HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sato, A. Articles by Kaminogawa, S. Search for Related Content PubMed PubMed Citation Articles by Sato, A. Articles by Kaminogawa, S.

CD11b⁺ Peyer’s Patch Dendritic Cells Secrete IL-6 and Induce IgA Secretion from Naive B Cells¹

Ayuko Sato^2,*, Masaaki Hashiguchi^3,*, Etsuko Toda^*, Akiko Iwasaki, Satoshi Hachimura^4,* and Shuichi Kaminogawa^*,

^* Department of Applied Biological Chemistry, University of Tokyo, Tokyo, Japan; Department of Epidemiology and Public Health, Yale University School of Medicine, New Haven, CT 06520; and Department of Food Science and Technology, College of Bioresource Sciences, Nihon University, Kanagawa, Japan

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Peyer’s patch (PP) dendritic cells (DCs) have been shown to exhibit a distinct capacity to induce cytokine secretion from CD4⁺ T cells compared with DCs in other lymphoid organs such as the spleen (SP). In this study, we investigated whether PP DCs are functionally different from DCs in the SP in their ability to induce Ab production from B cells. Compared with SP DCs, freshly isolated PP DCs induced higher levels of IgA secretion from naive B cells in DC-T cell-B cell coculture system in vitro. The IgA production induced by PP DCs was attenuated by neutralization of IL-6. In addition, the induction of IgA secretion by SP DCs, but not PP DCs, was further enhanced by the addition of exogenous IL-6. Finally, we demonstrated that only PP CD11b⁺ DC subset secreted higher levels of IL-6 compared with other DC subsets in the PP and all SP DC populations, and that PP CD11b⁺ DC induced naive B cells to produce higher levels of IgA compared with SP CD11b⁺ DC. These results suggest a unique role of PP CD11b⁺ DCs in enhancing IgA production from B cells via secretion of IL-6.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Although it is known that Ags and microorganisms from the intestinal lumen are transported into the Peyer’s patch (PP)⁵ by M cells present in the follicle-associated epithelium, the details of immune regulation within the PP are just beginning to be understood. PP represents the primary site for Ag processing and presentation in the intestine. Previous studies have suggested that dendritic cells (DCs) are the major APC population in the subepithelial dome (SED) of the PP, the region just beneath the follicle-associated epithelium (1, 2, 3). In addition, isolated DCs from the PP have been shown to stimulate T cells in MLR (4, 5) and to support IgA B cell differentiation (6, 7, 8, 9). Moreover, it was reported that freshly isolated PP DCs are functionally distinct from DCs from the spleen (SP) with regard to their capacity to induce Th cell differentiation in vitro (5); PP DCs were shown to prime naive CD4⁺ Ag-specific T cells to secrete IL-10, IL-4, and IFN-, whereas SP DCs predominantly primed CD4⁺ T cells to secrete IFN-. In addition, only PP DCs were shown to produce significant levels of IL-10 after stimulation in vitro with recombinant CD40 ligand (5). These prior studies have provided important information concerning the particular role of DCs in Ag presentation in the PP.

More recently, Iwasaki and Kelsall (10, 11) identified three distinct subsets of DCs in murine PP by immunohistochemical analysis. One population of DCs, CD11b⁺/CD8^- DCs, reside in SED region, whereas another subset, CD11b^-/CD8⁺ DCs, reside in the T cell-rich interfollicular region (IFR), and DCs that lack expression of CD11b or CD8 (double negative) are present in both the SED and IFR. All DC subpopulations maintained their surface phenotype upon maturation in vitro. These data suggested that these DC subsets are derived from different lineages. CD11b⁺ DCs from PP produce high levels of IL-10 upon in vitro stimulation. In contrast, CD8⁺ and double negative DCs, but not CD11b⁺ DCs, produce IL-12p70 following microbial stimulation. Moreover, CD11b⁺ DCs from PP were found to induce T cell differentiation to secrete high levels of IL-4 and IL-10, when compared with those from nonmucosal sites, whereas CD8⁺ and double negative DCs from all tissues induced IFN- production. This suggested that CD11b⁺ DCs from PP have unique immune inductive capacities.

Immune responses at the mucosal surfaces including the intestinal epithelium are characterized by the protection mediated by the secreted IgA. The PP has been considered to be the major inductive site for IgA. The following studies demonstrated that PP DCs play an important role in the induction of IgA production. Induction of significant polyclonal IgA secretion both by PP and SP B cells was shown in coculture of B cells with DC-T cell mixtures derived from murine PP but not SP (7). Nontransformed pre-B cells were induced to differentiate in vitro to secrete IgA with only DCs and Ag-stimulated T lymphocytes (8). This study showed that PP DCs induced high levels of IgA in combination with T cells derived from either the SP or the PP, and that both the isotype of Ab secreted and the extent of pre-B cell differentiation were determined by the lymphoid tissue source of DC, not of T cells. The importance and sufficiency of PP DCs in IgA induction from B cells were demonstrated in a study by George et al. (9), in which PP DCs, but not Th cells, were able to promote IgA secretion from B cells in a microculture. Although these data suggested the importance of PP DCs in the generation of IgA responses in the intestine, the mechanisms of induction of IgA production by PP DCs still remain unclear.

Previous studies have shown that cytokines, such as TGF- (12, 13, 14), IL-5 (15, 16, 17, 18, 19, 20), and IL-6 (19, 21, 22) are important factors for the development of IgA-producing B cells. PP is not the only inductive site for IgA-secreting plasma cells in the gut. Several studies have identified that up to 40% of IgA cells in the intestinal lamina propria arise from a pool of B-1 precursors derived from the peritoneal cavity (23), which are distinct from the conventional PP precursors by their anatomical origin, function, and expression of the CD5 surface marker (24, 25). CD5⁺ peritoneal cavity B cells are able to produce IgA in vitro in the absence of IL-6 (26). Therefore, more than one pathway exists for IgA production, and that by CD5⁺ B-1 cells is IL-6 independent. On the other hand, the potential role of IL-6 in IgA production by B-2 cells, which are the predominant B cells in the PP, has been supported by analysis of IL-6-deficient mice (22).

