Shin Makita

CD4+CD25bright T Cells in Human Intestinal Lamina Propria as Regulatory Cells

It is well known that immune responses in the intestine remain in a state of controlled inflammation, suggesting that not only active suppression by regulatory T cells plays an important role in the normal intestinal homeostasis, but also its dysregulation leads to the development of inflammatory bowel disease. In this study, we demonstrate that the CD4+CD25bright T cells reside in the human intestinal lamina propria (LP) and functionally retain regulatory activities. All human LP CD4+ T cells regardless of CD25 expression constitutively expressed CTLA-4, glucocorticoid-induced TNFR family-related protein, and Foxp3 and proliferate poorly. Although LP CD4+CD25− T cells showed an activated and anergic/memory phenotype, they did not retain regulatory activity. In LP CD4+CD25+ T cells, however, cells expressing CD25 at high levels (CD4+CD25bright) suppressed the proliferation and various cytokine productions of CD4+CD25− T cells. LP CD4+CD25bright T cells by themselves produced fewer amounts of IL-2, IFN-γ, and IL-10. Interestingly, LP CD4+CD25bright T cells with regulatory T activity were significantly increased in patients with active inflammatory bowel disease. These results suggest that CD4+CD25bright T cells found in the normal and inflamed intestinal mucosa selectively inhibit the host immune response and therefore may contribute to the intestinal immune homeostasis.

MyD88-deficient mice develop severe intestinal inflammation in dextran sodium sulfate colitis.

BackgroundGut commensal microbes affect the development and activation of the mucosal and systemic immune systems. However, the exact molecular mechanism of these microbes that is involved in the development of colitis remains unclear.MethodsThe present study was conducted to determine the distinct role of the innate immune system in the development of a dextran sulfate sodium (DSS) colitis model in MyD88−/− mice, because myeloid differentiation protein (MyD88) is a major adaptor molecule essential for signaling via Toll-like receptors (TLRs). To this end, MyD88−/− and wild-type (WT) mice received sterile distilled water containing 1.2% DSS for 8 days. The survival rate, total clinical score (body weight loss, stool consistency, and rectal bleeding), colon length, and histological score were assessed. The expression of surface markers (F4/80 and CD4) on infiltrating lamina propria mononuclear cells was analyzed immunohistochemistrically.ResultsMyD88−/− mice exhibited increased susceptibility to DSS-induced colitis, as reflected by significantly higher lethality and higher clinical and histological scores, and more severe colonic shortening compared to WT mice. Immunohistochemical analysis revealed a significant increase of both F4/80+ macrophages and CD4+ T cells in the inflamed mucosa in DSS-fed MyD88−/− mice compared to DSS-fed WT mice.ConclusionsThese findings suggest that, via MyD88 signaling, the innate immune system in the gut plays an important protective role in colitis.

Blockade of B7-H1 Suppresses the Development of Chronic Intestinal Inflammation

A newly identified costimulatory molecule, programmed death-1 (PD-1), provides a negative signal that is essential for immune homeostasis. However, it has been suggested that its ligands, B7-H1 (PD-L1) and B7-dendritic cells (B7-DC; PD-L2), could also costimulate T cell proliferation and cytokine secretion. Here we demonstrate the involvement of PD-1/B7-H1 and B7-DC interaction in the development of colitis. We first examined the expression profiles of PD-1 and its ligands in both human inflammatory bowel disease and a murine chronic colitis model induced by adoptive transfer of CD4+CD45RBhigh T cells to SCID mice. Second, we assessed the therapeutic potential of neutralizing anti-B7-H1 and/or B7-DC mAbs using this colitis model. We found significantly increased expression of PD-1 on T cells and of B7-H1 on T, B, and macrophage/DCs in inflamed colon from both inflammatory bowel disease patients and colitic mice. Unexpectedly, the administration of anti-B7-H1, but not anti-B7-DC, mAb after transfer of CD4+CD45RBhigh T cells suppressed wasting disease with colitis, abrogated leukocyte infiltration, and reduced the production of IFN-γ, IL-2, and TNF-α, but not IL-4 or IL-10, by lamina propria CD4+ T cells. These data suggest that the interaction of PD-1/B7-H1, but not PD-1/B7-DC, might be involved in intestinal mucosal inflammation and also show a possible role of interaction between B7-H1 and an as yet unidentified receptor for B7-H1 in inducing T cell activation.

