IL-10–treated dendritic cells decrease airway hyperresponsiveness and airway inflammation in mice

IL-10–treated dendritic cells decrease airway hyperresponsiveness and airway inflammation in mice Toshiyuki Koya, MD, PhD, Hiroyuki Matsuda, MD, PhD, Katsuyuki Takeda, MD, PhD, Shigeki Matsubara, PhD, Nobuaki Miyahara, MD, PhD, Annette Balhorn, BS, Azzeddine Dakhama, PhD, and Erwin W. Gelfand, MD Denver, Colo

Key words: IL-10–treated dendritic cells, eosinophils, airway hyperresponsiveness, regulatory T cells

From the Division of Cell Biology, Department of Pediatrics, National Jewish Medical and Research Center, Denver. Supported by National Institutes of Health grants HL-36577 and HL-61005 and by US Environmental Protection Agency grant R825702. Disclosure of potential conflict of interest: The authors have declared that they have no conflict of interest. Received for publication August 24, 2006; revised January 29, 2007; accepted for publication January 30, 2007. Available online March 15, 2007. Reprint requests: Erwin W. Gelfand, MD, National Jewish Medical and Research Center, 1400 Jackson St, Denver, CO 80206. E-mail: gelfande@ njc.org. 0091-6749/$32.00 Ó 2007 American Academy of Allergy, Asthma & Immunology doi:10.1016/j.jaci.2007.01.039

Abbreviations used AHR: Airway hyperresponsiveness APC: Antigen-presenting cell BAL: Bronchoalveolar lavage BMDC: Bone marrow–derived dendritic cell DC: Dendritic cell MCh: Methacholine MNC: Mononuclear cell OVA: Ovalbumin PAS: Periodic acid-Schiff RL: Lung resistance

T lymphocytes that secrete TH2 cytokines, such as IL-4, IL-5, and IL-13, in response to allergen play a major role in the pathogenesis of allergic asthma.1 A critical step in the induction and regulation of the T-cell immune response is the uptake, processing, and presentation of antigen by antigen-presenting cells (APCs). Among the different types of APCs, dendritic cells (DCs) play an important role in antigen presentation in the airways.2,3 DCs are distributed throughout the body and act as sentinel cells in monitoring and regulating immune responses. Immature DCs actively take up and process antigens by means of endocytosis or pinocytosis4; can be differentiated into mature DCs after stimulation with proinflamatory cytokines (TNF-a, IL1b, and IL-6), prostaglandin E2, LPS, or CD40 ligand; and express several costimulatory factors and MHC class II.5,6 Mature DCs also express the chemokine receptor CCR7, the ligand of which is abundant in lymph nodes, and expression of this receptor contributes to the migration of DCs to lymph nodes, where DCs encounter naive T cells and initiate their priming.7,8 In animal models of airway allergic inflammation, DCs play critical roles in both the sensitization9 and challenge10,11 phases. The immunoregulatory cytokine IL-10 was identified originally as an inhibitor of IFN-g and IL-2 synthesis in TH2 cells12 and is known to be an inhibitor of proliferative and cytokine responses in T cells. IL-10 is produced by numerous cell types, including macrophages, natural killer cells, and both TH1- and TH2-type T cells.13-15 The role of IL-10 in the development of allergic airway inflammation is controversial. In mouse lung allergic inflammatory models, IL-10 was shown to be essential for the development of airway hyperresponsiveness (AHR), regardless of the level of eosinophilic inflammation.16-18 In contrast, 1241

