IL-10–Producing Regulatory B10 Cells Inhibit Intestinal Injury in a Mouse Model
2011; Elsevier BV; Volume: 178; Issue: 2 Linguagem: Inglês
10.1016/j.ajpath.2010.10.022
ISSN1525-2191
AutoresKoichi Yanaba, Ayumi Yoshizaki, Yoshihide Asano, Takafumi Kadono, Thomas F. Tedder, Shinichi Sato,
Tópico(s)T-cell and B-cell Immunology
ResumoB cells mediate multiple functions that influence immune and inflammatory responses. In mice, the addition of dextran sulfate sodium (DSS) to drinking water leads to immediate intestinal injury. Dextran sulfate sodium–induced intestinal injury serves as an experimental animal model for human ulcerative colitis. The contribution of B cells to DSS-induced intestinal injury is unclear. In this study, we show that DSS-induced intestinal injury was more severe in CD19-deficient (CD19−/−) mice than in wild-type mice. These inflammatory responses were negatively regulated by a unique IL-10–producing CD1dhiCD5+ regulatory B cell subset (B10 cells) that was absent in CD19−/− mice and represented only 1% to 2% of splenic B220+ cells in wild-type mice. Remarkably, adoptive transfer of these B10 cells from wild-type mice reduced inflammation in CD19−/− mice in an IL-10–dependent manner. These results demonstrate that IL-10 production from regulatory B10 cells regulates DSS-induced intestinal injury. These findings may provide new insights and therapeutic approaches for treating ulcerative colitis. B cells mediate multiple functions that influence immune and inflammatory responses. In mice, the addition of dextran sulfate sodium (DSS) to drinking water leads to immediate intestinal injury. Dextran sulfate sodium–induced intestinal injury serves as an experimental animal model for human ulcerative colitis. The contribution of B cells to DSS-induced intestinal injury is unclear. In this study, we show that DSS-induced intestinal injury was more severe in CD19-deficient (CD19−/−) mice than in wild-type mice. These inflammatory responses were negatively regulated by a unique IL-10–producing CD1dhiCD5+ regulatory B cell subset (B10 cells) that was absent in CD19−/− mice and represented only 1% to 2% of splenic B220+ cells in wild-type mice. Remarkably, adoptive transfer of these B10 cells from wild-type mice reduced inflammation in CD19−/− mice in an IL-10–dependent manner. These results demonstrate that IL-10 production from regulatory B10 cells regulates DSS-induced intestinal injury. These findings may provide new insights and therapeutic approaches for treating ulcerative colitis. Ulcerative colitis (UC) is an inflammatory bowel disease characterized by pathological mucosal damage and ulceration, which can involve the rectum and extend proximally.1Fiocchi C. Inflammatory bowel disease: etiology and pathogenesis.Gastroenterology. 1998; 115: 182-205Abstract Full Text Full Text PDF PubMed Scopus (1827) Google Scholar Although the etiology and pathogenesis of UC have not yet been identified, inappropriate activation of the mucosal immune system has played an important role in the pathogenesis of mucosal inflammation. At sites of intestinal inflammation, granulocytes and macrophages produce high levels of proinflammatory cytokines, including IL-1β, IL-6, and tumor necrosis factor-α,2Hibi T. Ogata H. Novel pathophysiological concepts of inflammatory bowel disease.J Gastroenterol. 2006; 41: 10-16Crossref PubMed Scopus (110) Google Scholar, 3Sands B.E. Inflammatory bowel disease: past, present, and future.J Gastroenterol. 2007; 42: 16-25Crossref PubMed Scopus (191) Google Scholar that are directly involved in the pathogenesis of UC. Oral administration of dextran sulfate sodium (DSS) solution to rodents is widely used as a model of human UC because it can cause an acute inflammatory reaction and ulceration in the entire colon, similar to that observed in patients with UC.4Okayasu I. Hatakeyama S. Yamada M. Ohkusa T. Inagaki Y. Nakaya R. A novel method in the induction of reliable experimental acute and chronic ulcerative colitis in mice.Gastroenterology. 