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Published ahead of print on August 20, 2007, doi:10.1165/rcmb.2007-0237OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 38, pp. 161-167, 2008
© 2008 American Thoracic Society
DOI: 10.1165/rcmb.2007-0237OC

Lung Dendritic Cells Have a Potent Capability to Induce Production of Immunoglobulin A

Tateaki Naito1, Takafumi Suda1, Kenichiro Suzuki1, Yutaro Nakamura1, Naoki Inui1, Jun Sato1, Kingo Chida1 and Hirotoshi Nakamura1

1 Second Division, Department of Internal Medicine, Hamamatsu University School of Medicine, Hamamatsu, Japan

Correspondence and requests for reprints should be addressed to Takafumi Suda, M.D., Ph.D., Second Division, Department of Internal Medicine, Hamamatsu University School of Medicine,1-20-1 Handayama, Higashiku, Hamamatsu, 431-3192, Japan. E-mail: suda{at}hama-med.ac.jp


   Abstract
Top
 Abstract
CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
The mucosal immune system provides the first line of defense against inhaled pathogens in the lung. This system is largely mediated by immunoglobulin A (IgA) locally produced by plasma cells, which originate from homing IgA-committed B cells. It has not been determined what types of antigen-presenting cells (APCs) primarily induce B cell differentiation for IgA production in the lung. In addition, although mucosal dendritic cells (DCs) are functionally distinct from DCs in other tissues, it is unclear whether IgA-inducing capability differs between mucosal lung DCs (LDCs) and nonmucosal DCs. The present study was conducted to identify APCs principally responsible for IgA induction in the lung, and to determine potential differences in IgA-inducing capacity between LDCs and nonmucosal DCs. We measured immunoglobulin and cytokine production in a coculture system containing naive IgD+ B cells, naive T cells from ovalbumin-specific T cell–receptor transgenic mice, and APCs including LDCs, alveolar macrophages (AMs), or spleen DCs (SDCs). LDCs induced significantly greater levels of IgA, IgG1, IL-6, and TGF-β than AMs and SDCs, whereas no differences were found in the production of IgM or IgG2a. In addition, the IgA percentage of total class-switched immunoglobulin was highest in cocultures with LDCs (38.4%) when compared with those with AMs (15.1%) and SDCs (22.7%). Neutralizing TGF-β, but not IL-6, significantly decreased IgA induction by LDCs and SDCs, but not by AMs. This study suggests that LDCs are the primary APCs introducing IgA to the lung, and have a more potent IgA-inducing capacity than nonmucosal DCs.

Key Words: immunoglobulin A • lung dendritic cell • alveolar macrophage • TGF-β • interleukin-6



   CLINICAL RELEVANCE
Top
 Abstract
 CLINICAL RELEVANCE
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Our study demonstrates that lung dendritic cells are the principal antigen-presenting cells introducing immunoglobulin (Ig)A to the lung, and that they have a more potent capacity to induce IgA production than nonmucosal dendritic cells.

 
The mucosal immune system provides the first line of defense against inhaled and ingested microbial pathogens. This defense system is to a large extent mediated through the actions of secretory immunoglobulin A (sIgA), which is the most abundantly produced immunoglobulin isotype in the body (1, 2). In the lung, mucosal immune defense of the upper and lower conducting airways has been shown to rely largely on locally produced IgA (3, 4). The primary role of mucosal IgA at the airway surface is to neutralize inhaled bacteria or viruses by interfering with their motility or by inhibiting their adherence to epithelial cells (5). The majority of IgA in the lung is produced by plasma cells that are densely distributed in the mucosal subepithelium of the airway (6). These mucosal plasma cells originate mostly from homing IgA-committed B cells, which undergo µ to {alpha} isotype class switching at inductive sites of mucosal immunity such as Peyer's patches (PPs) and nasopharynx-associated lymphoid tissue (NALT) (7, 8). This process of B cell differentiation, including the class switch, is essential for inducing IgA expression at the mucosal surface. It is principally generated by interaction with helper T cells and antigen-presenting cells (APCs) in the presence of major IgA inducible cytokines such as TGF-β (9–11) and IL-6 (12–14). In the lung, however, it has not been fully determined what types of APCs generate B cell differentiation for IgA induction.