Basic and clinical immunology

Background: IL-10 affects dendritic cell (DC) function, but the effects on airway hyperresponsiveness (AHR) and inflammation are not defined. Objective: We sought to determine the importance of IL-10 in regulating DC function in allergen-induced AHR and airway inflammation. Methods: DCs were generated from bone marrow in the presence or absence of IL-10. In vivo IL-10–treated DCs from IL-101/1 and IL-102/2 donors pulsed with ovalbumin (OVA) were transferred to naive or sensitized mice before challenge. In recipient mice AHR, cytokine levels, cell composition of bronchoalveolar lavage (BAL) fluid, and lung histology were monitored. Results: In vitro, IL-10–treated DCs expressed lower levels of CD11c, CD80, and CD86; expressed lower levels of IL-12; and suppressed TH2 cytokine production. In vivo, after transfer of OVA-pulsed IL-10–treated DCs, naive mice did not have AHR, airway eosinophilia, TH2 cytokine increase in BAL fluid, or goblet cell metaplasia when challenged, and in sensitized and challenged mice IL-10–treated DCs suppressed these responses. Levels of IL-10 in BAL fluid and numbers of lung CD41IL-101 T cells were increased in mice that received OVA-pulsed IL-10–treated DCs. Transfer of IL-10–treated DCs from IL-10–deficient mice were ineffective in suppressing the responses in sensitized and challenged mice. Conclusions: These data demonstrate that IL-10–treated DCs are potent suppressors of the development of AHR, inflammation, and TH2 cytokine production; these regulatory functions are at least in part through the induction of endogenous (DC) production of IL-10. Clinical implications: Modification of DC function by IL-10 can attenuate lung allergic responses, including the development of AHR. (J Allergy Clin Immunol 2007;119:1241-50.)

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Oh et al reported that T cells secreting IL-10 in the airways reduced TH2-type inflammation and AHR.19 We previously reported that repeated antigen challenge with ovalbumin (OVA) increased IL-10 levels in bronchoalveolar lavage (BAL) fluid in association with decreases in AHR.20 Taken together, the findings suggest that IL-10 might play different roles in the regulation of allergic responses at different stages of the response. IL-10 has been demonstrated to modify the phenotypic and functional maturation of in vitro–propagated myeloid DCs and confers tolerogenic properties in human cells.21,22 IL-10 inhibits the upregulation of expression of MHC class II, the DC maturation marker CD83, and several costimulatory molecules on in vitro–generated human and mouse DCs.21,23-25 Functionally, IL-10 treatment of DCs suppresses IL-12 production,26 and bone marrow–derived murine DCs treated with IL-10 show impaired antigen presentation in vitro.25,27-29 Likewise, IL-10–treated human DCs decreased T-cell stimulatory capacity and induced anergy in responding T cells.22,30,31 The effects of IL-10– treated DCs on TH2-mediated inflammation are nonetheless somewhat inconsistent. Some have reported that IL-10–treated DCs inhibit TH1 cytokine production from T cells, whereas they triggered increased levels of TH2 cytokines.32,33 On the other hand, IL-10–treated DCs suppressed TH2 cytokine production in T cells from allergic patients.34 The purpose of the present study was to define the effects of IL-10–treated DCs on allergen-induced AHR, airway eosinophilia, and TH2 cytokine production by using an adoptive transfer model in both naive and sensitized and challenged mice.

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anti-CD11c (HL3), anti-Gr-1 (RB6-8C5), anti-CD80 (16-10A1), and anti-CD86 (GL1; all obtained from BD Pharmingen, San Diego, Calif). For control staining, similarly labeled isotype-matched control antibodies were used.

Cell preparations and cultures of DCs Spleens were removed from OVA-sensitized mice, tissue was dispersed into single-cell suspensions, and mononuclear cells (MNCs) were purified by means of Histopaque gradient centrifugation (Sigma-Aldrich). Cells were washed, counted, and suspended at 4 3 106 cells/mL in RPMI 1640 (Life Technologies, Gaithersburg, Md) tissue culture medium containing heat-inactivated 10% FCS, 100 U/mL penicillin, 100 mg/mL streptomycin, 5 mmol/L L-glutamine, and 50 mmol/L 2-mercaptoethanol. Spleen T cells were purified from spleen MNCs by using the Mouse T Cell Recovery Column Kit (purity, >95%; Cederlane, Hornby, Ontario, Canada). Spleen T cells were cultured with DCs and OVA (10 mg/mL) for 72 hours. After coculture, culture supernatants were assayed for cytokine levels. IL-101 DCs or IL-10–nontreated DCs (IL-102 DCs, 2 3 105 cells) were incubated in 96-well culture plates with or without OVA (100 mg/mL). After 24 hours, culture supernatants were harvested.