1990; 98: 694-702Abstract PubMed Google Scholar, 5Cooper H.S. Murthy S.N. Shah R.S. Sedergran D.J. Clinicopathologic study of dextran sulfate sodium experimental murine colitis.Lab Invest. 1993; 69: 238-249PubMed Google Scholar Mice exposed to DSS in drinking water develop inflammation only in the large intestine and show signs such as diarrhea, hematochezia, and body weight loss with histological findings, including inflammatory cell infiltration, erosion, ulceration, and crypt abscesses. Furthermore, increased production of proinflammatory cytokines, including interferon-γ, tumor necrosis factor-α, and ILs-1, -6, -12, and -17, has been found in the colon of mice with DSS-induced intestinal injury.6Melgar S. Karlsson A. Michaelsson E. Acute colitis induced by dextran sulfate sodium progresses to chronicity in C57BL/6 but not in BALB/c mice: correlation between symptoms and inflammation.Am J Physiol Gastrointest Liver Physiol. 2005; 288: G1328-G1338Crossref PubMed Scopus (398) Google Scholar, 7Egger B. Bajaj-Elliott M. MacDonald T.T. Inglin R. Eysselein V.E. Buchler M.W. Characterisation of acute murine dextran sodium sulphate colitis: cytokine profile and dose dependency.Digestion. 2000; 62: 240-248Crossref PubMed Scopus (304) Google Scholar B cells play a central role in humoral immunity and regulate CD4+ T-cell responses to foreign and self-antigens,8Bouaziz J.D. Yanaba K. Venturi G.M. Wang Y. Tisch R.M. Poe J.C. Tedder T.F. Therapeutic B cell depletion impairs adaptive and autoreactive CD4+ T cell activation in mice.Proc Natl Acad Sci U S A. 2007; 104: 20882-20887Crossref Scopus (250) Google Scholar, 9Xiu Y. Wong C.P. Hamaguchi Y. Wang Y. Pop S. Tisch R.M. Tedder T.F. B lymphocytes depletion by CD20 monoclonal antibody prevents diabetes in NOD mice despite isotype-specific differences in FcγR effector functions.J Immunol. 2008; 180: 2863-2875PubMed Google Scholar function as antigen-presenting cells,10Constant S. Schweitzer N. West J. Ranney P. Bottomly K. B lymphocytes can be competent antigen-presenting cells for priming CD4+ T cells to protein antigens in vivo.J Immunol. 1995; 155: 3734-3741PubMed Google Scholar produce cytokines,11Harris D.P. Haynes L. Sayles P.C. Duso D.K. Eaton S.M. Lepak N.M. Johnson L.L. Swain S.L. Lund F.E. Reciprocal regulation of polarized cytokine production by effector B and T cells.Nat Immunol. 2000; 1: 475-482Crossref PubMed Scopus (643) Google Scholar provide co-stimulatory signals,12Linton P.J. Bautista B. Biederman E. Bradley E.S. Harbertson J. Kondrack R.M. Padrick R.C. Bradley L.M. Costimulation via OX40L expressed by B cells is sufficient to determine the extent of primary CD4 cell expansion and Th2 cytokine secretion in vivo.J Exp Med. 2003; 197: 875-883Crossref PubMed Scopus (194) Google Scholar and promote naïve CD4+ T-cell differentiation into T-helper 1 or 2 subsets.11Harris D.P. Haynes L. Sayles P.C. Duso D.K. Eaton S.M. Lepak N.M. Johnson L.L. Swain S.L. Lund F.E. Reciprocal regulation of polarized cytokine production by effector B and T cells.Nat Immunol. 2000; 1: 475-482Crossref PubMed Scopus (643) Google Scholar Abnormal B-cell function can also drive the development of autoimmunity.13Sato S. Ono N. Steeber D.A. Pisetsky D.S. Tedder T.F. CD19 regulates B lymphocyte signaling thresholds critical for the development of B-1 lineage cells and autoimmunity.J Immunol. 