Mucosal T lymphocyte numbers are selectively reduced in integrin alpha E (CD103)-deficient mice.

The mucosal lymphocyte integrin alpha E(CD103)beta 7 is thought to be important for intraepithelial lymphocyte (IEL) localization or function. We cloned the murine integrin gene encoding alpha E, localized it to chromosome 11, and generated integrin alpha E-deficient mice. In alpha E-/- mice, intestinal and vaginal IEL numbers were reduced, consistent with the known binding of alpha E beta 7 to E-cadherin expressed on epithelial cells. However, it was surprising that lamina propria T lymphocyte numbers were diminished, as E-cadherin is not expressed in the lamina propria. In contrast, peribronchial, intrapulmonary, Peyer's patch, and splenic T lymphocyte numbers were not reduced in alpha E-deficient mice. Thus, alpha E beta 7 was important for generating or maintaining the gut and vaginal T lymphocytes located diffusely within the epithelium or lamina propria but not for generating the gut-associated organized lymphoid tissues. Finally, the impact of alpha E deficiency upon intestinal IEL numbers was greater at 3-4 wk of life than in younger animals, and affected the TCR alpha beta+ CD8+ T cells more than the gamma delta T cells or the TCR alpha beta+ CD4+CD8- population. These findings suggest that alpha E beta 7 is involved in the expansion/recruitment of TCR alpha beta+ CD8+ IEL following microbial colonization. Integrin alpha E-deficient mice will provide an important tool for studying the role of alpha E beta 7 and of alpha E beta 7-expressing mucosal T lymphocytes in vivo.     

Striking phenotypic and functional differences in lamina propria lymphocytes from the large and small intestine of mice

Although intraepithelial T lymphocytes of the large intestine (LI) are known to differ from those of the small intestine (SI) in phenotype and function, differences in LI and SI lamina propria (LP) lymphocyte populations have not been clearly established. In this work we found striking phenotypic differences between SI and LI LP lymphocyte populations from Balb/c mice analyzed by flow cytometry. In the LI most lymphocytes were B cells and the predominant T cells were TCR-alpha beta+, CD8+. In contrast, in the SI most T lymphocytes were CD4+ expressing TCR-alpha beta+, although a higher proportion expressed TCR-gamma delta+ than in the LI. In T cells the expression of adhesion molecules and cytokines was also different between SI and LI. The proportion of LP T cells expressing alpha4beta7 and L-selectin was higher in the LI than in the SI; whereas a greater proportion of cells expressing alpha(E)beta7 were detected in the SI than in LI. Higher proportions of T cells expressing L-selectin and alpha4beta1 were detected in the intraepithelial compartment of the LI than that of the SI, whereas the number of T cells expressing alpha(E)beta7 was much higher in the SI than in the LI. The proportion of T cells spontaneously producing IL-2, IFN gamma, and IL-4 at the intraepithelial and lamina propria, in the small and large intestine, was different indicating that distinctive functional features exist in the lymphocyte populations residing at the different intestinal compartments.     

Colitis-inducing potency of CD4+ T cells in immunodeficient, adoptive hosts depends on their state of activation, IL-12 responsiveness, and CD45RB surface phenotype