The lung contains several types of professional APCs, including dendritic cells (DCs) and macrophages. DCs are the most potent APCs that play a central role in initiating the primary immune response (15). In the lung, DCs are ideally localized within and beneath the airway epithelium, and also in alveolar septae to perform a sentinel function (16–18). Lung DCs (LDCs) have an excellent capability for uptake and processing of inhaled antigens, and they migrate into regional lymph nodes for the induction of antigen-specific responses (19). Many studies, including ours (20, 21), have shown that LDCs are involved in a variety of pathological processes in the lung. On the other hand, alveolar macrophages (AMs) are highly specialized mononuclear phagocytic cells located in the alveolar space. Our previous studies demonstrated that AMs are different from other macrophages in functional expression of Toll-like receptors (TLRs) and peroxisome proliferator–activated receptors (PPARs) (22, 23). AMs have been reported to have a poor antigen-presenting function when compared with LDCs; they actually down-regulate the immune response in the lung (24). However, AMs were also shown to stimulate T cells in the secondary immune response of the lung and to migrate to regional lymph nodes (25). To date, little is known about the roles of LDCs and AMs in inducing mucosal IgA expression in the airway. There has been only one study comparing the immunoglobulin (Ig)-inducing capacity of LDCs with that of AMs (26). Interestingly, that study showed that human AMs potently induced IgA as well as IgG expression in response to alloantigen, but human LDCs failed to induce either, suggesting that LDCs, despite their potent ability to induce T cell proliferation, are ineffective stimulators of IgA synthesis. However, allogeneic responses usually occur during allogeneic transplantation, and Ig production against alloantigens may not be applicable to that against common foreign antigens. To determine the precise role of LDCs and AMs in IgA induction in response to inhaled foreign antigens, a syngeneic coculture system that includes syngeneic APCs, B cells, and T cells in the presence of a foreign antigen must be employed.

Elements of the tissue microenvironment such as the cytokine milieu influence the phenotypic and functional properties of DCs. DCs at the mucosal surfaces have been reported to be functionally distinct from DCs in other tissues with regard to their profiles of cytokine production and capability for T cell polarization (27). For examples, mucosal DCs in the intestine, particularly DCs obtained from Peyer's patches, preferentially stimulate T cells to produce larger amounts of Th2 cytokines (28). In the same way, mucosal LDCs and nonmucosal DCs may differ in the capacity to induce IgA. However, there have been no studies comparing the IgA-inducing capacity of LDCs with that of nonmucosal DCs.

In the present study, to clarify the types of APCs that are primarily responsible for IgA induction in the lung, we examined the ability of LDCs and AMs to induce IgA production in a coculture system using APCs, syngeneic naive B cells, and naïve T cells from ovalbumin (OVA)-specific T cell-receptor (TCR) transgenic (Tg) mice in the presence of OVA. Additionally, to determine whether LDCs are different from nonmucosal DCs with regard to their IgA-inducing capacity, we compared IgA induction by LDCs with induction by nonmucosal, spleen DCs (SDCs).


   MATERIALS AND METHODS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Mice
Experiments were performed on 10-week-old male BALB/c mice and OVA-specific TCR Tg mice (DO11.10) on a BALB/c background (Nippon SLC, Shizuoka, Japan).