Adoptive transfer of DCs into naive mice OVA-pulsed IL-101 DCs or IL-102 DCs (1 3 106 cells) were administered intratracheally to naive mice, and 10 days later, animals were challenged with nebulized OVA (1% in saline) for 20 minutes on 3 consecutive days. Forty-eight hours after the last OVA challenge, AHR was assessed, and BAL fluid, serum, and tissues were obtained for further analyses.

Adoptive transfer of DCs into OVA-sensitized mice OVA-pulsed or nonpulsed IL-101 DCs (1 3 106) or IL-102 DCs (1 3 106) were injected intravenously into OVA-sensitized mice on day 26. Two days after DC transfer, animals were challenged with nebulized OVA (1% in saline) for 20 minutes on 3 consecutive days.

METHODS

OVA-induced allergic airway inflammation

Animals

Mice were sensitized on days 1 and 14 by means of intraperitoneal injection of 20 mg of OVA premixed with 2.25 mg of Al(OH)3 (Pierce, Rockford, Ill) in 100 mL of PBS. After sensitization, animals were exposed to aerosolized OVA (1% in saline) for 20 minutes per day on days 28, 29, and 30 (OVA/OVA). Forty-eight hours after the last OVA challenge (day 32), AHR was assessed, and BAL fluid, serum, and tissues were obtained for further analyses. Control groups consisted of nonsensitized but OVA-challenged animals (PBS/OVA).

Female C57BL/6 mice and IL-10–deficient mice were purchased at 8 to 12 weeks of age from Jackson Laboratories (Bar Harbor, Me) and housed under specific pathogen-free conditions. IL-10–deficient mice on a B6.129P2-IL10tm1Cgn/J background were backcrossed for more than 10 generations onto the C57BL/6 strain to yield mice with C57BL/6-defined background. The animals were maintained on an OVA-free diet. Experiments were conducted under a protocol approved by the Institutional Animal Care and Use Committee of the National Jewish Medical and Research Center. Basic and clinical immunology

Generation of bone marrow–derived dendritic cells Bone marrow–derived DCs (BMDCs) were generated from bone marrow cells of a naive C57BL6 mouse or IL-10–deficient mice, as described previously, in the presence of GM-CSF and IL-4 (10 ng/ mL; R&D Systems, Minneapolis, Minn).20 IL-10–treated DCs (IL101 DCs) were obtained by culturing bone marrow cells with recombinant murine GM-CSF (10 ng/mL) and recombinant murine IL-10 (20 ng/mL, R&D Systems). On day 8, cells were pulsed or not with OVA (200 mg/mL, grade V, Sigma-Aldrich, St Louis, Mo) for 24 hours and washed 3 times with PBS.

Flow cytometry The surface phenotype of BMDCs was analyzed with mAbs, as described previously.20 The mAbs included anti-CD11b (M1/70),

Determination of airway responsiveness Airway function was assessed, as previously described, by measuring changes in lung resistance (RL) in response to increasing doses of inhaled methacholine (MCh).35 Data are expressed as the percentage change from baseline RL obtained after inhalation of saline.

BAL Immediately after assessment of AHR, lungs underwent lavage through the tracheal tube with 1 mL of HBSS at room temperature. Total leukocyte numbers were measured (Coulter Counter; Coulter Corp, Hialeah, Fla). Cytospin slides were stained with Leukostat (Fisher Diagnostics, Pittsburgh, Pa) and differentiated by using standard hematologic procedures in a blinded fashion.

Lung cell isolation Lung cells were isolated as previously described20 by using collagenase digestion. Cells were resuspended in HBSS, and MNCs were purified with 35% Percoll (Sigma-Aldrich).