In this report, we demonstrate that PP DCs, particularly the CD11b⁺ DCs found in the SED region, can produce higher levels of IL-6 compared with SP DCs. Moreover, we demonstrate that upon in vitro culture, PP DCs, especially the CD11b⁺ subset, have a particular capacity to induce IgA production compared with SP DCs. This effect of the PP DCs is largely diminished upon addition of IL-6 neutralizing Ab in the DC-T-B coculture. These data suggest that PP DCs have a unique ability to secrete IL-6 and contribute to the establishment of IgA secretion in the intestinal mucosa.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

Female BALB/c mice were purchased from CLEA Japan (Tokyo, Japan) and used at 6–7 wk of age. OVA-specific TCR transgenic (Tg) mice (OVA 23–3 (27) and DO11.10 TCR Tg mice (28) were maintained by back-crossing to BALB/c mice. Female Tg mice were used at 8–22 wk of age.

Culture medium

Cells were cultured in RPMI 1640 medium supplemented with 5% heat-inactivated FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 3 mM L-glutamine, and 50 µM 2-ME

RT-PCR

Total RNA was isolated from sorted DC populations by RNeasy (Qiagen, Hilden, Germany). Single-stranded cDNA was synthesized using SuperScript preamplification system (Life Technologies, Gaithersburg, MD). PCR was conducted for 40 cycles using primer pairs for IL-6, forward, 5'-TGGAGTCACAGAAGGAGTGGCTAAG-3'; reverse, 5'-TCTGACCACAGTGAGGAATGTCCAC-3' and GAPDH, forward, 5'-TGAACGGGAAGCTCACTGG-3'; reverse, 5'-TCCACCACCCTGTTGCTGTA-3' and for fluorophore-labeled hybridization probes for IL-6, forward, 5'-LC-AACCTAGTGCGTTATGCCTAAGC-3'; reverse, 5'-AAGTCACTTTGAGATCTACTCGGC-FITC-3' and GAPDH, forward, 5'-LC-TTCAACAGCAACTCCCACTCTTCCACC-3'; reverse, 5'-CTGAGGACCAGGTTGTTGTCTCCTGCGA-FITC-3' with the LightCycler (Roche Diagnostics, Rotkreuz, Switzerland).

Antibodies

Surface phenotype of DCs was analyzed with purified mAb as follows. Hamster anti-mouse CD11c was purified from hybridoma, N418 obtained from the American Type Culture Collection (HB224). Rat anti-mouse CD8 (53-6.7) was purchased from BD PharMingen (San Diego, CA). Purified monoclonal rat anti-mouse CD11b (M1/70) was purchased from Caltag Laboratories (Burlingame, CA). Before staining, Fc receptors (FcRIII/II) were blocked using anti-mouse CD16/CD32 (2.4G2). Naive T cells from OVA TCR Tg mice were stained with anti-mouse CD4-FITC (H129.19) and anti-mouse CD62L-biotin (MEL-14) and Streptavidin-PE. For isolation of naive B cells, anti-mouse IgD (Southern Biotechnology Associates, Birmingham, AL) was used. For stimulation to DC, hamster anti-mouse CD40 (HM40-3) and anti-hamster IgM (G188-2) were purchased from BD PharMingen.

Preparation of whole DCs and subpopulation of DCs

DCs were prepared from PP and SP of naive 6-wk-old mice in parallel. PP and SP were digested with collagenase type I (1 mg/ml; Sigma-Aldrich, St. Louis, MO), and a single cell suspension was prepared. The cells were incubated with anti-mouse CD11c-coated magnetic beads (Miltenyi Biotec, Auburn, CA) and selected on MACS separation columns (Miltenyi Biotec). Cells selected on the basis of CD11c expression were stained with biotinylated hamster anti-mouse CD11c Ab, streptavidin-PE and FITC-labeled anti-B220 Ab. CD11c⁺/B220^- cells were purified by flow cytometric sorting performed using a FACSVantage (BD Immunocytometry Systems, Mountain View, CA). For isolation of DC subpopulations, cells selected on the basis of CD11c expression by MACS were stained with biotinylated hamster anti-mouse CD11c Ab and streptavidin-Red670 (Life Technologies) and FITC-labeled anti-B220 Ab and either PE-labeled anti-CD11b or PE-labeled CD8, and then CD11b⁺/CD11c⁺/B220^- and CD8⁺/CD11c⁺/B220^- DCs were sorted by using FACSVantage. Sorted DC populations were routinely 97–100% positive for the surface marker of interest.

Stimulation of DCs in vitro

FACS-purified DC populations were preincubated with hamster anti-mouse CD40 (50 µg/ml) and then incubated with anti-hamster IgM (5 µg/ml) to cross-link anti-CD40. Alternatively, DCs were incubated with PMA (50 ng/ml) and Ca²⁺ ionophore (A23187, 250 ng/ml), or with LPS (20 µg/ml). Supernatants were harvested at 48 h, and IL-6 levels were measured by ELISA.

Stimulation of TCR Tg T Cells by DCs or DC populations

Naive SP CD4⁺ CD62L^high T cells from OVA-specific TCR Tg mice (OVA 23–3) were prepared using MACS. Isolated naive T cells were routinely 95–97% positive for CD4⁺ CD62L^high.

Primary stimulation cultures were established by coincubation of MACS-purified naive OVA TCR Tg CD4⁺ T cells from SP (1 x 10⁵ per well) and sorted CD11c⁺/B220^-, CD11b⁺/CD11c⁺/B220^-, or CD8⁺/CD11c⁺/B220^- DCs from SP or PP (1 x 10⁴ per well). Cells were incubated with OVA (1 mg/ml) in 96-well plates for 48 h. Supernatants were collected, and then IL-2, IL-4, IL-5, IL-6, IL-10, and IFN- levels were measured by ELISA.