Regulation of Murine Inflammatory Bowel Disease by CD25+ and CD25−CD4+ Glucocorticoid-Induced TNF Receptor Family-Related Gene Regulatory T Cells

CD4+CD25+ regulatory T cells in normal animals are engaged in the maintenance of immunological self-tolerance and prevention of autoimmune disease. However, accumulating evidence suggests that a fraction of the peripheral CD4+CD25− T cell population also possesses regulatory activity in vivo. Recently, it has been shown glucocorticoid-induced TNFR family-related gene (GITR) is predominantly expressed on CD4+CD25+ regulatory T cells. In this study, we show evidence that CD4+GITR+ T cells, regardless of the CD25 expression, regulate the mucosal immune responses and intestinal inflammation. SCID mice restored with the CD4+GITR− T cell population developed wasting disease and severe chronic colitis. Cotransfer of CD4+GITR+ population prevented the development of CD4+CD45RBhigh T cell-transferred colitis. Administration of anti-GITR mAb-induced chronic colitis in mice restored both CD45RBhigh and CD45RBlow CD4+ T cells. Interestingly, both CD4+CD25+ and CD4+CD25− GITR+ T cells prevented wasting disease and colitis. Furthermore, in vitro studies revealed that CD4+CD25−GITR+ T cells as well as CD4+CD25+GITR+ T cells expressed CTLA-4 intracellularly, showed anergic, suppressed T cell proliferation, and produced IL-10 and TGF-β. These data suggest that GITR can be used as a specific marker for regulatory T cells controlling mucosal inflammation and also as a target for treatment of inflammatory bowel disease.

Intestinal Lamina Propria Retaining CD4+CD25+ Regulatory T Cells Is A Suppressive Site of Intestinal Inflammation

It is well known that immune responses in the intestine remain in a state of controlled inflammation, suggesting that not only does active suppression by regulatory T (TREG) cells play an important role in the normal intestinal homeostasis, but also that its dysregulation of immune response leads to the development of inflammatory bowel disease. In this study, we demonstrate that murine CD4+CD25+ T cells residing in the intestinal lamina propria (LP) constitutively express CTLA-4, glucocorticoid-induced TNFR, and Foxp3 and suppress proliferation of responder CD4+ T cells in vitro. Furthermore, cotransfer of intestinal LP CD4+CD25+ T cells prevents the development of chronic colitis induced by adoptive transfer of CD4+CD45RBhigh T cells into SCID mice. When lymphotoxin (LT)α-deficient intercrossed Rag2 double knockout mice (LTα−/− × Rag2−/−), which lack mesenteric lymph nodes and Peyer’s patches, are transferred with CD4+CD45RBhigh T cells, they develop severe wasting disease and chronic colitis despite the delayed kinetics as compared with the control LTα+/+ × Rag2−/− mice transferred with CD4+CD45RBhigh T cells. Of note, when a mixture of splenic CD4+CD25+ TREG cells and CD4+CD45RBhigh T cells are transferred into LTα−/− × Rag2−/− recipients, CD4+CD25+ TREG cells migrate into the colon and prevent the development of colitis in LTα−/− × Rag2−/− recipients as well as in the control LTα+/+ × Rag2−/− recipients. These results suggest that the intestinal LP harboring CD4+CD25+ TREG cells contributes to the intestinal immune suppression.

