus resolved RSV infection. Spearman's coefficient was used for correlations.

    RESULTS

    Patient enrollment.

    Twenty-one patients with acute RSV infection, 8 with other acute viral respiratory infections, and 10 healthy control children were enrolled between December 2000 and February 2003 (table 1). All patients with acute RSV infection had symptoms of bronchiolitis requiring hospitalization. During the acute phase, they were treated symptomatically with oxygen, intravenous fluids, and -adrenergic agents for variable periods of time, but they did not receive antiviral agents or corticosteroids. Age distribution was similar between the 3 groups. Among the patients with other acute viral respiratory infections, 4 had parainfluenza and 3 had influenza (2 influenza B and 1 influenza A) respiratory infections requiring hospitalization. These patients manifested respiratory symptoms including cough, nasal congestion, hypoxia, and bronchiolitis. Another patient in this group was an asymptomatic infant initially enrolled in the control group whose nasopharyngeal viral culture later yielded cytomegalovirus (CMV). All patients with acute viral respiratory infections were enrolled during the same months of the year.

    mDCs, pDCs, monocytes, T cells, and B cells in nasal wash samples from patients with viral respiratory infections.

    Flow-cytometric analysis demonstrated that CD14+ monocytes, CD3+CD4- T cells, CD3+CD4+ T cells, and CD20+ and CD19+ B cells can be identified in nasal wash samples from patients with acute RSV infection (figure 1A1C). Neutrophils can also be found, as determined by morphological analysis of cytospins and Giemsa-stained cells (data not shown). This infiltrate was not due to bleeding, because (1) no bleeding was seen at the time samples were collected, (2) the red blood cell (RBC) : white blood cell (WBC) ratio was considerably lower in nasal wash samples than in blood samples (the highest nasal wash RBC : WBC ratio was 25 : 1, but most were much lower), and (3) immune effectors were abundant in nasal wash samples that contained no RBCs.

    Increase of mDCs in nasal wash samples and decrease in blood samples from children with acute viral respiratory infections.

    Using the parameters described above, we found that 27 of 29 evaluated patients with acute viral respiratory infections had mDCs in nasal wash samples (figure 2A), with a median of 2400 mDCs/nasal wash sample (range, 042,000 mDCs/nasal wash sample). In contrast, the healthy control children had very low numbers of mDCs in nasal wash samples (median, 45 mDCs/nasal wash sample; range, 01700 mDCs/nasal wash sample; P < .001, compared with all patients with viral infections). Of the 21 patients with acute RSV infection, 19 had mDCs in nasal wash samples (median, 1932 mDCs/nasal wash sample; range, 013,400 mDCs/nasal wash sample; P = .0041, compared with healthy control children). The 8 patients with other viral respiratory infections also had mDCs in nasal wash samples (median, 6665 mDCs/nasal wash sample; range, 57842,000 mDCs/nasal wash sample). The numbers were significantly different from those in healthy control children (P = .0014), but not from those in patients with RSV infection (P = .339).

    Increase of pDCs in nasal wash samples and decrease in blood samples from patients with acute viral respiratory infections.

    Twenty-three of 29 patients with acute viral respiratory infections had pDCs in nasal wash samples (median, 760 pDCs/nasal wash sample; range, 032,200 pDCs/nasal wash sample) (figure 3A). Five of 10 healthy control children also had pDCs in nasal wash samples, although at much lower numbers (median, 6 pDCs/nasal wash sample; range, 0680 pDCs/nasal wash sample; P = .0052, compared with patients with other viral respiratory infections). Fifteen of 21 patients with acute RSV infection had pDCs in nasal wash samples (median, 490 pDCs/nasal wash sample; range, 04480 pDCs/nasal wash sample; P = .043, compared with healthy control children). A positive correlation between the number of pDCs and the number of mDCs in nasal wash samples was identified (P < .01). All 8 patients with other viral respiratory infections had pDCs in nasal wash samples (median, 1094 pDCs/nasal wash sample; range, 10032,200 pDCs/nasal wash sample; P = .0003, compared with healthy control children).

    Increased concentrations of IL-6, IL-8, and macrophage inflammatory protein (MIP)1 in nasal wash samples from patients with RSV infection.

    We next analyzed the concentrations of several cytokines and chemokines in the respiratory tract. Concentrations of IL-6, IL-8, and MIP-1 were significantly increased in nasal wash samples from patients with RSV infection, compared with those in nasal wash samples from healthy control children (figure 4A). Concentrations of IL-8 were also significantly increased in nasal wash samples from patients with RSV infection, compared with those in nasal wash samples from patients with other viral respiratory infections. Among the cytokines and chemokines measured, only MIP-1 was significantly increased in nasal wash samples from patients with other viral respiratory infections, but a trend for increased concentrations of IL-6 was also observed. No significant differences in the concentrations of IL-4, IL-5, or IFN- were observed (figure 4B).

    Among the patients with RSV infection, there was a positive correlation between numbers of mDCs and concentrations of IL-6 in nasal wash samples (r = 0.5; P = .04). Although this correlation was not present in the control group, numbers of mDCs did strongly correlate with concentrations of MIP-1 (r = 0.77; P = .017). In patients with other acute viral respiratory infections, numbers of mDCs significantly correlated with concentrations of IL-8 (r = 0.88; P = .03), and there was a marked trend for increased concentrations of MIP-1 (r = 0.81; P = .058).

    The number of pDCs in nasal wash samples also increased after resolution of acute RSV infection. In a comparison of the patients with RSV infection as a group, the median number of pDCs in nasal wash samples increased from 760 pDCs/nasal wash sample during acute infection to 11,763 pDCs/nasal wash sample after resolution of infection, representing a 15-fold increase. By use of paired evaluations of only the patients with RSV infection from whom follow-up samples were available, the increase in the number of pDCs in nasal wash samples was significant (P = .047, Wilcoxon signed rank test; P = .03 [RSV-04 not included in analysis]). Only 1 patient (RSV-04) had a decreased number of pDCs in nasal wash samples after resolution of acute RSV infection. This patient was indeed receiving daily inhaled steroids after hospitalization for RSV infection. It is interesting to note that this patient still had an increase in mDCs in nasal wash samples, which is consistent with the finding that steroids block the generation of pDCs from hematopoietic progenitor cells more efficiently than that of mDCs (A.K.P., unpublished observations). No other evaluated patients received any form of steroids.