Preparation of AMs, LDCs, and SDCs
AMs and LDCs were obtained as described in our previous study (23). Briefly, AMs were harvested by whole lung lavage with 5 ml of ice-cold PBS. The purity of AMs was routinely greater than 98%, as determined by their morphology. LDCs were isolated using enzymatic digestion and immunomagnetic cell sorting. Briefly, a single lung cell suspension from lungs digested with collagenase and DNase was incubated for 90 minutes at 37°C, and nonadherent cells were discarded. Afterward, loosely adherent cells were harvested. To purify LDCs, CD45R (B220)-negative and I-Ad–positive cells were sorted by magnetic cell sorting (MACS; Miltenyi Biotech, Gladbach, Germany). The resulting DC preparation was more than 95% I-Ad+/CD11c+ cells, contained less than 1% F4/80+ cells and less than 1% B220+ cells as determined by flow cytometry, and showed the characteristic morphology of DCs (see online supplement for details). Spleens were digested with collagenase and then sorted using MACS with anti-CD11c mAb-conjugated magnetic beads. The splenic DC preparations were over 95% I-Ad+/CD11c+ cells and contained less than 2% F4/80+ cells, less than 3% CD19+ cells, and less than 2% B220+ cells as determined by flow cytometry.

Preparations of Naive B Cells and T Cells
Naive B cells were isolated from spleens of wild-type BALB/c mice. Of digested spleen cells, surface IgD+ B lymphocytes were separated using MACS (see online supplement for details). Isolated B cell populations were routinely 95 to 98% positive for the surface markers sIgD and B220. Naive T cells were obtained from OVA-specific TCR Tg mice (DO11.10). Spleen CD4+ CD62Lhigh T cells were prepared using MACS (see online supplement for details). Isolated naive TCR-Tg T cell populations were routinely 95 to 97% cells expressing CD4 and CD62Lhigh.

Induction of Ig Production
Ig production was induced with a modified method as previously described (29). Briefly, sIgD+ naive B cells (1.25 x 106/ml), naive DO11.10 T cells (1.0 x 106/ml), and either AMs, LDCs, or SDCs (5.0 x 104/ml) were cocultured in the presence or absence of 100 µg/ml of OVA (Sigma-Aldrich, St. Louis, MO) on flat- or round-bottom 96-well plates, or flat-bottom 24-well plates, for 7 days. Cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, 1% penicillin-streptomycin, and 3 mM L-glutamine. Neutralizing antibodies against IL-6 (10 µg/ml; BD PharMingen, San Diego, CA), TGF-β1 (50 µg/ml; R&D Systems, Minneapolis, MN), or rat IgG1 as an isotype control (10–50 µg/ml; BD PharMingen) were added to some cultures.

Measurement of Immunoglobulins and Cytokines
The levels of IgA, IgM, IgG1, IgG2a, and TGF-β in the supernatants obtained from 7-day cocultures were measured by enzyme-linked immunosorbent assay (IgA, IgM, IgG1, and IgG2a; Bethyl, Montgomery, TX; and TGF-β1; Promega, Madison, WI). The levels of cytokines in the supernatants obtained from the 7-day coculture were also measured by BD mouse cytometric bead array kits (IL-12p70, IFN-{gamma}, IL-4, IL-13, IL-10, and IL-6 flex bead set) according to the manufacturer's protocol (BD PharMingen).

Flow Cytometry
For measuring surface expression of immunostimulatory molecules, cells were analyzed with an EPICS XL flow cytometer (Beckman Coulter, Fullerton, CA). The cells were incubated with the appropriately diluted fluorescein isothiocyanate (FITC)-conjugated anti-I-Ad (Miltenyi Biotech), anti-IgD or anti-CD4 mAb, phycoerythrin (PE)-conjugated anti-CD11c or anti-B220 mAb, and biotin-conjugated anti-CD8{alpha}, anti-F4/80, anti-CD11b, anti-CD40 (BD PharMingen), anti-CD62L, anti-CD80, anti-CD86, anti-Gr-1 (Beckman Coulter) or anti-DEC205 mAb (Acris, Hiddenhausen, Germany), followed by incubation with streptavidin-conjugated Quantum Red (Sigma). The following Abs were used as controls: FITC-conjugated rat IgG2a or rat IgG2b, phycoerythrin-conjugated rat IgG2a (Beckman Coulter) or hamster IgG1 (BD PharMingen), and biotin-conjugated rat IgG2a or rat IgG2b (Beckman Coulter).