We studied the induction, severity and rate of progression of inflammatory bowel disease (IBD) induced in SCID mice by the adoptive transfer of low numbers of the following purified BALB/c CD4+ T cell subsets: 1) unfractionated, peripheral, small (resting), or large (activated) CD4+ T cells; 2) fractionated, peripheral, small, or large, CD45RBhigh or CD45RBlow CD4+ T cells; and 3) peripheral IL-12-unresponsive CD4+ T cells from STAT-4-deficient mice. The adoptive transfer into SCID host of comparable numbers of CD4+ T cells was used to assess the colitis-inducing potency of these subsets. Small CD45RBhigh CD4+ T lymphocytes and activated CD4+ T blasts induced early (6-12 wk posttransfer) and severe disease, while small resting and unfractionated CD4+ T cells or CD45RBlow T lymphocytes induced a late-onset disease 12-16 wk posttransfer. SCID mice transplanted with STAT-4-/- CD4+ T cells showed a late-onset IBD manifest > 20 wk posttransfer. In SCID mice with IBD transplanted with IL-12-responsive CD4+ T cells, the colonic lamina propria CD4+ T cells showed a mucosa-seeking memory/effector CD45RBlow Th1 phenotype abundantly producing IFN-gamma and TNF-alpha. In SCID mice transplanted with IL-12-unresponsive STAT-4-/- CD4+ T cells, the colonic lamina propria, mesenteric lymph node, and splenic CD4+ T cells produced very little IFN-gamma but abundant levels of TNF-alpha. The histopathologic appearance of colitis in all transplanted SCID mice was similar. These data indicate that CD45RBhigh and CD45RBlow, IL-12-responsive and IL-12-unresponsive CD4+ T lymphocytes and lymphoblasts have IBD-inducing potential though of varying potency.     

MHC-II-independent CD4+ T cells induce colitis in immunodeficient RAG-/- hosts

CD4(+) alpha beta T cells from either normal C57BL/6 (B6) or MHC-II-deficient (A alpha(-/-) or A beta(-/-)) B6 donor mice engrafted into congenic immunodeficient RAG1(-/-) B6 hosts induced an aggressive inflammatory bowel disease (IBD). Furthermore, CD4(+) T cells from CD1d(-/-) knockout (KO) B6 donor mice but not those from MHC-I(-/-) (homozygous transgenic mice deficient for beta(2)-microglobulin) KO B6 mice induced a colitis in RAG(-/-) hosts. Abundant numbers of in vivo activated (CD69(high)CD44(high)CD28(high)) NK1(+) and NK1(-) CD4(+) T cells were isolated from the inflamed colonic lamina propria (cLP) of transplanted mice with IBD that produced large amounts of TNF-alpha and IFN-gamma but low amounts of IL-4 and IL-10. IBD-associated cLP Th1 CD4(+) T cell populations were polyclonal and MHC-II-restricted when derived from normal B6 donor mice, but oligoclonal and apparently MHC-I-restricted when derived from MHC-II-deficient (A alpha(-/-) or A beta(-/-)) B6 donor mice. cLP CD4(+) T cell populations from homozygous transgenic mice deficient for beta(2)-microglobulin KO B6 donor mice engrafted into RAG(-/-) hosts were Th2 and MHC-II restricted. These data indicate that MHC-II-dependent as well as MHC-II-independent CD4(+) T cells can induce a severe and lethal IBD in congenic, immunodeficient hosts, but that the former need the latter to express its IBD-inducing potential.     

Cytokine production by mature and immature CD4-CD8- T cells. Alpha beta-T cell receptor+ CD4-CD8- T cells produce IL-4.

This study follows our previous investigation describing the production of four cytokines (IL-2, IL-4, IFN-gamma, and TNF-alpha) by subsets of thymocytes defined by the expression of CD3, 4, 8, and 25. Here we investigate in greater detail subpopulations of CD4-CD8- double negative (DN) thymocytes. First we divided immature CD25-CD4-CD8-CD3- (CD25- triple negative) (TN) thymocytes into CD44+ and CD44- subsets. The CD44+ population includes very immature precursor T cells and produced high titers of IL-2, TNF-alpha, and IFN-gamma upon activation with calcium ionophore and phorbol ester. In contrast, the CD44- subset of CD25- TN thymocytes did not produce any of the cytokines studied under similar activation conditions. This observation indicates that the latter subset, which differentiates spontaneously in vitro into CD4+CD8+, already resembles CD4+CD8+ thymocytes (which do not produce any of the tested cytokines). We also subdivided the more mature CD3+ DN thymocytes into TCR-alpha beta- and TCR-gamma delta-bearing subsets. These cells produced cytokines upon activation with solid phase anti-CD3 mAb. gamma delta TCR+ DN thymocytes produced IL-2, IFN-gamma and TNF-alpha, whereas alpha beta TCR+ DN thymocytes produced IL-4, IFN-gamma, and TNF-alpha but not IL-2. We then studied alpha beta TCR+ DN T cells isolated from the spleen and found a similar cytokine production profile. Furthermore, splenic alpha beta TCR+ DN cells showed a TCR V beta gene expression profile reminiscent of alpha beta TCR+ DN thymocytes (predominant use of V beta 8.2). These observations suggest that at least some alpha beta TCR+ DN splenocytes are derived from alpha beta TCR+ DN thymocytes and also raises the possibility that these cells may play a role in the development of Th2 responses through their production of IL-4.     