Induction of Ig production by DCs

Naive SP CD4⁺ CD62L^high T cells from splenocytes of TCR Tg mice (DO11.10 TCR Tg mice) were prepared using MACS. Naive B cells were isolated from SP of BALB/c mice. SP were digested with collagenase, and surface (s)IgD⁺ B lymphocytes were separated using MACS. Isolated B cells were routinely 95–98% positive for the surface marker of sIgD⁺/B220⁺. sIgD⁺ B lymphocytes (2.5 x 10⁵ per well) and MACS-purified OVA TCR Tg naive T cells from SP (2 x 10⁵ per well) and sorted CD11c⁺/B220^- DCs or CD11b⁺/CD11c⁺/B220^- or CD11b^-/CD11c⁺/B220^- DC subsets from SP or PP (1 x 10⁴ per well) were cultured in the presence or absence of OVA (100 µg/ml) on 96-well plates for a week. Recombinant IL-6 (10 ng/ml, BD PharMingen), neutralizing Ab against IL-6 (10 µg/ml, BD PharMingen), or rat IgG1 as isotype control (10 µg/ml, BD PharMingen) was added to some cultures. Supernatants were collected, and IgA, IgM, IgG1, and IgG2a levels were measured by ELISA.

Measurement of cytokine and Ig secretion

Cytokine and Ig productions in the culture supernatants were assayed by specific sandwich ELISA. A number of 96-well immunoplates (Nunc, Roskilde, Denmark) were coated with rat anti-mouse IL-2 (JES6-1A12, BD PharMingen), rat anti-mouse IL-4 (11B11, BD PharMingen), rat anti-mouse IL-5 (TRFK5, BD PharMingen), rat anti-mouse IL-6 (MP5-20F3, BD PharMingen), rat anti-mouse IFN- (R4-6A2, BD PharMingen), goat anti-mouse IgA (Zymed Laboratories, South San Francisco, CA), goat anti-mouse IgM (Organon Teknika, Durham NC) and goat anti-mouse IgG (Sigma-Aldrich). After washing and blocking the plates, samples and standards ware added. Bound cytokine was detected using biotin-labeled monoclonal anti-mouse IL-2 (JES6-5H4, BD PharMingen), rat anti-mouse IL-4 (BVD6-24G2, BD PharMingen), rat anti-mouse IL-5 (TRFK4, BD PharMingen), rat anti-mouse IL-6 (MP5-32C11, BD PharMingen) or rat anti-mouse IFN- (XMG1.2, BD PharMingen), respectively. This was followed by incubation with alkaline phosphatase-labeled streptavidin (Zymed). For Ig quantification, alkaline phosphatase-conjugated monoclonal anti-mouse IgA, IgM, IgG1, and IgG2a (Zymed) were used. The plates were then washed and the substrate (disodium p-nitrophenylphosphate) was added. Absorbance was determined at a wavelength of 405 nm. IL-10 was measured using the OptEIA set (BD PharMingen).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Induction of Ig production by DCs from PP and SP

In initial experiments, to address whether PP DCs have a particular capacity to induce IgA production, we compared Ig production levels in an in vitro culture system in which DCs from PP or SP were used as the APCs. Freshly isolated PP or SP DCs were purified on the basis of surface marker (CD11c⁺/B220^-) by flow cytometric sorting. Naive sIgD⁺ B lymphocytes isolated from SP of BALB/c mice, naive T cells from SP of OVA TCR Tg mice, and sorted CD11c⁺/B220^- DCs from either PP or SP were cocultured in the presence of OVA. Fig. 1 is representative data that depict the levels of IgA secreted in these DC-T-B coculture systems. Consistently, higher IgA production was observed when PP DCs were used as the APCs compared with those wells containing SP DCs (p < 0.01). In some experiments, we observed a tendency for PP DCs to induce higher levels of IgM production compared with SP DCs as shown in Fig. 1 (not statistically significant); however, in other experiments, there was no difference between cultures containing PP DCs or SP DCs (data not shown). IgG1 or IgG2a levels did not differ significantly between PP DCs and SP DC cultures either. To examine the importance of DC in directing IgA secretion from B cells, we have analyzed IgA production levels in either CD3-stimulated T cell-B cell culture or Ag-stimulated T cell-B cell culture. Only background levels of IgA production was observed from B cells cultured with T cells in the presence of OVA Ag (no DC in Fig. 1), or with anti-CD3 activated T cells (data not shown). These results showed that IgA production observed in DC-T-B culture was not merely the result of B cell help provided by T cells activated by the DCs, but revealed a specific function of the PP DCs.

View larger version (28K): [in this window] [in a new window]   FIGURE 1. Ig production induced by freshly isolated PP or SP. MACS-purified naive CD4+/CD62Lhigh T cells from SP of OVA TCR Tg mice (2 x 105 per well) and MACS-purified naive IgD+ B cells from SP of BALB/c mice (2.5 x 105 per well) were coincubated with DCs (1 x 104 per well) from PP or SP for 7 days in the presence or absence of OVA (100 µg/ml). Either rIL-6 (10 ng/ml), neutralizing Ab against IL-6 (10 µg/ml), or isotype control (10 µg/ml) was added to some cultures as indicated. Supernatants were harvested and Ig levels were measured by ELISA. Results for individual culture wells are indicated and the crossbars represent the mean value. Detection limits of IgG1 and IgG2a were 0.010 µg/ml. ND, not detected. Data are representative of five separate experiments producing similar results.