Statistical Analysis
One-way ANOVA was used for comparisons of groups. The Tukey-Kramer test was used for comparisons of each group. P values less than 0.05 were considered significant. All data are expressed as mean ± SEM.


   RESULTS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Phenotypes of LDCs and SDCs
We first examined the phenotypes of obtained LDCs and SDCs using flow cytometry. The expression of I-Ad, CD11c, DEC205, and CD40 by LDCs was almost comparable to that by SDCs (Figure 1). However, LDCs showed slightly, but significantly, higher expression of CD80 (mean fluorescence intensity of the LDC, 13.6 ± 4.1, versus SDC, 4.1 ± 0.6; P < 0.05, n = 5), and CD86 (LDC, 28.0 ± 0.5, versus SDC, 5.5 ± 0.8; P < 0.05, n = 5) than SDCs. No expression of B220 or Gr-1 was found on LDCs or SDCs (data not shown), indicating that these prepared DCs were not plasmacytoid.


Figure 1
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  		Figure 1. Phenotypes of lung dendritic cells (LDCs) and spleen dendritic cells (SDCs). Expression of surface molecules by LDCs and SDCs isolated by MACS (shaded histograms). Open histograms show isotype controls.

 
Coculture System for Evaluating Ig Production
In initial experiments, we attempted to establish appropriate coculture conditions including LDCs as APCs, naive sIgD+ B cells, and naive DO11.10 T cells for assessing the capacity of APCs to induce Ig production. As shown in Figure 2A, considerable levels of total Ig in the culture supernatant were detected when cells were cocultured in round-bottomed 96-well plates in the presence of OVA, but negligible amounts of total Ig were found when cultured in the absence of OVA or the presence of OVA in flat-bottom 96-well plates or 24-well plates. These results suggest that close cell-to-cell contact was critical for inducing Ig production and that production was antigen-dependent in this coculture system. In cultures with naive sIgD+ B cells plus naive DO11.10 T cells, naive sIgD+ B cells plus APCs, or naive sIgD+ B cells alone, the levels of total Ig production were negligible even in the presence of OVA, whereas high levels of Ig were produced in cultures with naive sIgD+ B cells, naive DO11.10 T cells, and APCs (Figure 2B). Thus, these three cell populations were necessary for optimal Ig production.


Figure 2
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  		Figure 2. Immunoglobulin (Ig) induction in various culture conditions. (A) Naive IgD+ B cells were cocultured with antigen-presenting cells (APC) and naive DO11.10 T cells in the presence or absence of ovalbumin (OVA) for 7 days. Round-bottom plates (96-well) or flat-bottom plates (96-well or 24-well) were used. Concentrations of Ig in the supernatants were measured by enzyme-linked immunosorbent assay (ELISA). Values are expressed as the mean ± SEM of five experiments. (B) Naive IgD+ B cells (B) were cocultured with or without APC or naive DO11.10 T cells (T) in the presence or absence of OVA in round-bottom 96-well plates for 7 days. Values are expressed as the mean ± SEM of five experiments. *P < 0.05, **P < 0.01.

 
Induction of Ig Production by LDCs, AMs, and SDCs
To clarify a role for LDCs and AMs in lung Ig induction, we compared the levels of IgA, IgM, IgG1, and IgG2a in culture supernatants taken from coculture systems with LDCs (LDC culture) and with AMs (AM culture). Strikingly, much higher levels of IgA were found in the LDC culture than in the AM culture, in which IgA levels were very low (Figure 3A). In contrast, the levels of IgM and IgG2a did not differ significantly between AM and LDC cultures (Figures 3B and 3D). In addition, the LDC culture produced significantly higher levels of IgG1 than the AM culture (Figure 3C). To address whether mucosal LDCs have a distinct ability to induce IgA production when compared with nonmucosal DCs, we compared Ig production of cocultures with LDCs to those with SDCs (SDC culture). As shown in Figure 3, LDCs induced significantly higher levels of IgA and IgG1 production than SDCs, whereas no difference was found in the levels of IgM and IgG2a between LDC and SDC cultures. In the absence of any type of APCs (control culture), IgA could not be induced. Collectively, these results suggest that LDCs have a potent capability to direct naive sIgD+ B cells toward IgA and IgG1 secretion when compared with AMs and SDCs.