    The pDC subset represented a larger percentage of the cellular infiltrate in the follow-up phase as well, although it was not as markedly increased as was the mDC subset (median, 0.11% after resolution of infection vs. 0.01% during acute infection; P = .14). There was a positive correlation between the increase in numbers of mDCs and pDCs in nasal wash samples after resolution of acute RSV infection (r = 0.93; P = .007). No evidence of RSV or other viruses was detected by routine viral cultures, DFA tests, or RSV plaque assays performed during the follow-up evaluations.

    Expression of RSV proteins by HLA-DRpositive cells from the respiratory tract.

    Next, we wondered whether nasal DCs would carry RSV. To investigate this question, we stained unsorted nasal and tracheal wash cells from RSV-infected patients with antibodies directed against HLA-DR and RSV fusion protein. Respiratory samples from 2 patients demonstrated HLA-DRpositive cells that also expressed RSV fusion protein (figure 7). A few HLA-DRnegative cells were found to be positive for RSV fusion protein, and not all HLA-DRpositive cells expressed RSV fusion protein.

    DISCUSSION

    RSV infection is a major cause of morbidity in infants and children, indicating that it is not efficiently controlled by the immune system. Pathogens affect different components of the immune system for their survival and replication. In particular, DCs are targeted by many viruses, including HIV [47], dengue virus [48, 49], and CMV [50]. This prompted the present study, since very little is known about the interactions between RSV and the DC system, most particularly in humans, the primary host of RSV.

    We initiated the present study by assessing what happens at the actual site of RSV entry/infection, the nasal cavity. We are naturally limited in the scope of our investigation by 2 parameters: (1) the young age of the patients (<15 months; median, 3 months in the RSV group); and (2) the small sample material to analyze (3-mL nasal wash samples), in which viscosity makes analysis of infiltrating cells challenging, and the limited volume of blood samples. Despite these limitations, several important conclusions can be made.

    First, as reported elsewhere [22], in nasal wash samples, we found virtually all the immune effectors, including T cells, B cells, monocytes, neutrophils, and, more importantly, both pDCs and mDCs, the focus of the present study. The results demonstrate that RSV attracts both mDCs and pDCs to the site of viral infection. The number of pDCs, and, to a lesser extent, that of mDCs, positively correlates with RSV load. The number of mDCs at the site of RSV infection (median, 1932 mDCs) is significantly higher than the number of pDCs (median, 490 pDCs). This may be due to the considerable phenotypic and morphological alterations that pDCs undergo after maturation. Unfortunately, no reagents are currently available that allow the identification of mature pDCs, and these cells likely remain uncounted. No CD83+ cells could be detected, suggesting that mature DCs might remain associated with the tissue. The attraction of both DC subsets to the nasal mucosa is not specific to RSV infection, as the 8 patients with other viral respiratory infections also had similar findings.

    With regard to mDCs, their increase in nasal wash samples was associated with a decrease in blood samples. This suggestsit is difficult to formally demonstrate without controlled challenge studiesthat the increase of mDCs in nasal mucosa results from their migration from the blood. The increased presence of mDCs in virus-infected nasal mucosa in humans is reported here for the first time. However, this finding is consistent with earlier studies performed in rats, in which Moraxella catarrhalis or Sendai virus recruited major histocompatibility complex (MHC) class IIpositive cells (putative DCs) into the airway epithelium [51, 52].

    In its consideration of pDCs, the present study is, to our knowledge, the first study to report their attraction to a mucosal site of viral replication. This could be viewed as an expected finding, since pDCs are considered to represent an early barrier to viral replication through their ability to secrete large amounts of IFN- in response to viral challenge. Attraction of pDCs to the nasal mucosa in humans has been reported under a different pathological conditionthat is, in allergic patients challenged with the relevant allergens [37]. Furthermore, studies of mice have reported the presence of cells with characteristics of pDCs in the bronchi of mice challenged with a leishmanial antigen [53]. These MHC class IIpositive cells were long lasting, which is consistent with our observation of long-lasting infiltration of DCs into the nasal mucosa as late as 8 weeks after resolution of acute RSV infection. More-recent studies have confirmed that pDCs have a long half-life in vivo [54], a finding that contrasts with their extreme propensity to apoptose after isolation [34]. In humans, pDCs have also been found to infiltrate cutaneous lesions caused by varicella-zoster virus [55]. Whether the infiltrating cells are long-lasting cells attracted at the time of acute infection or a set of new cells attracted by long-acting mechanisms initiated by viral infection represents a complex challenge to address in our patient population. Since we evaluated only patients with RSV infection after resolution of acute symptoms, we do not know whether the persistence of DCs is specific to RSV infection. It will be important to evaluate patients with other viral respiratory infections in future studies.

    DC subsets in blood are reduced and impaired in patients with HIV infection [5659]. The number of circulating pDCs correlated inversely with HIV load and the occurrence of opportunistic infections [58, 60]. In the present study, the number of pDCs in blood samples did not correlate with the RSV load measured in the respiratory tract. The decrease in pDCs in blood samples during acute RSV infection suggests that pDCs are recruited to the site of viral entry, the nasal mucosa. It is unknown whether RSV infects pDCs.

    We have made significant attempts to determine whether the DCs present in the nasal wash samples were functional. Unfortunately, it is difficult to isolate sufficient cell numbers from the small viscous nasal wash samples, to complete such studies. Therefore, we will be limited to in vitro studies to determine how RSV might affect the functions of human DCs. This will be important to investigate, since RSV may interfere with DC-induced T cell activation and differentiation in a fashion that leads to characteristics of disease. Indeed, it has recently been shown that RSV induces human cord bloodderived mDCs to secrete IL-10, IL-11, and prostaglandin E2, whereas influenza primarily induces generation of IL-12 in mDCs [61].