Intracellular cytokine staining Intracellular cytokine staining was performed as previously described.26 Briefly, lung MNCs were stimulated for 6 hours with phorbol 12–myrisytate 13–acetate (PMA; 10 ng/mL) and ionomycin (500 mg/mL) in the presence of brefeldin A (10 mg/mL). After washing, cells were stained with mAbs against CD3 (145-2C11, hamster IgG), CD4 (RM4-5, rat IgG2a), and CD8 (53-6.7, rat IgG2a). All fluorochrome-labeled mAbs, and isotype control IgGs were purchased from BD Pharmingen (San Diego, Calif). After fixation and permeabilization, cells were stained with phycoerythrin-conjugated anticytokine antibodies or similarly labeled isotype-matched control antibodies. Antibodies against IL-4 and IL-10 were purchased from BD Pharmingen, and anti-mouse Foxp3 was purchased from eBiosciences (San Diego, Calif). After washing, staining was analyzed by means of flow cytometry on a FACS Calibur with CellQuest software (BD Biosciences, Mountain View, Calif).

Histochemistry Lungs were fixed in 10% formalin and processed into paraffin. Mucus-containing goblet cells were detected by staining of paraffin sections (5 mm thick) with periodic acid-Schiff (PAS). Histologic analyses were carried out in a blinded manner by using light microscopy linked to an image system. Numbers of PAS-positive goblet cells were determined only in cross-sectional areas of the airway wall. Six to 10 different sections were evaluated per animal. The obtained measurements were averaged for each animal, and the mean values and SEs were determined for each group.

Measurement of culture supernatant or BAL fluid cytokines Culture or BAL supernatant cytokine levels were determined by using commercially available ELISAs according to the manufacturers’ instructions. ELISA kits for detection of IL-4, IL-5, IL-10, IL-12 (p70), and IFN-g in supernatants were obtained from BD Pharmingen. The IL-13 ELISA kit was purchased from R&D Systems. 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-g; and 1.5 pg/mL for IL-13.

Statistical analysis Mann-Whitney U tests were used to determine the levels of difference between all groups. The data were pooled from 3 independent experiments, with 4 mice per group in each experiment (n 5 12). Comparisons for all pairs were performed by using the KruskalWallis test. Significance was assumed at a P value of less than .05. Values for all measurements are expressed as means 6 SEMs.

RESULTS Effect of IL-10 on costimulatory molecule expression and cytokine production by BMDCs IL-10 was added to the culture medium during the differentiation of the cells to determine whether IL-10 modifies the phenotype of BMDCs in vitro. Pulsing of DCs with OVA had little effect (slightly increased) on expression of costimulatory molecules, whereas expression of the costimulatory molecules CD80 and CD86 was decreased significantly in the presence of IL-10 (see Fig E1, A, in this article’s Online Repository at www.jacionline. org). The production of IL-12 from OVA-pulsed IL-10– treated DCs (IL-101 DCs) was significantly lower than

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in IL-10–nontreated DCs (IL-102 DCs; see Fig E1, B, in the Online Repository at www.jacionline.org). OVApulsed IL-101 DCs produced significantly higher levels of IL-10 than IL-102 DCs (see Fig E1, B, in this article’s Online Repository at www.jacionline.org).