Figure 3
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  		Figure 3. Induction of immunoglobulin production by LDCs, AMs, and SDCs. LDCs, AMs, or SDCs were cocultured with naive IgD+ B cells and naive DO11.10 T cells in the presence of OVA for 7 days. The control group contained naive IgD+ B cells and naive DO11.10 T cells without APCs in the presence of OVA. The levels of (A) IgA, (B) IgM, (C) IgG1, and (D) IgG2a in the supernatants were measured by ELISA. Values are expressed as the mean ± SEM of eleven experiments. *P < 0.05, **P < 0.01.

 
Regarding the constitution of Ig isotypes induced by each APC, the highest percentage of IgA production (38.4%) was found in LDC culture, followed by SDC (22.7%) and AM cultures (15.1%) (Figure 4). In LDC cultures, the most abundant Ig was IgA, whereas in AM and SDC cultures, IgM was the most highly produced Ig isotype (Figure 4).


Figure 4
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  		Figure 4. Constitution of Ig isotypes induced by LDCs, AMs, or SDCs. LDCs, AMs, or SDCs were cocultured with naive IgD+ B cells and naive DO11.10 T cells in the presence of OVA for 7 days. The percentages of IgA, IgM, IgG1, or IgG2a of class-switched Ig produced in the supernatants are schematically represented. Values are expressed as the mean of 11 experiments and two replicates in each experiment.

 
Production of Cytokines in APC Cultures
Several cytokines, including IL-6, IL-10, and TGF-β, have been shown to be directly involved in IgA induction (6). To elucidate whether the potent capacity of LDCs to induce IgA production is associated with production of these IgA-inducible cytokines, we measured cytokine levels in the supernatant of each APC culture. Among lung APC cultures, significantly higher levels of IL-6 were found in LDC than in AM cultures, whereas there was no difference in the levels of IL-10 or TGF-β (Figure 5). When compared with SDC cultures, LDC produced significantly higher amounts of IL-6 and TGF-β, but not IL-10 (Figure 5). In addition to IgA-inducible cytokines, we also measured the levels of the IgG1-inducible cytokines IL-4 and IL-13, and the IgG2a-inducible cytokines IL-12p70 and IFN-{gamma}. Levels of IL-13 were significantly higher in LDC cultures than in AM and SDC cultures, whereas no significant differences were found in levels of the remaining cytokines (Figures 5).


Figure 5
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  		Figure 5. Cytokine production in coculture system. LDCs, AMs, or SDCs were cocultured with naive IgD+ B cells and naive DO11.10 T cells in the presence of OVA for 7 days. Levels of TGF-β, IL-6, IL-10, IL-4, IL-13, IL-12p70, and IFN-{gamma} were measured by ELISA or cytometric bead array. Values are expressed as the mean ± SEM of 11 experiments. *P < 0.05, **P < 0.01.

 
Effect of Neutralizing IL-6 and TGF-β on Ig Production
Because LDC cultures had significantly higher levels of IL-6 than AM cultures and higher levels of IL-6 and TGF-β than SDC cultures, we examined whether production of these cytokines was directly responsible for the potent capability of LDCs to induce IgA. We measured the levels of IgA production in cultures in the presence of anti-mouse IL-6 or TGF-β antibodies. Neutralization of TGF-β significantly decreased IgA production in LDC and SDC cultures, but not in AM cultures (Figure 6). In contrast, treatment with anti-mouse IL-6 mAb did not significantly change IgA production in LDC, AM, or SDC cultures (data not shown). These results suggest that IgA production of DC cultures, but not AM cultures, was TGF-β dependent, and that the potent capability of LDCs to produce IgA is partly associated with high levels of secreted TGF-β.