    We found significantly increased concentrations of IL-6, IL-8, and MIP-1 in nasal wash samples from patients with RSV infection. Despite the small number of patients in the group with other viral respiratory infections, the concentrations of MIP-1 in their nasal wash samples were also increased, and there was a clear trend for increased concentrations of IL-6. Concentrations of IL-8, however, were significantly greater in patients with RSV infection than in patients with other viral respiratory infections. In addition, the correlation between numbers of mDCs and concentrations of IL-6 was detected only in patients with RSV infection. Although it is difficult to draw conclusions from these correlations, we can speculate that the balance of cytokines/chemokines and DCs that exists in healthy control children is altered during RSV infection. Indeed, the correlation between numbers of mDCs in the mucosa and concentrations of MIP-1 observed in healthy control children was absent in patients with RSV infection.

    Although we were unable to address the functional status of the DCs in nasal wash samples in patients with RSV infection, we have demonstrated that RSV antigen can be detected within HLA-DRpositive cells with the morphology of DCs. Further studies will be required to confirm that these cells are indeed DCs and to determine whether RSV replicates within these cells or whether RSV particles are simply captured from the mucosal microenvironment.

    In conclusion, we have shown that RSV infection results in mobilization of DCs to the site of viral entry. How RSV affects the functions of these immune cells will be the focus of important future studies that may shed light on the pathogenesis of this common infection.

    Acknowledgments

    We extend special thanks to Virginia Pascual, for many discussions and for reviewing our data; Elizabeth Kraus and Sebastien Coquery, for their superb technical assistance with the flow-cytometric analysis of our patient samples; Sandra Clayton, for her expertise in confocal microscopy; and Jeanine Hatfield, for her efforts in viral quantitation and numerous other aspects of sample processing. We also thank Edsel Arce, for his assistance, and Lonnie Roy and Naveed Ahmed, for their input regarding the statistical analysis of the data. We also acknowledge Michael Ramsay, for his support of this project.

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    Division of Cell Biology, Department of Pediatrics, National Jewish Medical and Research Center, Denver, Colorado

    ABSTRACT

    Rationale: There is conflicting information about the development and resolution of airway inflammation and airway hyperresponsiveness (AHR) after repeated airway exposure to allergen in sensitized mice.

    Methods: Sensitized BALB/c and C57BL/6 mice were exposed to repeated allergen challenge on 3, 7, or 11 occasions. Airway function in response to inhaled methacholine was monitored; bronchoalveolar lavage fluid inflammatory cells were counted; and goblet cell metaplasia, peribronchial fibrosis, and smooth muscle hypertrophy were quantitated on tissue sections. Bone marrow–derived dendritic cells were generated after differentiation of bone marrow cells in the presence of growth factors.

    Results: Sensitization to ovalbumin (OVA) in alum, followed by three airway exposures to OVA, induced lung eosinophilia, goblet cell metaplasia, mild peribronchial fibrosis, and peribronchial smooth muscle hypertrophy; increased levels of interleukin (IL)-4, IL-5, IL-13, granulocyte-macrophage colony–stimulating factor, transforming growth factor-1, eotaxin-1, RANTES (regulated on activation, normal T-cell expressed and secreted), and OVA-specific IgG1 and IgE; and resulted in AHR. After seven airway challenges, development of AHR was markedly decreased as was the production of IL-4, IL-5, and IL-13. Levels of IL-10 in both strains and the level of IL-12 in BALB/c mice increased. After 11 challenges, airway eosinophilia and peribronchial fibrosis further declined and the cytokine and chemokine profiles continued to change. At this time point, the number of myeloid dendritic cells and expression of CD80 and CD86 in lungs were decreased compared with three challenges. After 11 challenges, intratracheal instillation of bone marrow–derived dendritic cells restored AHR and airway eosinophilia.

    Conclusions: These data suggest that repeated allergen exposure leads to progressive decreases in AHR and allergic inflammation, through decreases in myeloid dendritic cell numbers.

    Key Words: airway hyperresponsiveness  chronic asthma  cytokine  dendritic cells  eosinophil

    Chronic inflammation of the airways with variable airflow limitation is responsible for the altered airway responsiveness seen in individuals with asthma to a variety of stimuli (1). Elevated serum IgE levels and inflammatory cell infiltration, especially of eosinophils, mast cells, and CD4+ T cells, appear to be involved in the pathogenesis of the disease (2–4). Much of our understanding of asthma points to a complex and multifactorial condition involving interactions between genetic factors and environmental stimuli. It has been argued that repeated exposure to airborne allergens, such as house dust mite or animal dander, contributes to the initiation and persistence of airway inflammation, which is believed to be the central abnormality resulting in airway damage and dysfunction (5, 6). Several studies identified increased muscle mass and airway wall thickening as determinants of airway hyperresponsiveness (AHR) (7, 8). This chronic airway inflammatory response, which is characteristic of asthma, can lead to airway remodeling, and structural alterations that may underlie some of the persistent manifestations of asthma. Indeed, a variety of cells, including epithelial cells, fibroblasts, and smooth muscle cells, in the chronic stages of asthma likely contribute through the secretion of various cytokines and chemokines that result in the irreversible fibrosis and remodeling seen in the airways of individuals with asthma (9, 10).

    In allergic diseases, such as atopic dermatitis and allergic asthma, allergen-specific T cells express a helper T-cell type 2 (Th2) phenotype (11). In animal models of acute allergic inflammation, Th2 cell–derived cytokines, notably interleukin (IL)-4, IL-5, IL-9, and IL-13 and C-C chemokine family members such as RANTES (regulated on activation, normal T-cell expressed and secreted) and eotaxin-1, are pivotal in the induction of eosinophilic airway inflammation, antigen-specific IgE production, and AHR (12). Although airway inflammation and Th2 cytokines and chemokines are undoubtedly a cornerstone of asthma, it is clear that the asthmatic response is more complex. The relative importance of these factors may vary according to the stage or duration of asthma. Much less is known about the pathogenesis of chronic airway eosinophilic inflammation and cytokine/chemokine production in long-standing asthma. Because no satisfactory disease-modifying treatment is currently available for chronic asthma, evaluation of the therapeutic effects of the modulation of cytokine/chemokine production in chronic disease is an important area of investigation (13). In this regard, an essential first step is to characterize the profile and kinetics of cytokine/chemokine production in a model system where allergen challenges are incremental, to identify potential therapeutic targets.