In vitro coculture of DCs with spleen T cells Primed spleen T cells were cocultured with DCs in the presence of OVA to test whether IL-101 DCs have the potential to modulate cytokine production from antigenprimed and stimulated T cells. Cultures containing IL-101 DCs released significantly lower levels of IL-4, IL-5, and IL-13 than those cultures containing IL-102 DCs (see Fig E2 in this article’s Online Repository at www.jacionline.org). Transfer of IL-10–treated DCs into naive mice To determine the effect of IL-10 treatment of DCs in vivo, we first examined naive mice, which received either OVA-pulsed IL-101 DCs, IL-102 DCs, or no DCs, followed by 3 days of allergen (OVA) challenge. As shown in Fig 1, A, transfer of OVA-pulsed IL-101 DCs resulted in little increase in RL to inhaled MCh, with levels of RL similar to those seen in mice receiving no DCs. In contrast, transfer of IL-102 DCs that were pulsed with OVA led to significant increases in RL throughout the MCh dose-response curve. Results after transfer of nonOVA–pulsed DCs were similar to those in mice receiving no DCs (data not shown). The number of inflammatory cells in BAL fluid was determined 48 hours after the last allergen challenge. Administration of OVA-pulsed IL-102 DCs significantly increased the number of eosinophils, with a corresponding reduction in the number of macrophages in BAL fluid, whereas administration of OVA-pulsed IL-101 DCs did not induce airway eosinophilia, which is similar to what is seen in recipients of no DCs (Fig 1, B). Cytokine levels in BAL fluid were assayed in the naive recipients of DCs after allergen challenge. Challenge after transfer of OVA-pulsed IL-102 DCs resulted in significant increases in the levels of the TH2-type cytokines IL-4, IL-5, and IL-13 in the BAL fluid compared with those seen in recipients of no DCs (see Fig E3 in the Online Repository at www.jacionline.org). These cytokine levels were significantly lower in recipients of OVApulsed IL-101 DCs and not significantly different from those seen in mice receiving no cells. Levels of IFN-g in the BAL fluid of mice that received OVA-pulsed IL-102 DCs were significantly decreased compared with those of recipients of OVA-pulsed IL-101 DCs or PBS (no DCs). The degrees of goblet cell metaplasia and mucus hyperproduction were evaluated by means of PAS staining and quantification of PAS-stained cells. Transfer of OVA-pulsed IL-102 DCs to naive mice significantly increased the numbers of PAS-positive cells when challenged with allergen (Fig 1, C). In contrast, the transfer of OVA-pulsed IL-101 DCs had only a minimal effect in increasing goblet cell numbers.

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Basic and clinical immunology

FIG 1. Effect of transfer of IL-101 DCs into naive mice before challenge. OVA-pulsed IL-101 DCs (1 3 106), IL-102 DCs (1 3 106), or no cells were administered intratracheally to naive mice. Ten days after DC transfer, animals were challenged with OVA (1% in saline) for 20 minutes on 3 consecutive days. Forty-eight hours after the last OVA challenge, AHR was assessed, and BAL fluid and tissues were obtained for further analysis. Data represent means 6 SEMs from 3 separate experiments (n 5 12). A, Changes in lung resistance (RL). **P < .01 and *P < .05, comparing recipients of IL-102 DCs to IL-101 DCs or no cells. B, Cellular composition in BAL fluid. Mac, Macrophages; Lym, lymphocytes; Neu, neutrophils; Eos, eosinophils. *P < .05 and **P < .01, comparing recipients of IL-102 and IL-101 DCs or no DCs. C, Representative photomicrographs and quantitative analysis of PAS-positive cells in the lung tissues. The tissues were obtained 48 hours after the last challenge. Recipients of no cells (a), recipients of IL-102 DCs (b), recipients of IL-101 DCs (c) are shown. Quantitative analysis of PAS-positive cells in lung tissues (d) was performed as described in the Methods section. **P < .01 comparing recipients of IL-101 and IL-102 DCs or no DCs. BM, Basement membrane.

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FIG 2. Continued.

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Basic and clinical immunology