IL-7 Is Essential for the Development and the Persistence of Chronic Colitis

Although IL-7 has recently emerged as a key cytokine involved in controlling the homeostatic turnover and the survival of peripheral resting memory CD4+ T cells, its potential to be sustained pathogenic CD4+ T cells in chronic immune diseases, such as inflammatory bowel diseases, still remains unclear. In this study, we demonstrate that IL-7 is essential for the development and the persistence of chronic colitis induced by adoptive transfer of normal CD4+CD45RBhigh T cells or colitogenic lamina propria (LP) CD4+ memory T cells into immunodeficient IL-7+/+ × RAG-1−/− and IL-7−/− × RAG-1−/− mice. Although IL-7+/+ × RAG-1−/− recipients transferred with CD4+CD45RBhigh splenocytes developed massive inflammation of the large intestinal mucosa concurrent with massive expansion of Th1 cells, IL-7−/− × RAG-1−/− recipients did not. Furthermore, IL-7−/− × RAG-1−/−, but not IL-7+/+ × RAG-1−/−, mice transferred with LP CD4+CD44highCD62L−IL-7Rαhigh effector-memory T cells (TEM) isolated from colitic CD4+CD45RBhigh-transferred mice did not develop colitis. Although rapid proliferation of transferred colitogenic LP CD4+ TEM cells was observed in the in IL-7−/− × RAG-1−/− mice to a similar extent of those in IL-7+/+ × RAG-1−/− mice, Bcl-2 expression was significantly down-modulated in the transferred CD4+ T cells in IL-7−/− × RAG-1−/− mice compared with those in IL-7+/+ × RAG-1−/− mice. Taken together, IL-7 is essential for the development and the persistence of chronic colitis as a critical survival factor for colitogenic CD4+ TEM cells, suggesting that therapeutic approaches targeting IL-7/IL-7R signaling pathway may be feasible in the treatment of inflammatory bowel diseases.

Interleukin-18 overproduction exacerbates the development of colitis with markedly infiltrated macrophages in interleukin-18 transgenic mice

Background and Aim:  The authors have previously shown that production of interleukin (IL)‐18 was increased in the inflamed mucosa of patients with Crohns disease (CD) and blockade of IL‐18 ameliorated the murine model of CD. This demonstrated that IL‐18 plays a significant role during intestinal inflammation. However, the initial role of IL‐18 during intestinal inflammation was unclear; therefore the susceptibility of IL‐18 transgenic (Tg) mice to acute dextran sulfate sodium (DSS)‐induced colitis was examined.

Regulation of murine chronic colitis by CD4+CD25– programmed death‐1+ T cells

Naturally arising CD4+CD25+ regulatory T (TR) cells are engaged in the maintenance of self tolerance and prevention of autoimmune diseases. However, accumulating evidence suggests that a fraction of peripheral CD4+CD25– T cells also possesses regulatory activity. Programmed death‐1 (PD‐1) is a new member of the CD28/CTLA‐4 family, which has been implicated in the maintenance of peripheral self tolerance. Here, we identified a subpopulation of CD4+CD25–PD‐1+ T cells in the spleen of naive mice that constitutively expressed CTLA‐4 and FoxP3 and was hypoproliferative in response to anti‐CD3 antibody stimulation in vitro. However, the CD4+CD25–PD‐1+ T cells uniquely produced large amounts of IL‐4 and IL‐10 in response to anti‐CD3 and anti‐CD28 mAb stimulation, unlike the CD4+CD25+ TR cells. The CD4+CD25–PD‐1+ T cells exhibited a suppressor activity against the proliferation of anti‐CD3 antibody‐stimulated CD4+CD25–PD‐1– T cells in vitro, which was partially abrogated by anti‐CTLA‐4 mAb, but not by anti‐IL‐10 or anti‐PD‐1 mAb. Remarkably, the CD4+CD25–PD‐1+ T cells inhibited the development of colitis induced by adoptive transfer of CD4+CD45RBhigh T cells into C.B17‐scid/scid mice, albeit to a lesser extent than CD4+CD25+ TR cells, in a CTLA‐4‐dependent manner. These results indicate that the CD4+CD25–PD‐1+ T cells contain substantial amounts of TR cells that are involved in the maintenance of peripheral tolerance.