    Whereas the role of T cells has been fairly well described, much less information is available on the role of dendritic cells (DCs) in allergic airway inflammation. DCs have been thought to be an important cell type in the initiation of T-cell–mediated inflammation by priming naive T cells (14, 15). Several reports have identified a critical role for DCs in allergic airway inflammation (16, 17). DCs are subdivided on the basis of surface antigen expression. Each DC subtype may induce different stimulatory signals on T cells and on the development of allergic inflammation. For example, bone marrow–derived DCs (BMDCs) expressing a myeloid DC (mDC) phenotype prime T cells to specific antigen and induce their expansion (18). On the other hand, plasmacytoid DCs (pDCs) inhibit the development of effector T cells (19). The purpose of this study was to delineate the characteristics of allergic airway responses after repeated allergen challenge and the role of DCs in regulating these responses.

    METHODS

    Animals

    Female BALB/c and C57BL/6 mice were purchased at 8 to 12 wk of age from Jackson Laboratories (Bar Harbor, ME) and housed under specific pathogen–free conditions. The animals were maintained on an ovalbumin (OVA)-free diet. Experiments were conducted under a protocol approved by Institutional Animal Care and Use Committee of the National Jewish Medical and Research Center (Denver, CO).

    Protocols for OVA-induced Allergic Airway Inflammation

    Mice were sensitized on Days 1 and 14 by intraperitoneal injection of 20 μg of OVA (grade V; Sigma-Aldrich, St. Louis, MO) premixed with 2.25 mg of Al(OH)3 (Pierce Biotechnology, Rockford, IL) in 100 μl of phosphate-buffered saline (PBS). After sensitization, animals were exposed to aerosolized OVA (1% in saline) for 20 min/d on Days 28, 29, and 30 (3 challenges, short-term challenge), or continued to receive airway challenges on Days 36, 40, 44, and 48 (7 challenges), as well as on Days 52, 56, 60, and 64 (11 challenges; Figure 1). Forty-eight hours after the last OVA challenge (Day 32, 50, or 66) AHR was assessed and bronchoalveolar lavage (BAL) fluid, serum, and tissues were obtained for further analyses. Control groups consisted of nonsensitized but OVA-challenged animals.

    Assessment of Airway Function

    Airway function was assessed as previously described by measuring changes in lung resistance (RL) and dynamic compliance (Cdyn) in response to increasing doses of inhaled methacholine (MCh) (20).

    BAL

    Immediately after assessment of airway function, lungs were lavaged via the tracheal tube with 1 ml of Hanks' balanced salt solution at room temperature. Total leukocyte numbers were measured (Coulter Counter; Beckman Coulter, Fullerton, CA). Cytospin slides were stained with Leukostat (Fisher Diagnostics, Pittsburgh, PA) and differentiated by standard hematologic procedures in a blinded fashion.

    Lung Histopathology and Morphometric Analyses

    Lungs were fixed in 10% formalin and processed into paraffin. Mucus-containing goblet cells were detected by staining of paraffin sections (5-μm thick) with periodic acid–Schiff (PAS). Airway fibrosis was detected by staining of collagen with Sirius red. Airway smooth muscle cells were stained with immunoperoxidase, using anti–-smooth muscle actin monoclonal antibody (Sigma-Aldrich). Tissue-infiltrating eosinophils were identified by immunofluorescence staining of major basic protein as previously described using rabbit anti-mouse major basic protein (kindly provided by J. J. Lee, Mayo Clinic, Scottsdale, AZ) (20). CD4+ T cells were detected by immunoperoxidase staining of frozen lung tissue sections with a rat anti-mouse CD4 antibody (L3T4; BD Biosciences Pharmingen, San Diego, CA). As a control, rat IgG was used. Histologic analyses were performed in a blinded manner by light microscopy linked to an image capture system. The data were analyzed by quantitative morphometry, using Scion Image analysis software available as public domain software from the U.S. National Institutes of Health (http://rsb.info.nih.gov/nih-image/). Numbers of PAS-positive goblet cells, airway tissue–infiltrating eosinophils, and collagen (Sirius red staining) were determined only in cross-sectional areas of the airway wall. Tangential sections and airway wall areas adjacent to large blood vessels were excluded from the analysis. For smooth muscle and collagen analysis, the slice density function of the software was used to highlight pixels belonging to the staining, excluding intercellular space and unstained tissue structures, which fall within the stained collagen or smooth muscle areas, and expressed as square micrometers. To allow for comparisons, all measurements were normalized to the length of the basement membrane of the adjacent epithelium for each corresponding airway. Airways 250 to 500 μm in internal diameter were evaluated and 10 different sections were evaluated per animal. The obtained measurements were averaged for each animal and the mean values and standard errors were determined for each group.

    Measurement of BAL Fluid Cytokines and Chemokines

    Levels of cytokines and chemokines were determined by commercial ELISAs in accordance with the manufacturer instructions. ELISA kits for detection of IL-4, IL-5, IL-10, IL-12 (p70), and IFN- in supernatants were obtained from BD Biosciences Pharmingen. IL-13, transforming growth factor (TGF)-1, granulocyte-macrophage colony–stimulating factor (GM-CSF), eotaxin-1 and RANTES ELISA kits were purchased from R&D Systems (Minneapolis, MN). Before the TGF-1 assay, BAL fluid samples were acidified to activate any latent TGF-1 (21). The limits of detection for each assay were as follows: 4 pg/ml for IL-4 and IL-5; 10 pg/ml for IL-10, IL-12, and IFN-; 1.5 pg/ml for IL-13 and GM-CSF; 3 pg/ml for eotaxin-1 and RANTES; and 7 pg/ml for TGF-1.

    Measurement of OVA-specific Antibodies

    Serum levels of OVA-specific IgE, IgG1, and IgG2a were measured by ELISA as previously described (22).