FIG 2. Effect of transfer of IL-101 DCs into OVA-sensitized mice before airway challenge. OVA-pulsed or nonpulsed IL-101 DCs or IL-102 DCs (1 3 106) were injected intravenously into OVA-sensitized mice before OVA challenge (OVA/OVA). Two days after DC transfer, animals were challenged with OVA (1% in saline) for 20 minutes on 3 consecutive days. Forty-eight hours after the last OVA challenge, AHR was assessed, and BAL fluid and tissue were obtained for further analysis. Control animals were nonsensitized but challenged with OVA (PBS/OVA). Data represent means 6 SEMs from 3 separate experiments (n 5 12). A, Changes in lung resistance (RL). *P < .05 and **P < .01 between groups indicated. B, Cellular composition in BAL fluid. Mac, Macrophages; Lym, lymphocytes; Neu, neutrophils; Eos, eosinophils. *P < .05 between recipients of nonpulsed and pulsed IL-101 DCs. C, Cytokine levels in BAL fluid. *P < .05 between recipients of nonpulsed and pulsed DCs. D, Representative photomicrographs and quantitative analysis of PAS-positive cells in the lung tissues. The tissues were obtained 48 hours after the last challenge. Shown are representative photomicrographs from the PBS/OVA group (a), OVA/OVA recipients of no cells (b), OVA/OVA recipients of non-pulsed IL-101 DCs (c), and OVA/OVA recipients of pulsed IL-101 DCs (d). Quantitative analysis of PAS-positive cells in lung tissues (e) is also shown. *P < .05 between recipients of nonpulsed and pulsed IL-101 DCs. BM, Basement membrane. E, The percentage of IL-10–producing CD41 T cells in the lung was determined by means of intracellular cytokine staining, as described in the Methods section. *P < .05 between recipients of nonpulsed and pulsed DCs.

Transfer of DCs into OVA-sensitized mice We next compared the in vivo effects of DC transfer in sensitized mice just before allergen challenge. OVAsensitized mice received OVA-pulsed IL-101 DCs, nonpulsed IL-101 DCs, IL-102 DCs, or no DCs 2 days before the first of 3 daily airway allergen challenges. As shown in Fig 2, A, transfer of OVA-pulsed IL-101 DCs resulted in significant decreases in RL to inhaled MCh when

compared with the transfer of nonpulsed IL-101 DC, IL102 DC, or no DC administration (Fig 2, A). In parallel, when BAL cell composition was examined, transfer of OVA-pulsed IL-101 DCs, but not nonpulsed IL-101 DCs, significantly reduced the number of eosinophils in BAL fluid, whereas the numbers of macrophages and lymphocytes in the BAL fluid were not altered significantly (Fig 2, B).

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FIG 3. Effect of transfer of IL-101 DCs from IL-10–deficient mice. A, Changes in lung resistance (RL) to inhaled MCh; B, BAL cellular composition. OVA-pulsed IL-101 DCs from bone marrow of wild-type or IL-10–deficient mice were administered to OVA-sensitized mice before airway challenge. The data are expressed as means 6 SEMs from 3 separate experiments (n 5 12). *P < .05 or **P < .01 between recipients of DCs from IL-101/1 and IL-102/2 donors.

DCs from IL-10–deficient mice fail to alter AHR and airway eosinophilia DCs from IL-10–deficient mice were differentiated in the presence of IL-10, pulsed with OVA, and transferred

into OVA-sensitized mice before the first of 3 airway challenges to determine whether endogenous IL-10 from the DCs was essential to the downregulation of AHR and airway eosinophilia. As shown in Fig 3, A, transfer of OVA-pulsed IL-10–treated DCs from IL-10–deficient mice to sensitized and challenged recipients failed to significantly alter the development of AHR (Fig 3, A) or airway eosinophilia (Fig 3, B) compared with that seen in sensitized and challenged recipients of pulsed IL-101– treated DCs from IL-101/1 donors. Levels of IL-10 in the BAL fluid of recipients of pulsed IL-101–treated DCs from IL-10–deficient mice (15 6 7 pg/mL) were not significantly different from those found in recipients of no DCs (18 6 6, n 5 12) but were significantly lower than in the BAL fluid of recipients of IL-101–treated DCs from IL-101/1 mice (36 6 11 pg/mL, n 5 12, P < .05).