Higher levels of IL-6 are induced in cultures with PP DCs as the APCs

Because it was likely that PP DCs regulate IgA responses through stimulation of T cells, we investigated the induction of cytokine production by PP DCs or SP DCs as the APCs using naive CD4⁺ T cells from OVA TCR Tg mice. As shown in Fig. 2, we observed that higher levels of IFN- and IL-6 were produced in T cell stimulation cultures with PP DCs as APCs compared with cultures with SP DCs. IL-4, IL-5, and IL-10 were not detected by ELISA.

View larger version (15K): [in this window] [in a new window]   FIGURE 2. Cytokine productions induced by the Ag presentation of sorted DC from PP and SP during primary culture. MACS-purified naive CD4+/CD62Lhigh T cells from SP of OVA TCR Tg mice (1 x 105 per well) were coincubated with DCs (1 x 104 per well) from PP or SP for 48 h in the presence of OVA (1 mg/ml). Supernatants were harvested and cytokine levels were measured by ELISA. ND, not detected. Detection limits were 0.25 ng/ml. Data are representative of two separate experiments producing similar results.

View larger version (19K): [in this window] [in a new window]   FIGURE 3. IL-6 production by sorted PP DCs after stimulation. Sorted DCs (5 x 104 per well) from BALB/c mice were incubated in the presence of stimulators. Cells were preincubated with hamster anti-mouse CD40 mAb (50 µg/ml), and then incubated with anti-hamster IgM (5 µg/ml) to cross-link the anti-CD40 mAb, incubated with PMA (50 ng/ml)+A23187 (250 ng/ml), or incubated with LPS (2 µg/ml). Supernatants were harvested and the cytokine levels were measured by ELISA at 48 h. Data are representative of five separate experiments producing similar results.

We investigated which subpopulation of the PP DCs produce IL-6. Freshly sorted DC subpopulations from PP and SP, namely CD11b⁺/CD11c⁺/B220^- and CD8⁺/CD11c⁺/B220^- populations (Fig. 4, A and B), were stimulated with either anti-CD40 and anti-hamster IgM, PMA and Ca²⁺ ionophore (A23187), or LPS. As shown in Fig. 4C, CD11b⁺ DCs from the PP secreted higher levels of IL-6 compared with CD8⁺ DCs from the PP and SP DCs. To confirm this finding, we also compared the production by sorted CD11b⁺ and CD11b^- DCs (Fig. 4D), or CD8⁺ and CD8^- DCs from the PP (Fig. 4E). In response to CD40 cross-linking, CD11b⁺ DCs from the PP secreted higher levels of IL-6 compared with CD11b^- DCs, and CD8^- DCs from the PP secreted higher levels of IL-6 compared with CD8⁺ DCs. Therefore, CD11b⁺/CD8^- DCs from the PP can secrete higher levels of IL-6 compared with the splenic counterpart. Neither CD8⁺/CD11b^- DCs nor CD8^-/CD11b^- double negative DCs from either PP or SP secreted appreciable levels of IL-6.

View larger version (31K): [in this window] [in a new window]   FIGURE 4. The FACS profiles defining the sorting gates used to isolate CD8+ or CD11b+ DCs. A, Sorting gates used to isolate DC subsets. B, Purified DC subsets. IL-6 productions by sorted PP DC subpopulations after stimulation. Sorted DC subpopulations (2 x 104 per well) from BALB/c mice were incubated in the presence of stimulators. C, CD8+ or CD11b+ DCs. D, CD11b+ or CD11b- DCs. E, CD8+ or CD8- PP DCs. Cells were stimulated as described in Fig. 3. Supernatants were harvested and IL-6 levels were measured by ELISA at 48 h. ND, not detected. Detection limits were 0.312 ng/ml (C) and 0.156 ng/ml (D). Data are representative of two separate experiments producing similar results.

View larger version (13K): [in this window] [in a new window]   FIGURE 5. IL-6 production induced by the Ag presentation of sorted DC populations from PP and SP during primary culture. MACS-purified naive CD4+/CD62Lhigh T cells from SP of OVA TCR Tg mice (1 x 105 per well) were coincubated with DCs (1 x 104 per well) from PP or SP for 48 h in the presence of OVA (1 mg/ml). Supernatants were harvested and IL-6 levels was measured by ELISA. ND, not detected. Detection limit was 0.62 ng/ml. Data are representative of two separate experiments producing similar results.

Finally, we investigated whether CD11b⁺ DCs isolated from PP induce higher levels of IgA production in DC-T-B cell coculture. As shown in Fig. 6, PP CD11b⁺ DCs were found to induce higher levels of IgA production in the presence of specific Ag compared with other DC populations, in particular, there was a statistically significant difference when compared with SP CD11b⁺ DCs (p < 0.05).

View larger version (20K): [in this window] [in a new window]   FIGURE 6. Ig production induced by freshly isolated DC populations from PP and SP. MACS-purified naive CD4+/CD62Lhigh T cells from SP of OVA TCR Tg mice (2 x 105 per well) and MACS-purified naive IgD+ B cells from SP of BALB/c mice (2.5 x 105 per well) were coincubated with DCs (1 x 104 per well) from PP or SP for 7 days in the presence of OVA (100 µg/ml). Supernatants were harvested and Ig levels were measured by ELISA. Results for individual culture wells are indicated and the crossbars represent the mean value. Data are representative of two separate experiments producing similar results.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

PP DCs were found to secrete high levels of IL-6 not only in response to anti-CD40 mAb but also to LPS. These results suggest that PP DCs secrete high levels of IL-6 during interaction with T cells, and in response to microbial components. Indeed, freshly sorted PP DCs expressed an IL-6 mRNA level higher than did SP DCs without stimulation (Table I). Because an enormous amount of food and microbial Ags are continuously being processed within the PP, PP DCs may be in a constant state of activation. This is supported by our observation that freshly isolated PP DCs possessed high levels of IL-6 mRNA not seen in SP DCs.