Figure 6
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  		Figure 6. Effect of anti–TGF-β antibody on the induction of IgA. LDCs, AMs, or SDCs were cocultured with naive IgD+ B cells and naive DO11.10 T cells in the presence of OVA for 7 days. Neutralizing antibody against TGF-β or isotype control was added as indicated. The levels of IgA in the supernatants were measured by ELISA. Values are expressed as the mean ± SEM of three experiments. **P < 0.01.

 

   DISCUSSION
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
The present study examined the capability of LDCs to induce IgA production, and also compared this capability with that of AMs and SDCs. We found that LDCs induced significantly larger levels of IgA production than AMs and SDCs. In addition, LDCs secreted high levels of IgA-inducible cytokines such as TGF-β, which may be associated with their potent IgA-inducing capacity. These data suggest that LDCs are the APCs primarily responsible for IgA induction in the lung, and have a more powerful capacity to induce IgA than nonmucosal DCs.

To examine induction of Ig production by APCs, we first set up a coculture system using APCs, naive sIgD+ B cells, and naive OVA-Tg T cells. In vivo, direct interaction among APCs, B cells, and T cells in the germinal centers is considered to be essential for the generation of memory B cells in response to T cell–dependent antigens, affinity maturation of the B cell receptor (BCR), and Ig-class switching (30). In this context, the coculture system we used was an appropriate model to assess Ig induction, because Ig production in this system was highly dependent on T cell help, as well as the presence of antigen and close cell-to-cell interaction (30). Using this coculture system, we examined the Ig-inducing capacity of lung APCs, LDCs, and AMs. Interestingly, we found that LDCs induced much greater production of IgA and IgG1 than AMs, whereas IgM and IgG2a induction levels remained similar across culture types. In contrast to our results, Wilkes and Weissler reported that human LDCs were incapable of inducing IgG or IgA in response to an alloantigen, whereas AMs were potent stimulators of Ig production (26). They concluded that the principal lung APCs responsible for Ig induction, including that of IgA, were AMs but not DCs. The reason for this discrepancy between the Wilkes and Weissler study and ours is unknown. One possible explanation is a difference in the antigen used. Wilkes and Weissler examined Ig production against alloantigens in cultures with APCs and allogeneic peripheral blood mononuclear cells. In contrast, we assessed Ig production in culture with APCs, syngeneic naive B cells, and naive TCR Tg T cells in the presence of a foreign antigen, OVA. Alloantigens are defined as antigens that are a part of a self-recognition system involved in transplant rejection. Thus, alloantigens are thought to have their own distinct mechanism of stimulating immunity from foreign antigens, which may cause the discrepancy in Ig induction between the Wilkes and Weissler study and ours. In the lung, inhaled antigens inducing the immune response are generally foreign antigens such as bacteria and viruses. Thus, our coculture system may be more appropriate in assessing the capacity of lung APCs to induce Ig production under physiologic conditions. Another possible explanation for the discrepancy is a difference in the maturation status of LDCs. LDCs used in the present study were relatively mature in terms of their expression of costimulatory molecules, but Wilkes and Weissler did not describe the expression of these molecules in their LDCs. Thus, we could not directly compare the maturation status of our LDCs with that of the LDCs used in the Wilkes and Weissler study, and LDC maturation status may be a factor affecting the results. Regarding the production of IgA-inducible cytokines in the coculture system, LDC cultures contained significantly higher levels of IL-6, which augments IgA production from IgA-committed B cells (12, 14), than did AM cultures. Thus, we hypothesized that high levels of IL-6 production in LDC cultures might be partly responsible for their strong IgA-inducing ability. However, treatment with an anti–IL-6 antibody did not significantly reduce the IgA level in LDC cultures, suggesting that other factors are associated with the difference in IgA induction between LDCs and AMs. Collectively, our data suggest that the two major lung APCs, LDCs and AMs, differ in their profiles of induced-Ig isotypes, and that LDCs are the primary APCs responsible for IgA induction in the lung.