1996; 157: 4371-4378PubMed Google Scholar Recently, it has been demonstrated that B cells and specific B-cell subsets can also negatively regulate immune responses in mice, validating the existence of regulatory B cells.14Bouaziz J.D. Yanaba K. Tedder T.F. Regulatory B cells as inhibitors of immune responses and inflammation.Immunol Rev. 2008; 224: 201-214Crossref PubMed Scopus (349) Google Scholar A potent subset of regulatory B cells with a phenotype of CD1dhiCD5+ regulates T-cell–dependent contact hypersensitivity and experimental autoimmune encephalomyelitis (EAE) in an IL-10–dependent manner.15Yanaba K. Bouaziz J.-D. Haas K.M. Poe J.C. Fujimoto M. Tedder T.F. A regulatory B cell subset with a unique CD1dhiCD5+ phenotype controls T cell-dependent inflammatory responses.Immunity. 2008; 28: 639-650Abstract Full Text Full Text PDF PubMed Scopus (931) Google Scholar, 16Matsushita T. Yanaba K. Bouaziz J.D. Fujimoto M. Tedder T.F. Regulatory B cells inhibit EAE initiation in mice while other B cells promote disease progression.J Clin Invest. 2008; 118: 3420-3430PubMed Google Scholar This regulatory B-cell subset is known as B10 cells to distinguish it from other possible regulatory B-cell subsets and to identify the cells as the predominant source of B-cell IL-10 production. B10 cell regulatory functions are antigen restricted in vivo, and the adoptive transfer of antigen-primed B10 cells reduces inflammation during contact hypersensitivity responses and ameliorates the severity of EAE.15Yanaba K. Bouaziz J.-D. Haas K.M. Poe J.C. Fujimoto M. Tedder T.F. A regulatory B cell subset with a unique CD1dhiCD5+ phenotype controls T cell-dependent inflammatory responses.Immunity. 2008; 28: 639-650Abstract Full Text Full Text PDF PubMed Scopus (931) Google Scholar, 16Matsushita T. Yanaba K. Bouaziz J.D. Fujimoto M. Tedder T.F. Regulatory B cells inhibit EAE initiation in mice while other B cells promote disease progression.J Clin Invest. 2008; 118: 3420-3430PubMed Google Scholar The severity of chronic colitis is increased by B-cell deficiency in T-cell receptor (TCR)-α−/− mice.17Mizoguchi A. Mizoguchi E. Smith R.N. Preffer F.I. Bhan A.K. Suppressive role of B cells in chronic colitis of T cell receptor α mutant mice.J Exp Med. 1997; 186: 1749-1756Crossref PubMed Scopus (289) Google Scholar The neutralization of IL-10 by monoclonal antibody (mAb) treatment also enhances the severity of colitis.18Mizoguchi A. Mizoguchi E. Takedatsu H. Blumberg R.S. Bhan A.K. Chronic intestinal inflammatory condition generates IL-10-producing regulatory B cell subset characterized by CD1d upregulation.Immunity. 2002; 16: 219-230Abstract Full Text Full Text PDF PubMed Scopus (721) Google Scholar Furthermore, IL-10 deficiency results in the development of spontaneous chronic colitis in mice.19Kuhn R. Lohler J. Rennick D. Rajewsky K. Muller W. Interleukin-10-deficient mice develop chronic enterocolitis.Cell. 1993; 75: 263-274Abstract Full Text PDF PubMed Scopus (3536) Google Scholar Thus, B cells and IL-10 play important inhibitory roles in the development of colitis. The phenotypically unique regulatory B10 cell subset is found within the spleen of naïve wild-type mice at 1% to 2% of the total B-cell count, whereas CD19-deficient (CD19−/−) mice have few, if any, B10 cells.15Yanaba K. Bouaziz J.-D. Haas K.M. Poe J.C. Fujimoto M. Tedder T.F. A regulatory B cell subset with a unique CD1dhiCD5+ phenotype controls T cell-dependent inflammatory responses.Immunity. 2008; 28: 639-650Abstract Full Text Full Text PDF PubMed Scopus (931) Google Scholar Therefore, we examined the importance of regulatory B cells in a DSS-induced UC model in CD19−/− and wild-type mice. Wild-type C57BL/6 and IL-10−/− (B6.129P2-Il10tm/cgn/J) mice19Kuhn R. Lohler J. Rennick D. Rajewsky K. Muller W. Interleukin-10-deficient mice develop chronic enterocolitis.Cell. 1993; 75: 263-274Abstract Full Text PDF PubMed Scopus (3536) Google Scholar were purchased from Jackson Laboratory (Bar Harbor, ME). CD19−/− (C57BL/6 × 129) mice were generated as previously described20Engel P. Zhou L.