IL-6 markedly and selectively enhances IgA production in vitro by isotype-committed B cells (21, 29). It has been reported that mice with targeted disruption of the gene encoding IL-6 mount very poor IgA responses in the intestine and lungs, but that this defect can be overcome following vector-directed IL-6 gene therapy (22). These mice are also markedly deficient in IgA-producing plasma cells in the mucosa. We hypothesized that PP DCs enhance IgA production by inducing high levels of IL-6 secretion from CD4⁺ T cells. To address the issue, we investigated Ig production induced by PP or SP DCs in the DC-T-B coculture. We observed that PP DCs, especially the CD11b⁺ subset, induced higher levels of IgA production from naive B cells in the in vitro culture compared with SP DCs (Figs. 1 and 6). We extend previous findings that PP DCs induce IgA production (7, 8, 9), using freshly isolated, highly purified DCs subsets. Further, IgA production stimulated by PP DCs was largely suppressed by adding anti-IL-6 Ab to the culture. Consistent with the role of IL-6 in IgA production, IgA levels were enhanced in the presence of exogenous IL-6 in the culture when SP DCs were used as the APCs, albeit not restoring it to the levels induced by PP DCs. These data suggest that although IL-6 in the cultures of PP DCs was critical for IgA production, there are factors other than IL-6 that contribute to the observed IgA induction by PP DCs. PP DCs may induce IgA production via another cytokine, such as TGF- (5), or costimulatory molecules, in addition to IL-6. Several studies have demonstrated that IL-6 is an important factor for the terminal differentiation of IgA B cells in vitro (19, 21) and in vivo (22). Ramsay et al. (22) reported that in mice with targeted disruption of the gene that encodes IL-6, greatly reduced numbers of IgA-producing cells were observed at mucosae. Beagley et al. (26) reported high level IgA-producing B-1 cells poorly responded to additional stimulation with IL-6 in vitro and that a higher frequency of IgA⁺ B-1 cells was present in the intestinal lamina propria of IL-6^-/- mice as compared with wild-type mice. These results suggest that IL-6 is critical for IgA production by B-2 cells, but not B-1 cells, and IgA production levels are reduced in IL-6^-/- mice; however, low IgA production is observed in IL-6^-/- mice because B-1 cells induce IgA production in an IL-6 independent manner. On the other hand, Bromander et al. (30) reported that normal total IgA levels in serum and frequencies of IgA plasma cells were found in IL-6^-/- mice and they observed no significant difference in frequencies and distribution of B-1 cells in intestine-associated tissues between IL-6^-/- mice and wild-type mice. Taken together, our data and that of Bromander et al. (30) suggest that there may be several mechanisms of IgA induction including an IL-6-dependent IgA response induced by PP CD11b⁺ DCs, as well as an IL-6 independent pathway, perhaps occurring in other inductive sites.

PP CD11c⁺/B220^- DCs were found to express slightly higher levels of MHC class II, CD86 and CD40 compared with SP DCs (5). In the present study we found that DEC-205 expression was higher on PP DCs compared with SP DCs, particularly in the case of CD11b⁺ DCs (data not shown). The enhanced expression of these costimulatory molecules and DEC-205 has been shown to correlate with maturation of DCs, so this result suggests that there are a greater number of mature CD11b⁺ DCs in PP compared with SP. Indeed, PP DCs were found to have enhanced T cell proliferative capacity compared with SP DCs (5), supporting the heightened state of activation of the PP DCs. Because PP CD11b⁺ DCs are present in the SED, the region just beneath the follicle-associated epithelium containing M cells, they may be constantly being stimulated with the incoming Ags that enter via M cells into PP and are continuously undergoing maturation. PP CD11b⁺ DCs were capable of secreting higher levels of IL-6 compared with PP CD8⁺ DCs and all SP DC populations. It is possible that the high IL-6 secretion was related to their maturation status within the CD11b⁺ subset.

Our results suggest that CD11b⁺ DCs may be stimulated by interaction with T cells in the SED and secrete IL-6. Alternatively, a microbial product may induce migration to other sites such as the IFR, as postulated in other studies (10). The migration of CD11b⁺ DCs may enable these cells to stimulate IgA production more productively in vivo, particularly within the B cell follicles and in the germinal center.

The regulation of IL-6 gene expression in PP DCs is unknown. It has been reported that IL-6 production involves the protein kinase A (31, 32, 33, 34), NFAT (31, 35), mitogen-activated protein kinase (31), and AP-1 (31) pathways. Several transcription factors, which bind to the promoter site of IL-6, have been identified. The respective binding of IL-1 inducible factors, NFIL6 (36), NF-B (36, 37), and IFN regulatory factor (37) to IL-1 responsive element, and the binding of transcriptional factor Sp1 to three repeats of the CCACC element of the IL-6 promoter (36) were found to induce the transcription of IL-6. Therefore, we speculate that the expression or activation of these transcription factors within the PP CD11b⁺ DCs may be different from other DC populations.

London et al. (38, 39) reported that gut mucosal infection with reovirus stimulates the appearance of virus-specific CTL precursors in the PP. The generation of a CTL response in PP may be important in preferentially repopulating mucosal tissues with effector CTLs that could result in the local containment of infections in the gut. IL-6 was found to function as a late-acting killer helper factor in the differentiation of CTLs (40). It is possible that CD11b⁺ DCs presenting in SED efficiently enhance not only IgA induction but also CTL induction by secretion of IL-6.

In conclusion, this study demonstrates that PP CD11b⁺ DCs have a distinct ability to secrete higher levels of IL-6 compared with PP CD8⁺ DCs or SP DC populations. Moreover, we demonstrated that IL-6 is a critical factor in PP DCs’ ability to induce IgA production. Future studies must investigate the in vivo role of IL-6 secreted by PP DCs in IgA production, including the analysis of in situ IL-6 production by CD11b⁺ PP DCs during an ongoing PP B cell response involving IgA secretion, and the confirmation of the strict requirement for IL-6 in this process using IL-6 deficient mice.