When we compared the IgA-inducing capacity of LDCs with that of nonmucosal SDCs, we found that LDCs induced significantly higher levels of IgA and IgG1 than SDCs, whereas no significant difference was observed in the levels of IgM or IgG2a. In particular, the amount of IgA production was much larger (3-fold) in LDC cultures than in SDC cultures. In addition, LDCs induced significantly higher levels of TGF-β and IL-6 production than SDCs. TGF-β has been shown to be a crucial IgA class-switch factor for activated B cells, whereas IL-6 is important for expansion and terminal differentiation of IgA-committed B cells (14, 31–33). To determine whether enhanced IgA production in LDC cultures can be explained by increased secretion of TGF-β or IL-6, we performed blocking experiments with neutralizing antibodies for TGF-β and IL-6. Interestingly, treatment with the anti–TGF-β antibody markedly decreased IgA production in both LDC and SDC cultures. Conversely, treatment with the anti–IL-6 antibody did not significantly decrease IgA production in LDC or SDC cultures. Thus, the potent capability of LDCs to induce IgA was associated, in part, with their ability to promote TGF-β secretion.

Regarding maturation status, LDCs showed relatively higher expression of costimulatory molecules, including CD80 and CD86, than did SDCs. Thus, there is a possibility that the difference in maturation state between LDCs and SDCs might affect their IgA-inducing capacity. To address this issue, we examined IgA induction by SDCs that had been matured by treatment with LPS or CpG to induce comparable mean fluorescence intensity of CD80 and CD86 to that of LDCs (data not shown). LPS- or CpG-stimulated DCs secreted abundant amounts of a variety of cytokines, including IL-6, IL-10, and IL-12, and dramatically increased the production of all Ig isotypes. Thus, we could not used LPS- or CpG-stimulated SDCs as matured SDCs because LPS- and CpG-activated SDCs were not comparable with LDCs in terms of their ability to produce cytokines. It is unlikely that maturation is the sole factor responsible for enhanced IgA induction by LDCs because the profile of Ig istotypes produced did not change when using LPS- or CpG-matured SDCs (data not shown). Taken together, these results indicate that LDCs have a distinct ability to induce high levels of IgA production when compared with nonmucosal SDCs.

Recently, Mora and colleagues reported that the DC from gut-associated lymphoid tissue (GALT) could directly induce IgA secretion from the naive B cells without T cell help (34). The T cell–independent IgA induction was mainly mediated by the synergetic effect of the retinoic acid with IL-6 or IL-5, which were derived from the DC from the GALT. Furthermore, they showed that TGF-β1 decreased the synergism of retinoic acid and IL-5 or IL-6 on IgA secretion, indicating that there would be a different role of TGF-β1 in T cell–independent IgA induction compared with that in T cell–dependent IgA induction. It would be meaningful to study whether the airway mucosal immune system has the similar T cell–independent IgA induction mechanisms and to disclose the relationship among the mediators including IL-5, IL-6, retinoic acid, and TGF-β1 in B cell priming for IgA response in the lung. In addition, because the present study was conducted in vitro, it would give us more important knowledge to elucidate a role of LDCs in in vivo IgA induction in the lung. Future studies to explore in vivo IgA production against foreign antigens using mice conditionally depleted of DC, such as CD11c-DTR mice (35), will clarify this.

In conclusion, the present study demonstrates that LDCs have a potent capability to induce IgA production when compared with AMs, suggesting that LDCs are the primary APCs responsible for stimulating the production of IgA at the mucosal surface. Moreover, LDCs promoted higher levels of IgA production than SDCs, indicating that mucosal LDCs functionally differ from nonmucosal SDCs in their ability to induce IgA. Because IgA plays a crucial role in the mucosal defense against invading pathogens, our data provide important knowledge that contributes to the further understanding of LDC function in the mucosal immunity of the lung.


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

Originally Published in Press as DOI: 10.1165/rcmb.2007-0237OC on August 20, 2007

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

Received in original form June 22, 2007

Accepted in final form August 6, 2007


   References
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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