    Lung Cell Isolation

    Lungs were dissected into small pieces and exposed to an enzymatic digestion solution containing collagenase V (175 IU/ml; Sigma-Aldrich, St. Louis, MO) in Hanks' balanced salt solution for 60 min. After enzymatic digestion, total lung leukocytes were further enriched by 35% Percoll (Sigma-Aldrich) gradient centrifugation. The pellets were resuspended in 5 ml of red blood cell lysing buffer (Sigma-Aldrich) for 10 min on ice. The cells were then washed twice and resuspended in PBS containing 1% bovine serum albumin.

    Flow Cytometry

    The surface phenotype of lung cells was analyzed with monoclonal antibodies (mAbs) in conjunction with a three- or four-color immunofluorescence test. To minimize nonspecific binding, 5 x 105 cells were incubated with 0.25 μg of Fc-blocking solution (CD16/CD32; clone 2.4G2) for 10 min and subsequently treated with fluorochrome-labeled mAbs. The mAbs included fluorescein isothiocyanate-, phycoerythrin-, peridinin chlorophyll protein-, or allophycocyanin-conjugated anti-CD11b (M1/70), anti-CD11c (HL3), anti–Ly-6G and anti–Ly-6C (Gr-1 RB6–8C5), anti-CD80 (16–10A1), anti-CD86 (GL1), and anti-CD45R/B220 (RA3–6B2; all obtained from BD Biosciences Pharmingen). For staining pDCs, anti–mPDCA-1 (JF05–1C2.4.1) was obtained from Miltenyi Biotec (Auburn, CA). For control staining, similarly labeled isotype-matched control antibodies were used. After washing, staining was analyzed by flow cytometry on a FACSCalibur using CellQuest software (BD Biosciences, Mountain View, CA).

    Generation and Transfer of BMDCs

    DCs were generated from bone marrow cells of naive BALB/c mice according to the procedure described by Inaba and coworkers (23), with some modification. In brief, bone marrow cells obtained from femurs and tibias of mice were placed in 75-cm2 flasks at 37°C in DC culture medium (RPMI 1640 containing 10% heat-inactivated fetal calf serum, 50 μM 2-mercaptoethanol, 2 mM L-glutamine, penicillin [100 U/ml], streptomycin [100 μg/ml; GIBCO; Invitrogen, Carlsbad, CA], recombinant mouse GM-CSF [10 ng/ml], and recombinant mouse IL-4 [10 ng/ml; R&D Systems]). Nonadherent cells were collected by aspirating the medium and transferred into fresh flasks. On Day 8, cells were pulsed with OVA (200 μg/ml) for 24 h and washed three times with PBS. More than 90% of the cells were mDCs (CD11c+CD11b+Gr-1–). For DC transfer after sensitization and 11 challenges with OVA, under anesthesia 1 x 106 OVA-pulsed BMDCs in 40 μl of PBS were instilled to BALB/c mice through the trachea, using fiberoptic illumination. Control groups of mice received PBS. Six days after DC transfer, mice were exposed to aerosolized OVA (1% in saline) for 20 min/d for 3 consecutive d. Forty-eight hours after the last challenge, AHR was assessed and BAL fluid and lung tissues were obtained.

    Statistical Analysis

    Mann-Whitney U tests were used to determine the levels of difference between all groups. Data were pooled from three independent experiments with four mice per group in each experiment (n = 12). Comparisons for all pairs were performed by Kruskal-Wallis test. Significance was assumed at p values less than 0.05. Values for all measurements were expressed as means ± SEM.

    RESULTS

    Airway Hyperresponsiveness

    Figure 2 illustrates the changes in lung resistance and dynamic compliance in response to inhaled MCh in both strains of mice after repeated allergen exposure. Exposure of sensitized mice to aerosolized allergen results in AHR to inhaled MCh. In sensitized BALB/c mice, short-term allergen exposure (i.e., three daily challenges) resulted in the development of increased AHR, detected by significant increases in lung resistance (Figure 2A) and decreases in dynamic compliance (Figure 2B), compared with similar challenge of nonsensitized animals. These results were strikingly different when sensitized mice were subjected to additional exposures of aerosolized allergen—in these mice there was little persistent alteration in airway function after seven allergen challenges. In fact, the results were similar to those of nonsensitized mice exposed to seven challenges alone. This absence of AHR was also observed after additional allergen exposure (11 challenges). In sensitized C57BL/6 mice the results were similar, with AHR developing after short-term exposure, but progressively declining with long-term allergen exposures (Figures 2C and 2D). As seen previously (24), the amounts of MCh required to elicit maximum responses in C57BL/6 mice were higher than needed in BALB/c animals.

    Inflammatory Cell Composition

    BAL fluid was examined in sensitized mice after 3, 7, and 11 inhalational challenges. In both BALB/c (Figure 3A) and C57BL/6 (Figure 3B) mice, significant increases in numbers of BAL fluid eosinophils were seen after three challenges. These numbers further increased in BALB/c mice exposed to seven challenges. However, after 11 challenges eosinophil numbers were reduced significantly in both strains. In both strains of mice, BAL fluid lymphocyte numbers were also increased after short-term challenge and the increases were sustained even after 11 challenges; in BALB/c mice the increases were also greater after long-term challenge. There were only small changes in neutrophil numbers. These results revealed some dissociation between AHR and the persistence of BAL fluid eosinophilia, at least after seven challenges of sensitized mice.

    Lung Tissue Analysis

    In parallel to the analysis of BAL fluid, lung tissue sections were immunolabeled with antibody to major basic protein to identify eosinophils in the lung tissue. As shown in Figures 4A and 4B, few eosinophils were detected in nonsensitized but challenged mice. In sensitized mice, after both short-term (three) and long-term (seven) challenges, lung eosinophilia developed and persisted. Interestingly, when these mice were monitored for an additional 2 wk (11 challenges), tissue eosinophilia was significantly decreased in the lung.

    When these same tissues were stained for the presence of CD4+ T lymphocytes, both short- and long-term challenge protocols resulted in marked increases in cell numbers, compared with the nonsensitized controls (Figures 4C and 4D). These increases in CD4+ T cells persisted through 11 challenges in both sensitized strains of mice.