	Acknowledgments

 
We thank Drs. Sonoko Habu and Takehito Sato for providing OVA-specific TCR Tg mice (OVA 23–3).

	Footnotes

 
¹ This work was supported in part by a grant-in-aid for Creative Scientific Research (13GS0015), a grant-in-aid for Scientific Research, and a grant-in-aid for Young Scientists, the Japan Society for the Promotion of Science, and by a program for the Promotion of Basic Research Activities for Innovative Biosciences.

² Current address: Department of Epidemiology and Public Health, Yale University School of Medicine, New Haven, CT 06520.

³ Current address: Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA 30912.

⁴ Address correspondence and reprint requests to Dr. Satoshi Hachimura, Department of Applied Biological Chemistry, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. E-mail address: ahachi{at}mail.ecc.u-tokyo.ac.jp

⁵ Abbreviations used in this paper: PP, Peyer’s patch; DC, dendritic cell; SED, subepithelial dome; IFR, interfollicular region; SP, spleen; Tg, transgenic.

Received for publication December 4, 2002. Accepted for publication July 22, 2003.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Mayrhofer, G., C. W. Pugh, A. N. Barclay. 1983. The distribution, ontogeny and origin in the rat of Ia-positive cells with dendritic morphology and of Ia antigen in epithelia, with special reference to the intestine. Eur. J. Immunol. 13:112.[Medline]
2. Spencer, J., T. Finn, P. G. Isaacson. 1986. Human Peyer’s patches: an immunohistochemical study. Gut 27:405.[Abstract/Free Full Text]
3. Witmer-Pack, M. D., D. A. Hughes, G. Schuler, L. Lawson, A. McWilliam, K. Inaba, R. M. Steinman, S. Gordon. 1993. Identification of macrophages and dendritic cells in the osteopetrotic (op/op) mouse. J. Cell Sci. 104:1021.[Abstract]
4. Pavli, P., C. E. Woodhams, W. F. Doe, D. A. Hume. 1990. Isolation and characterization of antigen-presenting dendritic cells from the mouse intestinal lamina propria. Immunology 70:40.[Medline]
5. Iwasaki, A., B. L. Kelsall. 1999. Freshly isolated Peyer’s patch, but not spleen, dendritic cells produce interleukin 10 and induce the differentiation of T helper type 2 cells. J. Exp. Med. 190:229.[Abstract/Free Full Text]
6. Spalding, D. M., W. J. Koopman, J. H. Eldridge, J. R. McGhee, R. M. Steinman. 1983. Accessory cells in murine Peyer’s patch. I. Identification and enrichment of a functional dendritic cell. J. Exp. Med. 157:1646.[Abstract/Free Full Text]
7. Spalding, D. M., S. I. Williamson, W. J. Koopman, J. R. McGhee. 1984. Preferential induction of polyclonal IgA secretion by murine Peyer’s patch dendritic cell-T cell mixtures. J. Exp. Med. 160:941.[Abstract/Free Full Text]
8. Spalding, D. M., J. A. Griffin. 1986. Different pathways of differentiation of pre-B cell lines are induced by dendritic cells and T cells from different lymphoid tissues. Cell 44:507.[Medline]
9. George, A., J. J. Cebra. 1991. Responses of single germinal-center B cells in T-cell-dependent microculture. Proc. Natl. Acad. Sci. USA 88:11.[Abstract/Free Full Text]
10. Iwasaki, A., B. L. Kelsall. 2000. Localization of distinct Peyer’s patch dendritic cell subsets and their recruitment by chemokines macrophage inflammatory protein (MIP)-3, MIP-3, and secondary lymphoid organ chemokine. J. Exp. Med. 191:1381.[Abstract/Free Full Text]
11. Iwasaki, A., B. L. Kelsall. 2001. Unique functions of CD11b⁺, CD8⁺, and double-negative Peyer’s patch dendritic cells. J. Immunol. 166:4884.[Abstract/Free Full Text]
12. Sonoda, E., R. Matsumoto, Y. Hitoshi, T. Ishii, M. Sugimoto, S. Araki, A. Tominaga, N. Yamaguchi, K. Takatsu. 1989. Transforming growth factor induces IgA production and acts additively with interleukin 5 for IgA production. J. Exp. Med. 170:1415.[Abstract/Free Full Text]
13. Coffman, R. L., D. A. Lebman, B. Shrader. 1989. Transforming growth factor specifically enhances IgA production by lipopolysaccharide-stimulated murine B lymphocytes. J. Exp. Med. 170:1039.[Abstract/Free Full Text]
14. van Ginkel, F. W., S. M. Wahl, J. F. Kearney, M. N. Kweon, K. Fujihashi, P. D. Burrows, H. Kiyono, J. R. McGhee. 1999. Partial IgA-deficiency with increased Th2-type cytokines in TGF-1 knockout mice. J. Immunol. 163:1951.[Abstract/Free Full Text]
15. Beagley, K. W., J. H. Eldridge, H. Kiyono, M. P. Everson, W. J. Koopman, T. Honjo, J. R. McGhee. 1988. Recombinant murine IL-5 induces high rate IgA synthesis in cycling IgA-positive Peyer’s patch B cells. J. Immunol. 141:2035.[Abstract]
16. Harriman, G. R., D. Y. Kunimoto, J. F. Elliott, V. Paetkau, W. Strober. 1988. The role of IL-5 in IgA B cell differentiation. J. Immunol. 140:3033.[Abstract]
17. Lebman, D. A., R. L. Coffman. 1988. The effects of IL-4 and IL-5 on the IgA response by murine Peyer’s patch B cell subpopulations. J. Immunol. 141:2050.[Abstract]
18. Schoenbeck, S., D. T. McKenzie, M. F. Kagnoff. 1989. Interleukin 5 is a differentiation factor for IgA B cells. Eur. J. Immunol. 19:965.[Medline]
19. Kunimoto, D. Y., R. P. Nordan, W. Strober. 1989. IL-6 is a potent cofactor of IL-1 in IgM synthesis and of IL-5 in IgA synthesis. J. Immunol. 143:2230.[Abstract]