DISCUSSION We examined the functional activity of IL-10–treated BMDCs on the development of lung allergic responses in both naive mice exposed to limited airway challenge and in sensitized and challenged mice. In vitro IL-10–treated DCs, which expressed lower levels of CD80 and CD86, were shown to suppress the production of IL-4, IL-5, and IL-13 in cultures of spleen T cells from OVA-sensitized mice. These IL-10–treated DCs released lower levels of IL-12 but significantly more IL-10. Unlike the transfer of DCs not differentiated in the presence of IL-10, in naive mice the transfer of IL-10–treated DCs prevented the development of AHR, airway eosinophilia, increased TH2 cytokines levels in BAL fluid, and goblet cell metaplasia when challenged with allergen. In previously sensitized mice, when administered before challenge, IL-10–treated

Basic and clinical immunology

After challenge of sensitized mice, increased levels of the TH2-type cytokines IL-5 and IL-13 in the BAL fluid were observed compared with those found in nonsensitized mice; levels of IL-4 were only marginally increased. These cytokine levels were decreased significantly after transfer of OVA-pulsed IL-101 DCs, whereas levels of IL-10 in BAL fluid were increased in mice that received OVA-pulsed IL-101 DCs (Fig 2, C). Levels of IL-12 or IFN-g were not altered by OVA-pulsed IL-101 DC treatment (data not shown). Transfer of IL-101 DCs that were not pulsed with OVA had no significant effect on cytokine levels. In allergen-sensitized and allergen-challenged mice, transfer of OVA-pulsed IL-101 DCs also significantly reduced the numbers of PAS-positive cells, whereas transfer of nonpulsed IL-101 DCs had little effect on the numbers of goblet cells (Fig 2, D). Intracellular cytokine staining of lung T cells was performed to determine whether the phenotype of lung T cells was altered after transfer of IL-101 DCs into sensitized and challenged recipients. The percentage of CD41IL-101 T cells in the CD41 T-cell fraction was significantly increased in mice that received OVA-pulsed IL101 DCs compared with percentages found in recipients of nonpulsed IL-101 DCs (Fig 2, E). However, the percentage of CD41CD251IL-101 T cells in the CD41 T-cell subset was not significantly different from that in the group that received OVA-pulsed IL-101 DCs (15.7% 6 3.4%) and recipients of nonpulsed IL-101 DCs (10.3% 6 2.2%). There were no differences in the total number of CD41IL-41 or CD41Foxp31 T cells in either group (data not shown).

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Basic and clinical immunology

DCs similarly suppressed AHR, airway eosinophilia, TH2 cytokine increases, and goblet cell metaplasia. In these recipient mice the levels of IL-10 in BAL fluid were increased, as were the numbers of IL-10–producing CD41 T cells in the lung. These studies demonstrate that IL-10 treatment of DCs has profound negative regulatory effects on the development of lung allergic responses in recipient mice. Moreover, the negative regulatory effects exhibited by the IL-10–treated DCs were dependent on endogenous production of IL-10 because DCs from IL-10–deficient mice, despite differentiation in the presence of IL-10, failed to exhibit the negative regulatory effects in any of these assays. Nakagome et al36 reported that IL-10 gene delivery suppressed OVA-induced eosinophilic airway inflammation and AHR by means of a mechanism suggested to be through downregulation of lung APC function and reduced TH2 responses. They also described that mice that received IL-10–treated spleen DCs primed with OVA had a milder airway allergic inflammatory response compared with that found in control animals. In the present study adoptive transfer of IL-10–treated BMDCs before OVA challenge either failed to induce in naive mice or significantly suppressed in sensitized mice the development of AHR and airway allergic inflammation, confirming the important functional differences between IL-10– treated and nontreated DCs. How IL-10 treatment of DCs directs their ‘‘regulatory’’ role is not well understood. Among the possible explanations for the negative regulatory activities of IL-10–treated DCs on AHR and lung allergic responses is that IL-10 treatment might influence DC maturation. DCs are essential to the priming of naive T cells and the initiation of cellular immune responses. However, DCs residing in the mucosa present antigen quite inefficiently because of their immaturity.6 IL-10–treated human or mouse DCs are reported to induce antigen-specific anergy in several studies.22,29,30,34 As a result, IL-10–treated DCs might have induced anergy/apoptosis in these antigen-specific T cells, leading to the failure of AHR and airway allergic inflammation development, increases in TH2 cytokine levels, and goblet cell metaplasia. In this study transfer of OVA-pulsed DCs that were not treated with IL-10 induced AHR and airway allergic inflammation after allergen challenge. Lambrecht et al9 and Koya et al20 previously reported that transferred DCs migrated to draining lymph nodes and primed naive T cells. In contrast, OVA-pulsed IL-10–treated DCs failed to induce AHR or airway inflammation. Because there was no evidence of T-cell apoptosis or anergy, it is suggested that the failure of transferred DC interaction with recipient T cells was, at least in part, the result of decreased expression of MHC and costimulatory molecules on the IL-10– treated DCs. A second explanation for the inhibitory effects of IL10–treated DCs might be through their ability to induce the expression and functional activation of regulatory T-cell populations. Several studies have shown that IL10–treated DCs have the potential to induce CD41 T cells