-J. Ord D.C. Sato S. Koller B. Tedder T.F. Abnormal B lymphocyte development, activation and differentiation in mice that lack or overexpress the CD19 signal transduction molecule.Immunity. 1995; 3: 39-50Abstract Full Text PDF PubMed Scopus (479) Google Scholar and backcrossed 7 to 12 generations onto the C57BL6 background before use in this study. Lack of cell surface CD19 expression was verified by two-color immunofluorescence staining with flow cytometric analysis. All mice were bred in a specific pathogen-free barrier facility and used at the age of 8 to 12 weeks. All studies were approved by the Committee on Animal Experimentation of Nagasaki University Graduate School of Medical Sciences, Nagasaki, Japan. Three percent (w/v) DSS (molecular mass, 36 to 50 kDa; Sigma, St Louis, MO) was dissolved in purified water and administered to mice in place of normal drinking water for 7 days.21Wirtz S. Neufert C. Weigmann B. Neurath M.F. Chemically induced mouse models of intestinal inflammation.Nat Protoc. 2007; 2: 541-546Crossref PubMed Scopus (1069) Google Scholar The volume of water intake was measured daily to determine the amount of DSS consumed per mouse; this was comparable between treatment groups in all experiments. In some experiments, mice were treated with IL-10 receptor (1B1.3a; BioLegend, San Diego, CA) or control mAb (250 μg) on days 0 and 3 after DSS administration. To determine the effect of recombinant murine IL-10 (rIL-10; Sigma) administration, rIL-10 (400 ng) was injected intraperitoneally on days 0, 2, 4, and 6 after the induction of intestinal injury. The clinical scoring of the disease activity index (DAI) for DSS-induced intestinal injury was based on weight loss, stool consistency, and bleeding, as previously described.22Murthy S.N. Cooper H.S. Shim H. Shah R.S. Ibrahim S.A. Sedergran D.J. Treatment of dextran sulfate sodium-induced murine colitis by intracolonic cyclosporin.Dig Dis Sci. 1993; 38: 1722-1734Crossref PubMed Scopus (417) Google Scholar The DAI was scored on a scale from 0 to 4 for each clinical parameter and then averaged for each group. Weight changes were based on the starting weight of each mouse at the initiation of DSS treatment. Weight loss scores were determined as follows: 0, no weight loss; 1, 1% to 5% weight loss; 2, 6% to 10% weight loss; 3, 11% to 15% weight loss; and 4, greater than 15% weight loss. Stool samples were collected from each mouse at all points. Stool scores were determined as follows: 0, normal stools; 2, loose stools; and 4, diarrhea. (Grades 1 and 3 do not exist in this scale.) Fecal blood testing kits (Shionogi, Osaka, Japan) were used to check the stools for the presence of blood. Bleeding scores were determined as follows: 0, no bleeding; 1, guaiac occult blood test, resulting in minimal color change to green; 2, guaiac occult blood test, resulting in maximal color change to blue; 3, blood visibly present in the stool and no clotting on the anus; and 4, gross bleeding from the anus with clotting present. The mice were sacrificed 5 days after the induction of intestinal injury. Colon samples were removed and segments were fixed in 10% buffered formalin. After paraffin embedding, 5-μm-thick sections were cut and stained with H&E. Histological scoring was based on a previously described method.21Wirtz S. Neufert C. Weigmann B. Neurath M.F. Chemically induced mouse models of intestinal inflammation.Nat Protoc. 