TH1/TH2-mediated colitis induced by adoptive transfer of CD4+CD45RBhigh T lymphocytes into nude mice

Background: Transfer of CD4+CD45RBhigh T cells from normal donors to SCID/Rag‐1, 2‐deficient mice, which lack T and B cells, leads to the development of a TH1‐mediated inflammatory bowel disease (IBD)‐like syndrome characterized by extensive mononuclear cell infiltrates and epithelial cell hyperplasia. Because it is well known that B cells are also involved in a multitude of mechanistic pathways in human IBD, this study attempts to establish a new model of colitis in nude mice. Methods: We transferred CD4+CD45RBhigh T cells into athymic nude mice, which lack thymus‐dependent T cells but retain normal B cells, to establish and investigate a B cell‐involving chronic colitis model. As a control, CD4+CD25− T cells were also used. Results: Mice reconstituted with CD4+CD45RBhigh but not CD4+CD25− T cells developed a wasting disease, with severe infiltrates of B cell aggregates as well as T cells, macrophages, and dendritic cells into the colon and elevated levels of interferon‐&ggr;, tumor necrosis factor‐&agr;, interleukin (IL)‐4, IL‐5, and IL‐10, by 7 weeks after T cell transfer. Furthermore, the infiltrated lamina propria B cells in colitic nude mice consisted predominantly of massive aggregated immunoglobulin (Ig) M‐ and scattered IgG‐positive cells, but not IgA‐positive cells. In contrast, mice reconstituted with CD4+CD45RBhigh and CD4+CD45RBlow did not develop wasting disease or colitis. Conclusions: Collectively, the power of the colitis model induced by the adoptive transfer of CD4+CD45RBhigh T cells into nude mice is that one can investigate the roles of TH2‐type cells and B cells in a regulatory T cell‐depleted condition.

Extracorporeal elimination of TNF‐α‐producing CD14dullCD16+ monocytes in leukocytapheresis therapy for ulcerative colitis

Background In recent years leukocytapheresis using a leukocyte removal filter (known as lymphocytapheresis, LCAP) has been applied to the treatment of various autoimmune diseases including ulcerative colitis (UC). In the present study we aimed to clarify how LCAP therapy modifies inflammatory responses by modulating circulating TNF‐&agr;‐producing monocytes. Methods Mononuclear cells were obtained from blood before and after the first treatment, and the expression profiles of various immune cells (naive versus. memory, regulatory CD4+CD25bright versus non‐regulatory CD4+CD25− T cells, and CD14+CD16− versus CD14dullCD16+ monocytes) were assessed. To evaluate immunological differences between CD14+CD16− and CD14dullCD16+ monocytes, the expression of TNF‐&agr;, IL‐6, IL‐12, IL‐10, IL‐18, surface toll‐like receptor 2 (TLR2), TLR4, and other activation markers including HLA‐DR, CD80 and CD86, as well as cytokine profiles, were analyzed. Results LCAP treatment selectively removed CD14dullCD16+ monocytes, which preferentially produce TNF‐&agr; and IL‐12 and express HLA‐DR, CD80, CD86, and TLR2, compared with the major fraction of CD14+CD16− monocytes, which conversely produce a higher amount of IL‐10. In addition, the CD4+CD45RO+CD62L−/CD4+CD45RO+CD62L+ ratio was significantly lower after LCAP therapy. However, the CD4+CD25bright/total CD4+ ratio did not change. Conclusions The present findings revealed the real target of proinflammatory CD14dullCD16+ monocytes removed during LCAP treatment of UC and that LCAP might be used as an extracorporeal anti‐TNF‐&agr; therapy, expanding the clinical applications of this procedure to include the treatment of Crohns disease. (Inflamm Bowel Dis 2006)