20. Kopf, M., F. Brombacher, P. D. Hodgkin, A. J. Ramsay, E. A. Milbourne, W. J. Dai, K. S. Ovington, C. A. Behm, G. Kohler, I. G. Young, K. I. Matthaei. 1996. IL-5-deficient mice have a developmental defect in CD5⁺ B-1 cells and lack eosinophilia but have normal antibody and cytotoxic T cell responses. Immunity 4:15.[Medline]
21. Beagley, K. W., J. H. Eldridge, F. Lee, H. Kiyono, M. P. Everson, W. J. Koopman, T. Hirano, T. Kishimoto, J. R. McGhee. 1989. Interleukins and IgA synthesis. Human and murine interleukin 6 induce high rate IgA secretion in IgA-committed B cells. J. Exp. Med. 169:2133.[Abstract/Free Full Text]
22. Ramsay, A. J., A. J. Husband, I. A. Ramshaw, S. Bao, K. I. Matthaei, G. Koehler, M. Kopf. 1994. The role of interleukin-6 in mucosal IgA antibody responses in vivo. Science 264:561.[Abstract/Free Full Text]
23. Kroese, F. G., E. C. Butcher, A. M. Stall, P. A. Lalor, S. Adams, L. A. Herzenberg. 1989. Many of the IgA producing plasma cells in murine gut are derived from self-replenishing precursors in the peritoneal cavity. Int. Immunol. 1:75.[Abstract/Free Full Text]
24. Herzenberg, L. A., A. M. Stall, P. A. Lalor, C. Sidman, W. A. Moore, D. R. Parks. 1986. The Ly-1 B cell lineage. Immunol. Rev. 93:81.[Medline]
25. Staal, F. J., M. Roederer, L. A. Herzenberg. 1992. Glutathione and immunophenotypes of T and B lymphocytes in HIV-infected individuals. Ann. NY Acad. Sci. 651:453.[Medline]
26. Beagley, K. W., S. Bao, A. J. Ramsay, J. H. Eldridge, A. J. Husband. 1995. IgA production by peritoneal cavity B cells is IL-6 independent: implications for intestinal IgA responses. Eur. J. Immunol. 25:2123.[Medline]
27. Sato, T., T. Sasahara, Y. Nakamura, T. Osaki, T. Hasegawa, T. Tadakuma, Y. Arata, Y. Kumagai, M. Katsuki, S. Habu. 1994. Naive T cells can mediate delayed-type hypersensitivity response in T cell receptor transgenic mice. Eur. J. Immunol. 24:1512.[Medline]
28. Murphy, K. M., A. B. Heimberger, D. Y. Loh. 1990. Induction by antigen of intrathymic apoptosis of CD4⁺CD8⁺TCR^lo thymocytes in vivo. Science 250:1720.[Abstract/Free Full Text]
29. Fujihashi, K., J. R. McGhee, C. Lue, K. W. Beagley, T. Taga, T. Hirano, T. Kishimoto, J. Mestecky, H. Kiyono. 1991. Human appendix B cells naturally express receptors for and respond to interleukin 6 with selective IgA1 and IgA2 synthesis. J. Clin. Invest. 88:248.
30. Bromander, A. K., L. Ekman, M. Kopf, J. G. Nedrud, N. Y. Lyck. 1996. IL-6-deficient mice exhibit normal mucosal IgA responses to local immunizations and Helicobacter felis infection. J. Immunol. 156:4290.[Abstract]
31. Ray, A., S. B. Tatter, L. T. May, P. B. Sehgal. 1988. Activation of the human "₂-interferon/hepatocyte-stimulating factor/interleukin 6" promoter by cytokines, viruses, and second messenger agonists. Proc. Natl. Acad. Sci. USA 85:6701.[Abstract/Free Full Text]
32. Zhang, Y. H., J. X. Lin, Y. K. Yip, J. Vilcek. 1988. Enhancement of cAMP levels and of protein kinase activity by tumor necrosis factor and interleukin 1 in human fibroblasts: role in the induction of interleukin 6. Proc. Natl. Acad. Sci. USA 85:6802.[Abstract/Free Full Text]
33. Zhang, Y., J. X. Lin, J. Vilcek. 1988. Synthesis of interleukin 6 (interferon-₂/B cell stimulatory factor 2) in human fibroblasts is triggered by an increase in intracellular cyclic AMP. J. Biol. Chem. 263:6177.[Abstract/Free Full Text]
34. Zhang, Y. H., J. X. Lin, J. Vilcek. 1990. Interleukin-6 induction by tumor necrosis factor and interleukin-1 in human fibroblasts involves activation of a nuclear factor binding to a B-like sequence. Mol. Cell. Biol. 10:3818.[Abstract/Free Full Text]
35. Sehgal, P. B., Z. Walther, I. Tamm. 1987. Rapid enhancement of ₂-interferon/B-cell differentiation factor BSF-2 gene expression in human fibroblasts by diacylglycerols and the calcium ionophore A23187. Proc. Natl. Acad. Sci. USA 84:3663.[Abstract/Free Full Text]
36. Kang, S. H., D. A. Brown, I. Kitajima, X. Xu, O. Heidenreich, S. Gryaznov, M. Nerenberg. 1996. Binding and functional effects of transcriptional factor Sp1 on the murine interleukin-6 promotor. J. Biol. Chem. 271:7330.[Abstract/Free Full Text]
37. Faggioli, L., M. Merola, J. Hiscott, A. Furia, R. Monese, M. Tovey, M. Palmieri. 1997. Molecular mechanisms regulating induction of interleukin-6 gene transcription by interferon-. Eur. J. Immunol. 27:3022.[Medline]
38. London, S. D., D. H. Rubin, J. J. Cebra. 1987. Gut mucosal immunization with reovirus serotype 1/L stimulates virus-specific cytotoxic T cell precursors as well as IgA memory cells in Peyer’s patches. J. Exp. Med. 165:830.[Abstract/Free Full Text]
39. London, S. D., D. H. Rubin, J. J. Cebra. 1987. Murine Peyer’s patches are capable of generating a cytotoxic T cell (CTL) response to nominal antigens. Adv. Exp. Med. Biol. 216A:267.
40. Okada, M., M. Kitahara, S. Kishimoto, T. Matsuda, T. Hirano, T. Kishimoto. 1988. IL-6/BSF-2 functions as a killer helper factor in the in vitro induction of cytotoxic T cells. J. Immunol. 141:1543.[Abstract]