2007; 2: 541-546Crossref PubMed Scopus (1069) Google Scholar Briefly, H&E-stained cross sections of the descending colon tissue were scored microscopically in a blinded fashion on a scale from 0 to 4, based on the following histological criteria: 0, no change from normal tissue; 1, low level of inflammation with scattered infiltrating mononuclear cells (foci, 1 to 2); 2, moderate inflammation with multiple foci; 3, high level of inflammation with increased vascular density and marked wall thickening; and 4, maximal severity of inflammation with transmural leukocyte infiltration and loss of goblet cells. An average of four fields of view per colon was evaluated for each mouse. These scores were averaged for each group and recorded as the histopathological score. For immunohistochemistry, frozen tissue sections of the colon samples were acetone fixed and incubated with 10% normal rabbit serum in phospate-buffered saline (for 10 minutes at 37°C) to block nonspecific staining. Sections were then incubated with rat mAbs specific for mouse CD3 (BD PharMingen, San Diego, CA), B220 (BD PharMingen), and macrophages (F4/80; American Type Culture Collection, Rockville, MD). Rat IgG (Southern Biotechnology Associates Inc., Birmingham, AL) was used as a control for nonspecific staining. Sections were then incubated sequentially (for 20 minutes at 37°C) with a biotinylated rabbit anti–rat IgG and then a horseradish peroxidase–conjugated avidin-biotin complex (Vectastain ABC kit; Vector Laboratories, Burlingame, CA). Sections were developed with 3,3′-diaminobenzidine tetrahydrochloride and hydrogen peroxide and then counterstained with methyl green. Stained cells were counted in 10 random grids under high-magnification fields (×400) using a light microscope. Each section was examined independently by two investigators (K.Y. and A.Y.) in a blinded manner. Single-cell suspensions of splenic and mesenteric lymph node were generated by gentle dissection. Intestinal Peyer's patches were isolated as previously described.23Venturi G.M. Tu L. Kadono T. Khan A.I. Fujimoto Y. Oshel P. Bock C.B. Miller A.S. Albrecht R.M. Kubes P. Steeber D.A. Tedder T.F. Leukocyte migration is regulated by L-selectin endoproteolytic release.Immunity. 2003; 19: 713-724Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar Lamina propria lymphocytes were isolated by modification of a previously described protocol.24Taguchi T. McGhee J.R. Coffman R.L. Beagley K.W. Eldridge J.H. Takatsu K. Kiyono H. Analysis of Th1 and Th2 cells in murine gut-associated tissues: frequencies of CD4+ and CD8+ T cells that secrete IFN-γ and IL-5.J Immunol. 1990; 145: 68-77PubMed Google Scholar Peripheral blood mononuclear cells were isolated from heparinized blood after centrifugation over a discontinuous Lymphoprep (Axis-Shield PoC As, Oslo, Norway) gradient. B220 mAb–coated microbeads (Miltenyi Biotech, Auburn, CA) were used to purify B cells by positive selection, according to the manufacturer's instructions. When necessary, the cells were enriched a second time using a fresh MACS column (Miltenyi Biotech) to obtain more than 95% B220+ cell purity. Total RNA was isolated from purified B cells with spin columns (RNeasy; Qiagen, Crawley, UK). Total RNA from each sample was reverse transcribed into cDNA. The expression of IL-10 was analyzed using a real-time PCR quantification method according to the manufacturer's instructions (Applied Biosystems, Foster City, CA). Sequence-specific primers and probes were designed by assay reagents (Pre-Developed TaqMan) or assay (Assay-On-Demand) (Applied Biosystems for both). Real-time PCR (40 cycles of denaturing at 92°C for 15 seconds and annealing at 60°C for 60 seconds) was performed on a sequence detector (ABI Prism 7000; Applied Biosystems). Glyceraldehyde-3-phosphate dehydrogenase was used to normalize mRNA expression. The relative expression of real-time PCR products was determined using the ΔΔCT method25Meijerink J. Mandigers C. van de Locht