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expressing cytotoxic T lymphocyte–associated antigen 4 (CD152), which are implicated in the induction of tolerance in a cell-to-cell contact-dependent manner.25,31 Moreover, immature DCs, including IL-10–treated DCs, are potent inducers of IL-10–producing and nonproliferating regulatory T cells, which suppress proliferation and inflammatory cytokine production from memory T cells.25,37 Wakkach et al28 reported that among the IL10–treated DCs, a significant proportion expressed CD11clowB2202CD45RB1 and that LPS, but not CpG, stimulation enhanced the secretion of IL-10 from this subset of DCs. These IL-10–producing DCs play an important role in the induction of regulatory T cells, which secrete IL-10 and TGF-b and are unresponsiveness to antigen.38,39 In the present study IL-10–treated DCs produced considerable amounts of IL-10 when compared with nontreated DCs. As a corollary, we demonstrated that IL-10– treated DCs derived from IL-10–deficient mice were significantly less effective in the prevention of the development of AHR and airway eosinophilia. Of note, the levels of IL-10 in the BAL fluid of mice receiving IL101 DCs from IL-101/1 mice were higher than in mice that received IL-101 DCs from IL-10–deficient mice. Taken together, the data imply that IL-10 enhanced the release of endogenous IL-10 from IL-10–treated DCs, which in turn was essential for the negative regulatory effects on the development of AHR and allergic airway inflammation in both nonsensitized and sensitized recipients before airway allergen challenge. Regulatory T cells are diverse in phenotype and functional characteristics or mechanisms of suppression, as well as their ability to produce different cytokines.39-45 In some reports allergen exposure in the airways was shown to induce IL-10–producing pulmonary DCs, which induced IL-10–secreting regulatory T cells through, at least in part, IL-10 or inducible costimulator–inducible costimulator ligand signaling.38,46 CD41CD251 regulatory T cells have been shown to inhibit AHR and airway inflammation by altering pulmonary DC phenotype and function47 in an IL-10–dependent manner.39 In the present study the expression of Foxp3 in lung CD41 T cells, including CD41CD251 T cells, was not increased after transfer of IL-10–treated DCs compared with recipients of no cells or non-IL-10–treated DCs. Transfer of IL10–treated DCs did lead to increased numbers of CD41IL-101 T cells in the lung. Although Foxp32, these cells might have exerted negative regulatory effects on the allergen-induced responses, and this possibility is currently under investigation. In summary, IL-10–treated DCs expressing lower levels of the costimulatory molecules CD80 and CD86 and producing lower levels of IL-12 and higher levels of IL-10 suppressed the production of TH2 cytokines in vitro. Transfer of OVA-pulsed IL-10–treated DCs to naive recipients or sensitized recipients before challenge suppressed the development of AHR, airway eosinophilia, levels of IL-5 and IL-13 in BAL fluid, and goblet cell metaplasia. The requirement for the DCs themselves to release IL-10 was shown to be essential for their negative regulatory

effects. These data highlight the important role for IL-10 in (down)regulating DC function in the response to allergen challenge and identifies a novel therapeutic strategy for the regulation of allergen-induced alterations in airway responsiveness and inflammation. We are grateful for the expert help of Diana Nabighian in preparing the manuscript and Lynn Cunningham for performing the histologic studies.

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