This article has been cited by other articles:

				M. M. Sira, T. Yoshida, M. Takeuchi, Y. Kashiwayama, T. Futatani, H. Kanegane, A. Sasahara, Y. Ito, M. Mizuguchi, T. Imanaka, et al. A novel immunoregulatory protein in human colostrum, syntenin-1, for promoting the development of IgA-producing cells from cord blood B cells Int. Immunol., July 10, 2009; (2009) dxp067v1. [Abstract] [Full Text] [PDF]

				J. M. Thompson, M. G. Nicholson, A. C. Whitmore, M. Zamora, A. West, A. Iwasaki, H. F. Staats, and R. E. Johnston Nonmucosal Alphavirus Vaccination Stimulates a Mucosal Inductive Environment in the Peripheral Draining Lymph Node J. Immunol., July 1, 2008; 181(1): 574 - 585. [Abstract] [Full Text] [PDF]

				A. B. Blazquez and M. C. Berin Gastrointestinal Dendritic Cells Promote Th2 Skewing via OX40L J. Immunol., April 1, 2008; 180(7): 4441 - 4450. [Abstract] [Full Text] [PDF]

				T. Naito, T. Suda, K. Suzuki, Y. Nakamura, N. Inui, J. Sato, K. Chida, and H. Nakamura Lung Dendritic Cells Have a Potent Capability to Induce Production of Immunoglobulin A Am. J. Respir. Cell Mol. Biol., February 1, 2008; 38(2): 161 - 167. [Abstract] [Full Text] [PDF]

				K. A. Kadaoui and B. Corthesy Secretory IgA Mediates Bacterial Translocation to Dendritic Cells in Mouse Peyer's Patches with Restriction to Mucosal Compartment J. Immunol., December 1, 2007; 179(11): 7751 - 7757. [Abstract] [Full Text] [PDF]

				K. Takamura, S. Fukuyama, T. Nagatake, D.-Y. Kim, A. Kawamura, H. Kawauchi, and H. Kiyono Regulatory Role of Lymphoid Chemokine CCL19 and CCL21 in the Control of Allergic Rhinitis J. Immunol., November 1, 2007; 179(9): 5897 - 5906. [Abstract] [Full Text] [PDF]

				I. D. Iliev, G. Matteoli, and M. Rescigno The yin and yang of intestinal epithelial cells in controlling dendritic cell function J. Exp. Med., October 1, 2007; 204(10): 2253 - 2257. [Abstract] [Full Text] [PDF]

				L. Saurer, K. C. McCullough, and A. Summerfield In Vitro Induction of Mucosa-Type Dendritic Cells by All-Trans Retinoic Acid J. Immunol., September 15, 2007; 179(6): 3504 - 3514. [Abstract] [Full Text] [PDF]

				B. Corthesy Roundtrip Ticket for Secretory IgA: Role in Mucosal Homeostasis? J. Immunol., January 1, 2007; 178(1): 27 - 32. [Abstract] [Full Text] [PDF]

				J. R. Mora, M. Iwata, B. Eksteen, S.-Y. Song, T. Junt, B. Senman, K. L. Otipoby, A. Yokota, H. Takeuchi, P. Ricciardi-Castagnoli, et al. Generation of Gut-Homing IgA-Secreting B Cells by Intestinal Dendritic Cells. Science, November 17, 2006; 314(5802): 1157 - 1160. [Abstract] [Full Text] [PDF]

				F.-X. Hubert, C. Voisine, C. Louvet, J.-M. Heslan, A. Ouabed, M. Heslan, and R. Josien Differential Pattern Recognition Receptor Expression but Stereotyped Responsiveness in Rat Spleen Dendritic Cell Subsets J. Immunol., July 15, 2006; 177(2): 1007 - 1016. [Abstract] [Full Text] [PDF]

				F Lanzarotto, A Akbar, and S Ghosh Does innate immune response defect underlie inflammatory bowel disease in the Asian population? Postgrad. Med. J., August 1, 2005; 81(958): 483 - 485. [Full Text] [PDF]

				D. W. Smith and C. Nagler-Anderson Preventing Intolerance: The Induction of Nonresponsiveness to Dietary and Microbial Antigens in the Intestinal Mucosa J. Immunol., April 1, 2005; 174(7): 3851 - 3857. [Abstract] [Full Text] [PDF]

				J. I. Bleier, V. G. Pillarisetty, A. B. Shah, and R. P. DeMatteo Increased and Long-Term Generation of Dendritic Cells with Reduced Function from IL-6-Deficient Bone Marrow J. Immunol., June 15, 2004; 172(12): 7408 - 7416. [Abstract] [Full Text] [PDF]

				J. Rey, N. Garin, F. Spertini, and B. Corthesy Targeting of Secretory IgA to Peyer's Patch Dendritic and T Cells after Transport by Intestinal M Cells J. Immunol., March 1, 2004; 172(5): 3026 - 3033. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sato, A. Articles by Kaminogawa, S. Search for Related Content PubMed PubMed Citation Articles by Sato, A. Articles by Kaminogawa, S.