L. Tonnissen E. Goodsaid F. Raemaekers J. A novel method to compensate for different amplification efficiencies between patient DNA samples in quantitative real-time PCR.J Mol Diag. 2001; 3: 55-61Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar to compare target gene with housekeeping gene mRNA expression. One of the control samples was chosen as a calibrator sample. Anti–mouse mAbs with specificities against B220 (RA3-6B2), CD19 (1D3), CD5 (53–7.3), and CD1d (1B1) were obtained from BD PharMingen. Phycoerythrin-conjugated anti–mouse IL-10 mAb (JES5-16E3) was obtained from eBioscience (San Diego). Single-cell suspensions of splenic and mesenteric lymph node were generated by gentle dissection. Viable cells were counted using a hemocytometer, with relative lymphocyte percentages determined by flow cytometry. Single-cell leukocyte suspensions were stained on ice using predetermined optimal concentrations of each antibody for 20 to 60 minutes and fixed as previously described.13Sato S. Ono N. Steeber D.A. Pisetsky D.S. Tedder T.F. CD19 regulates B lymphocyte signaling thresholds critical for the development of B-1 lineage cells and autoimmunity.J Immunol. 1996; 157: 4371-4378PubMed Google Scholar Cells with the light scatter properties of lymphocytes were analyzed using two- to four-color immunofluorescence staining and flow cytometers (FACSCalibur; Becton Dickinson, San Jose, CA). Background staining was determined using unreactive isotype-matched control mAbs (Caltag Laboratories, San Francisco, CA), with gates positioned to exclude 98% or greater of unreactive cells. Briefly, isolated leukocytes or purified cells were resuspended (1 × 106 cells/ml) with lipopolysaccharide (10 μg/ml), phorbol 12-myristate 13-acetate (50 ng/ml, Sigma), ionomycin (500 ng/ml, Sigma), and monensin (2 μmol/L, eBioscience) for 5 hours. For IL-10 detection, Fc receptors were blocked with mouse Fc receptor–specific mAb (2.4G2, BD PharMingen) before cell surface staining; and then fixed and permeabilized using a kit (Cytofix/Cytoperm kit; BD PharMingen), according to manufacturer's instructions. Permeabilized cells were stained with phycoerythrin-conjugated IL-10 mAb. Splenic B cells were purified using B220 mAb–coupled microbeads. In addition, CD1dhiCD5+ B cells were selected using a flow cytometer (FACSAria; Becton Dickinson), with purities of approximately 85% to 95%. After isolation, 2 × 106 CD1dhiCD5+ or non–CD1dhiCD5+ B cells were transferred by intraveneous injection into CD19−/− mice before the induction of intestinal injury. All data are expressed as mean ± SEM. The Mann-Whitney U-test was used for determining the level of significance of differences in sample means, and the Bonferroni test was used for multiple comparisons. To assess whether CD19 expression plays a role in the pathogenesis of DSS-induced intestinal injury, we treated CD19−/− and wild-type mice with 3% DSS for 7 days and quantitatively evaluated the severity of intestinal injury by measuring body weight and DAI scores. The DAI scores were based on weight loss, stool consistency, and bleeding. Body weight loss was first observed in DSS-treated wild-type mice on day 6 (Figure 1A). By contrast, DSS-treated CD19−/− mice began to show significant body weight loss on day 3 and continued to lose weight until day 7. The CD19 deficiency in DSS-treated mice caused a significant decrease in body weight from day 3 to 7 compared with DSS treatment alone in wild-type mice. Furthermore, DAI scores in DSS-treated wild-type mice began to show a significant increase on day 3, whereas the increase in DAI scores in DSS-treated CD19−/− mice was first observed on day 2. The DAI scores were significantly higher in DSS-treated CD19−/− mice than in DSS-treated wild-type mice from day 