    Goblet cell metaplasia and mucus hyperproduction were evaluated by PAS staining and quantification of positively stained cells. In nonsensitized mice of either strain, few positive cells were detected (Figures 4E and 4F, and Figure 5A). This contrasted with staining of tissue from sensitized and short- and long-term challenged mice, where the number of PAS-positive cells increased and was maintained, at least after 7 challenges (Figures 4E and 4F, and Figures 5B and 5C); after 11 challenges, the numbers of PAS-positive cells were decreased (Figures 4E and 4F, and Figure 5D). Thus, despite the persistence of BAL fluid and lung tissue eosinophilia, mucus hyperproduction, and infiltration of CD4+ lymphocytes, seven challenges with allergen via the airways was nonetheless associated with reduced AHR.

    Sirius red staining of the same tissues revealed a prominent subepithelial deposition of collagen in sensitized BALB/c and C57BL/6 mice after three and seven OVA challenges. However, after 11 challenges, the areas of collagen detected in the airway wall of sensitized mice were significantly reduced to levels that were comparable to those seen in nonsensitized but challenged control mice (Figures 4G and 4H, and Figures 5E–5H).

    After short-term allergen challenge, the area of the peribronchial smooth muscle layer was also increased in both sensitized BALB/c and C57BL/6 mice, when compared with challenged-only mice. However, this increase in airway smooth muscle area declined progressively with increasing allergen challenge (Figures 4I and 4J, and Figures 5I–5L).

    Cytokine/Chemokine Levels in BAL Fluid

    After sensitization and short-term challenge of both strains of mice, levels of the Th2 cytokines IL-4, IL-5, and IL-13 were significantly increased in BAL fluid (Figures 6A–6F). As previously reported (25), the BAL fluid levels of these cytokines were consistently higher in BALB/c mice (Figures 6A–6C) than in C57BL/6 mice (Figures 6D–6F). In contrast to short-term challenge, the levels of these cytokines decreased after additional (7 or 11) challenges.

    As previously reported (26), IL-10 levels in BALB/c mice decreased in the BAL fluid of sensitized animals compared with controls after short-term allergen challenge (Figures 6G and 6J). In contrast, after 7 and 11 challenges, levels of IL-10 increased significantly, to levels above those in controls of both strains. IFN- levels showed significant decreases after short-term challenge (Figures 6H and 6K), whereas IL-12 levels increased after the long-term challenges, more markedly in BALB/c mice than in C57BL/6 mice (Figures 6I and 6L). GM-CSF levels increased in both strains and were maintained even after 11 challenges in C57BL/6 mice (Figures 6M and 6O).

    Levels of total TGF-1 in BAL fluid were also elevated after three challenges in BALB/c mice or after three and seven challenges in C57/BL6 mice. However, these levels declined to control baseline levels after 11 challenges (Figures 6N and 6P). Among the chemokines assayed, eotaxin-1 levels increased after three and seven challenges in both strains, although levels were much higher in C57BL/6 mice (Figures 6Q and 6S). After 11 challenges, these levels fell significantly. RANTES levels also increased after 3 and 7 challenges in both strains, but returned to control values after 11 challenges (Figures 6R and 6T).

    Serum Antibody Levels

    OVA-specific IgE levels paralleled those of the Th2 cytokines, especially in sensitized BALB/c mice, with increases after three challenges followed by a significant decrease after long-term challenge (Figures 7A and 7D). In both strains, levels of OVA-specific IgG1 were maintained through the 11 challenges, as were levels of OVA-specific IgG2a (Figures 7B, 7C, 7E, and 7F).

    Lung DCs

    To determine whether repeated allergen challenge altered the number of DCs, lung cells were stained with anti–Gr-1 and anti-CD11c. In mice exposed to 11 challenges, the percentage of mDCs (CD11c+Gr-1–) was significantly decreased compared with that of mice receiving three challenges, returning to control baseline levels (percentage mDCs: 6.5 ± 0.7, 1.4 ± 0.2, and 1.5 ± 0.3 for sensitized mice exposed to 3 challenges, sensitized mice exposed to 11 challenges, and nonsensitized mice exposed to 3 challenges, respectively; n = 6; Figure 8A). Furthermore, expression of CD80 and CD86 on mDCs was significantly lower after 11 challenges, as was the mean fluorescence intensity (Figures 8B and 8C). On the other hand, numbers of pDCs (CD11c+ Gr-1+B220+PDCA-1+) were not significantly different between the groups exposed to 3 and 11 challenges (data not shown).

    Transfer of BMDCs

    Given that repeated allergen challenge, which led to the attenuation of AHR and Th2-mediated inflammation, was associated with decreased numbers of mDCs and lower expression of costimulatory molecules, we investigated whether intratracheal administration of OVA-pulsed mDCs could restore AHR and airway inflammation in recipient mice previously exposed to 11 challenges with OVA. As shown in Figure 9, the transfer of antigen-pulsed BMDCs restored AHR (Figure 9A) and BAL fluid eosinophilia (Figure 9B) compared with control mice.

    DISCUSSION

    Airway inflammation and AHR are hallmarks of allergic asthma. There are many reports in humans and in animal models linking airway inflammation and altered airway function to increases in Th2 cytokine and/or chemokine levels produced by immune cells (27, 28); the common theme is that these factors produce AHR indirectly through the recruitment of inflammatory cells. However, there remains significant controversy as to which cytokine(s) or chemokine(s) are essential to one or both processes. Despite their importance, only limited data are available with respect to the kinetics of development and maintenance or persistence of airway immune/inflammatory responses and AHR after repetitive allergen exposure of sensitized hosts. The primary purpose of the present study was to examine the kinetics of the development and resolution of AHR and airway inflammation after repetitive allergen exposure and relate these changes, when possible, to cytokine and chemokine responses.

    A characteristic and consistent feature of the murine response to sensitization and short-term airway allergen challenge is the development of AHR in response to inhaled MCh. Changes in lung function, measured in a variety of ways, have been demonstrated in both BALB/c and C57BL/6 mice. Although there are strain-related differences in the extent, for example, of changes in lung resistance versus compliance (24), or MCh reactivity (24, 25), AHR remains a consistent outcome of sensitization and short-term airway challenge. Extending airway challenge with the addition of four or eight more exposures over an additional 2 to 4 wk resulted in a marked attenuation of AHR in both strains of mice. In BALB/c mice, AHR was virtually abolished after seven challenges, whereas C57BL/6 mice showed a progressive decrease in AHR with increasing challenges. These decreases in AHR were observed throughout the MCh dose–response curve and in measures of both RL and Cdyn.