3 to 7. Each element of the DAI score showed the same trend as the overall DAI score (Figure 1B). Thus, CD19−/− mice are more susceptible to DSS-induced intestinal injury. To further evaluate disease severity, the degree of intestinal injury was also assessed histopathologically. After the 7-day period of ingestion of 3% DSS or normal drinking water, the colons were removed for histopathological evaluation (Figure 2A). Although DSS treatment induced epithelial injury and increased mononuclear cell infiltration and inflammatory changes in submucosal tissues in both wild-type and CD19−/− mice, these changes were more severe in the CD19−/− mice. The pathological scores were significantly higher in DSS-treated CD19−/− mice than in DSS-treated wild-type mice (P < 0.01, Figure 2B). Furthermore, neutrophil and T-cell numbers were significantly increased in CD19−/− mice relative to wild-type mice (P < 0.05, Figure 2C). There were no significant differences in the numbers of B cells and macrophages between wild-type and CD19−/− mice. Thus, intestinal injury was more severe, both clinically and pathologically, in CD19−/− mice than in wild-type mice. Although cytoplasmic IL-10 production was not detected in resting B cells from wild-type mice, splenic B cells that are competent to express cytoplasmic IL-10 after 5 hours of in vitro stimulation with lipopolysaccharide, phorbol 12-myristate 13-acetate, ionomycin, and monensin were predominantly found within the CD1dhiCD5+ B cell subset in wild-type mice (Figure 3A), as previously described.15Yanaba K. Bouaziz J.-D. Haas K.M. Poe J.C. Fujimoto M. Tedder T.F. A regulatory B cell subset with a unique CD1dhiCD5+ phenotype controls T cell-dependent inflammatory responses.Immunity. 2008; 28: 639-650Abstract Full Text Full Text PDF PubMed Scopus (931) Google Scholar, 26Yanaba K. Bouaziz J.D. Matsushita T. Tsubata T. Tedder T.F. The development and function of regulatory B cells expressing IL-10 (B10 cells) requires antigen receptor diversity and TLR signals.J Immunol. 2009; 182: 7459-7472Crossref PubMed Scopus (372) Google Scholar By contrast, IL-10–producing B cells were less common within the non–CD1dhiCD5+ B-cell subset. After stimulation for 5 hours with lipopolysaccharide, phorbol 12-myristate 13-acetate, and ionomycin, the proportions and absolute numbers of splenic IL-10–producing B cells were 4.9- and 8.2-fold higher in wild-type than in CD19−/− mice, respectively (P < 0.01, Figure 3B), as previously described.15Yanaba K. Bouaziz J.-D. Haas K.M. Poe J.C. Fujimoto M. Tedder T.F. A regulatory B cell subset with a unique CD1dhiCD5+ phenotype controls T cell-dependent inflammatory responses.Immunity. 2008; 28: 639-650Abstract Full Text Full Text PDF PubMed Scopus (931) Google Scholar Furthermore, the proportions and absolute numbers of splenic CD1dhiCD5+ B cells were 8.1- and 6.1-fold higher in wild-type than in CD19−/− mice, respectively (P < 0.01, Figure 3C). There were no detectable splenic IL-10–producing or CD1dhiCD5+ B cells in CD19−/− mice. Thus, the proportions and numbers of B10 cells were inversely proportional to the severity of intestinal injury in wild-type and CD19−/− mice. B10 cells and splenic CD1dhiCD5+ B-cell subpopulations are significantly expanded in autoimmune-prone mice.26Yanaba K. Bouaziz J.D. Matsushita T. Tsubata T. Tedder T.F. The development and function of regulatory B cells expressing IL-10 (B10 cells) requires antigen receptor diversity and TLR signals.J Immunol. 