    These significant decreases in AHR occurred despite the maintenance of a significant increase in numbers of BAL fluid and lung tissue eosinophils through seven inhalation challenges with allergen. At face value, the data suggest the absence of a correlation between lung and BAL fluid eosinophilia and persistence of AHR. By 11 challenges, this dissociation was no longer apparent, as airway function normalized. The role of eosinophils in AHR is controversial in many species, including humans (29, 30) and mice (31, 32). Even after sensitization and short-term challenge, discrepancies have been reported (33). We and others (31, 34) have suggested that eosinophils and IL-5 are linked to AHR, in contrast to another report (35), at least when sensitization is followed by limited airway challenge (one to three intranasal or nebulized challenges). Moreover, with respect to the temporal sequence of these events, lung tissue eosinophilia has been better correlated with AHR than BAL fluid eosinophil numbers (24). Exceptions to the association of AHR and BAL fluid eosinophilia may be attributed to differences in the challenge protocol and readouts of lung function (36), although the exact reason(s) remain unclear. In a previous study, we also demonstrated that after secondary challenge, the role of eosinophils in the development of AHR became more complicated, and, to some extent, eosinophil independent (37).

    The persistence of lung and BAL fluid eosinophilia in the absence of AHR after seven challenges could be the result of several possibilities. One possibility is that the data simply reflect differences in the kinetics of individual responses. A second possibility is that eosinophilia is not linked to development of AHR; this is possible but less likely on the basis of the data discussed above. These discrepancies are reinforced by two contradictory reports using "eosinophil-less" mice (38, 39). A third alternative is that the eosinophils persist for longer periods in the lung tissue (34), but are no longer activated so as to result in AHR. As there are no good markers of eosinophil activation, this question remains open, and could explain the shift in kinetics or dissociation of eosinophilia and AHR. Since other factors (eotaxin-1, RANTES, and GM-CSF) affecting eosinophil recruitment, survival, and activation were elevated, at least after seven challenges, this may account for the persistent eosinophilia but an eosinophil activation factor present after short-term challenge, such as IL-5, was no longer available. However, our results showed that the levels of RANTES or GM-CSF in BAL fluid were not correlated to the persistence of AHR in mice after repeated OVA challenges.

    It is also striking that other responses implicated in the development of AHR also persisted after repetitive challenges despite the normalization of lung function in response to inhaled MCh. CD4+ T cells are thought to play a central role in the development of AHR and lung inflammation (40). Depletion of CD4+ T cells during the sensitization phase prevented the subsequent development of these responses (41). CD4+ T cells are also responsible for the production, at least in part, of the Th2 cytokines IL-4, IL-5, and IL-13 (11). Virtually all the pathophysiological manifestations of allergen-induced airway inflammation and AHR can be accounted for by these cytokines (42–44). After increasing numbers of challenges, the levels of these Th2 cytokines in BAL fluid were significantly reduced despite persistent elevation of CD4+ T cells in lung tissue. If CD4+ T cells are primarily responsible for Th2 cytokine production, one implication is that after long-term airway challenge, the functional activity of these lung T cells or their phenotype is altered. Although Th2 CD4+ T cells may be essential in the induction phase of allergic responses in the lung, their role over time may become modified (45). It is also possible that with repeated challenges they become "tolerized"—that is, no longer responsive to allergen— although we have no direct data supporting this conclusion.

    Despite the decreases in levels of IL-13 after seven challenges, goblet cell metaplasia/mucus hyperproduction persisted. However, after 11 challenges, this increase in PAS-positive cell numbers was also resolving. IL-13 appears essential for goblet cell metaplasia and mucus production (44). Rather than involving an undefined pathway maintaining this response over seven challenges, the results likely reflect a simple shift in kinetics, with levels of IL-13 falling more rapidly than the disappearance of PAS-positive cells. As observed with eosinophils, continued monitoring for an additional 2 wk showed a loss of these PAS-positive cells.

    In contrast to the decreases in Th2 cytokine production, increases were seen in other cytokine levels. IL-12 levels were increased in the BAL fluid of BALB/c and C57BL/6 mice, but with different kinetics. IL-12 is an important regulator of the balance between Th1 and Th2 cells (46). IL-12 induces production of IFN- (47), but we saw no concomitant increases in the levels of IFN- in BAL fluid. In similar murine models, IL-12 administration did inhibit lung eosinophilia and AHR, as well as decrease IL-4 and IL-5, and increase IFN- (48). IL-12 effects may also be IFN- independent (49). However, it seems unlikely that the dissociation between AHR and lung and BAL fluid eosinophilia after seven challenges was related to IL-12 levels. This dissociation was not seen after 11 challenges in both strains, and elevated IL-12 levels at this time point were associated with the inhibition of AHR as well as the attenuation of lung and BAL fluid eosinophilia.

    Common to both strains after long-term challenge was the increase in BAL fluid IL-10 levels. These IL-10 levels were sustained for a further 2 wk (11 challenges). In general, IL-10 is considered to be an antiinflammatory factor and the release of IL-10 in asthma serves to downregulate the inflammatory reaction associated with this disorder (50–52). IL-10 is produced by CD4+CD25+ regulatory T cells, which have been shown to play a critical role in several models of tolerance (53, 54). Although in our study it is not clear that tolerance was induced by long-term challenge, the increases in IL-10 with repeated challenge could have contributed to the loss of AHR, the reduction in lung BAL fluid eosinophilia, and perhaps the prevention of eosinophil activation. We also considered "tolerance" from the point of view of oral tolerance developing as a result of licking of the pelt over time with repeated exposures. Initial experiments using repeated intranasal challenges did not alter the character of the responses, but simply shifted the kinetics by 1 to 2 wk (data not shown).