2009; 182: 7459-7472Crossref PubMed Scopus (372) Google Scholar To determine whether B10 cells expand during DSS-induced intestinal injury, B10 cell numbers were quantified. Splenic IL-10–producing B-cell proportions and numbers were significantly increased on day 7 in DSS-treated wild-type mice compared with naïve wild-type mice (Figure 3B; P < 0.01 and P < 0.05, respectively). CD1dhiCD5+ B-cell proportions and numbers were also significantly increased on day 7 in wild-type mice after DSS administration (Figure 3C, P < 0.01). Increased B-cell IL-10 production paralleled CD1dhiCD5+ B-cell proportions. The administration of DSS did not affect the proportions and numbers of splenic IL-10–producing B cells and CD1dhiCD5+ B cells in CD19−/− mice. Thus, there was an increase in B10 cell numbers after DSS administration. To determine whether B-cell IL-10 production might regulate DSS-induced intestinal injury, IL-10 production by B cells was assessed in wild-type and CD19−/− mice. The number of splenic B cells was significantly decreased in CD19−/− mice relative to wild-type mice (P < 0.01, Table 1), as previously described.27Haas K.M. Poe J.C. Steeber D.A. Tedder T.F. B-1a and B-1b cells exhibit distinct developmental requirements and have unique functional roles in innate and adaptive immunity to S. pneumoniae.Immunity. 2005; 23: 7-18Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar B cells from spleen, mesenteric lymph node, Peyer's patches, and intestinal lamina propria were purified 5 days after the administration of DSS; and IL-10 mRNA expression was quantified by real-time PCR. There were no significant differences in B-cell numbers in mesenteric lymph node, Peyer's patches, and intestinal lamina propria between wild-type and CD19−/− mice. During the induction of intestinal injury, splenic B cells from wild-type mice expressed more IL-10 transcripts than naïve B cells (17.8-fold, P < 0.01, Figure 4A). By contrast, IL-10 transcripts in splenic B cells from DSS-treated CD19−/− mice were similar to those from naïve CD19−/− mice. Furthermore, IL-10 transcripts in DSS-treated wild-type B cells were significantly increased compared with those in DSS-treated CD19−/− B cells (19.4-fold, P < 0.01). Moreover, B-cell IL-10 mRNA expression in mesenteric lymph nodes, Peyer's patches, and intestinal lamina propria did not change during DSS-induced intestinal injury in both wild-type and CD19−/− mice. Thus, B-cell IL-10 production in the spleen, but not in mesenteric lymph nodes, Peyer's patches, and intestinal lamina propria, was increased during DSS-induced intestinal injury and was inversely proportional to the severity of the inflammatory response.Table 1B Cells in Wild-Type and CD19−/− Mice⁎Data are given as the mean ± SEM (n ≥ 4 mice).B220+ B-cells, ×10−5Mouse genotypeSpleenMesenteric lymph nodePeyer's patchLamina propriaWild type451 ± 4529 ± 56.1 ± 2.33.1 ± 1.6CD19−/−263 ± 41†P < 0.01 (versus wild-type mice).23 ± 54.3 ± 1.92.6 ± 1.1 Data are given as the mean ± SEM (n ≥ 4 mice).† P < 0.01 (versus wild-type mice). Open table in a new tab Splenic IL-10–producing B10 cells have a phenotype of CD1dhiCD5+ and inhibit contact hypersensitivity responses and EAE.15Yanaba K. Bouaziz J.-D. Haas K.M. Poe J.C. Fujimoto M. Tedder T.F. A regulatory B cell subset with a unique CD1dhiCD5+ phenotype controls T cell-dependent inflammatory responses.Immunity. 2008; 28: 639-650Abstract Full Text Full Text PDF PubMed Scopus (931) Google Scholar, 16Matsushita T. Yanaba K. Bouaziz J.D. Fujimoto M. Tedder T.F. Regulatory B cells inhibit EAE initiation in mice while other B cells promote disease progression.J Clin Invest. 2008; 118: 3420-3430PubMed Google Scholar To assess IL-10 production by the splenic B10-cell subset during DSS-induced intestinal injury, IL-10 transcripts were quantified by real-time PCR analysis. The IL-10 transcripts produced by the splenic CD1dhiCD5+ B-cell subset were increased 4.1-fold during DSS-induced intestinal injury compared with CD1dhiCD5+ B ce
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