    Considerable efforts have been made by several laboratories to develop a murine model of chronic allergic airway inflammation and AHR (55–60) (see Table E1 in the online supplement). The presumption in these studies is that chronic allergic airway inflammation, expressing an eosinophilic signature, is maintained by long-term (repetitive) airway allergen exposure, which leads to structural airway remodeling and, as a result, persistent AHR. Although the experimental designs varied considerably between the studies (i.e., strains of mice, type of allergen, route of sensitization, and duration or number of airway challenges), the endpoint measurements were common and included AHR, airway eosinophilia, and airway tissue remodeling (goblet cell metaplasia, collagen deposition, and thickness of the airway smooth muscle layer). Of significance in evaluating and comparing these studies is the implication of a specific component in the ability to elicit chronic and persistent changes. Thus, the strain of mouse, the specific allergen, the dose and number of allergen challenges, the route of administration, and the intervals between exposures all have been interpreted to explain a successful outcome. There are also considerable differences in what constitutes a persistent change in, for example, lung function or eosinophilic inflammation, where for the most part, all have shown attenuation of both of these responses with increasing allergen exposure, with rare exceptions (60). The demonstration of increased collagen deposition, smooth muscle hypertrophy, or limited airway eosinophilia with increasing allergen exposure, findings we did not observe, is also subject to interpretation. Careful morphometry, avoidance of tangential sections, and exclusion of areas around blood vessels are absolutely necessary to show such chronic changes, especially in the face of normalization of lung function.

    Concern has also been raised that commercial preparations of OVA are contaminated with endotoxin and that endotoxin coadministration with OVA creates a state of tolerance (61). Somewhat to the contrary, endotoxin contamination has also been stated to be necessary for Th2 responses, at least in some strains of mice (62). We have seen little difference in results when using preparations of OVA assayed to be "endotoxin free."

    A number of studies have focused on the functional diversity of DCs in the lung parenchyma and airways (63–66), and the role these cells play in the priming and activation of T lymphocytes (65, 66). The important contribution of DCs in asthma pathogenesis has also been described (16, 17). The role of DCs after chronic or repeated allergen challenge has not been well defined. In the present study, we determined whether alterations in DC function might account for the progressive decreases in AHR and airway inflammation after increasing challenges, while numbers and expression of CD80 and CD86 were significantly decreased after 11 versus 3 challenges. Further, we investigated whether there were changes in DC numbers or phenotype in the peribronchial lymph nodes, and none were detected. It is unclear at present whether the decrease in number and accessory molecule expression of lung mDCs was responsible for the T-cell unresponsiveness to allergen after increased challenges and whether this was linked to the increase in IL-10 seen after 11 challenges. A number of reports suggested that IL-10 attenuates the differentiation and maturation of DCs in vitro, especially mDCs (67–69). Immature mDCs, generated under these high IL-10 conditions, may induce T-cell tolerance (69, 70), thus playing a role in the resolution of AHR and airway allergic inflammation after repeated allergen challenge. de Heer and coworkers reported that pDCs provided intrinsic protection against inflammatory responses to antigen (19). However, in our experiments, pDCs (CD11c+Gr-1+B220+PDCA-1+) were not increased in the lung parenchyma or draining lymph nodes. To directly address the issue of the role of lung mDCs in the resolution of AHR and allergic airway inflammation, transfer experiments were performed. Transfer of antigen-pulsed BMDCs to mice receiving 11 allergen challenges restored responsiveness to allergen, with development of AHR and eosinophilic inflammation. These data suggest that DCs can sustain T-cell–mediated allergic responses in the airways. The mechanism(s) contributing to the restoration of AHR and airway eosinophilia by instillation of mDCs after repeated allergen challenges are not clear. One possibility is that transferred DCs reactivated previously stimulated, antigen-specific T cells. Alternatively, and perhaps more likely, transferred DCs could activate newly primed T cells. BMDCs have the potential to generate primed T cells from naive T cells. Another possibility is that DCs directly cause AHR. Several reports have demonstrated that myeloid DCs have the capacity to produce several cytokines, not only IL-12 and IL-10, but also IL-13, which may directly contribute to airway smooth muscle hypercontractility.

    In summary, this study demonstrates that repeated allergen challenge in two strains of mice results in the loss of AHR to inhaled MCh despite the persistence of airway eosinophilia, at least for a period beyond normalization of lung function. Together with the loss of AHR, there were significant decreases in Th2 cytokine production but consistent and sustained increases in IL-10 and IL-12 levels. Repeated challenge was associated with a reduction in the number of lung mDCs, and transfer of antigen-pulsed DCs reversed this attenuation of AHR and eosinophilic inflammation. This decrease in allergic responsiveness with repeated challenge has plagued the development of a true chronic model of allergen exposure in mice. Nonetheless, the data reveal potential avenues of intervention in preventing some of the long-term consequences of repeated allergen exposure.

    Acknowledgments

    The authors thank Diana Nabighian and Ayako Takahashi for their expert help in preparing the manuscript, and Lynn Cunningham for performing the immunolabeling studies.

    FOOTNOTES

    Supported by NIH grants HL-36577 and HL-61005, and by EPA grant R825702 (to E.W.G.).

    These authors contributed equally to this work.

    This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

    Originally Published in Press as DOI: 10.1164/rccm.200505-783OC on September 28, 2005

    Conflict of Interest Statement: None of the authors have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

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

    ABSTRACT

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

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

    INTRODUCTION

    The "Sentinel" Paradigm of Dendritic Cell Biology

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

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

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

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

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

    Defining Features of the Human DC Family

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

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

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

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

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

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

    DCs OF THE LUNG: LOCALIZATION AND PHENOTYPE

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

    LUNG DCs AND THE SHAPING OF THE PULMONARY IMMUNE RESPONSE

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

    DC Recruitment to the Lung Is Driven by Environmental Stimuli

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

    DCs IN HUMAN LUNG DISEASE: FRIEND OR FOE

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

    Allergic Asthma

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

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

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

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

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

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

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

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

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

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

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

    Chronic Obstructive Pulmonary Disease

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

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

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

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

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

    Pulmonary LCH

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

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

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

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

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

    Lung Cancer

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

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

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

    Lung Transplant Rejection

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

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

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

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

    CONCLUSIONS AND PERSPECTIVES

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

    Acknowledgments

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

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

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