The differentiated state of intestinal lamina propria CD4+ T cells results in altered cytokine production, activation threshold, and costimulatory requirements

Intestinal lamina propria (LP) CD4+ T cells are memory-like effector cells that proliferate at relatively low levels and require high levels of TCR signaling and costimulation for full activation in vitro. To study LP CD4+ T cell functional potential we used DO11.10 TCR transgenic (Tg) mice specific for the class II MHC-restricted OVA323-339 peptide and nontransgenic BALB/c mice. Activation of LP Tg+ T cells with Ag using mucosal explants induced high levels of IL-2, IL-4, and IFN-gamma. Culturing isolated LP cells with IL-12 enhanced IFN-gamma production and down-regulated IL-4 and IL-2, whereas addition of IL-4 maintained IL-4 production without inhibiting IFN-gamma production. Systemic administration of relatively high dose (HD; 100 nM) OVA323-339 peptide induced similar levels of bromodeoxyuridine (BrdU) incorporation by LP and splenic Tg+ T cells in vivo, whereas low dose (LD; 4.5 nM) peptide injections induced 4-fold greater levels of BrdU incorporation for LP compared with splenic Tg+ T cells. Coadministration of CTLA-4Ig reduced BrdU incorporation for splenic cells by 70% with HD and LD stimulation, but had little effect on LP responses to HD stimulation. Results of in vivo studies were confirmed in nontransgenic BALB/c mice using HD (200 microg) and LD (10 microg) anti-CD3 mAb+/- CTLA-4Ig. These results suggest that LP T cells are differentiated effector cells that respond at high levels when activated with relatively low levels of Ag- and B7-mediated costimulation in vivo. The reduced activation threshold of LP T cells may facilitate responses to low levels of Ag derived from mucosal pathogens.     

Systemic, but not intestinal, IL-7 is essential for the persistence of chronic colitis

We previously demonstrated that IL-7 is produced by intestinal goblet cells and is essential for the persistence of colitis. It is well known, however, that goblet cells are decreased or depleted in the chronically inflamed mucosa of animal colitis models or human inflammatory bowel diseases. Thus, in this study, we assess whether intestinal IL-7 is surely required for the persistence of colitis using a RAG-1/2-/- colitis model induced by the adoptive transfer of CD4+CD45RBhigh T cells in combination with parabiosis system. Surprisingly, both IL-7-/-xRAG-1-/- and IL-7+/+xRAG-1-/- host mice developed colitis 4 wk after parabiosis to a similar extent of colitic IL-7+/+xRAG-1-/- donor mice that were previously transferred with CD4+CD45RBhigh T cells. Of note, although the number of CD4+ T cells recovered from the spleen or the bone marrow of IL-7-/-xRAG-1-/- host mice was significantly decreased compared with that of IL-7+/+xRAG-1-/- host mice, an equivalent number of CD4+ T cells was recovered from the lamina propria of both mice, indicating that the expansion of CD4+ T cells in the spleen or in the bone marrow is dependent on IL-7, but not in the lamina propria. Development of colitis was never observed in parabionts between IL-7+/+xRAG-1-/- host and noncolitic IL-7-/-xRAG-1-/- donor mice that were transferred with CD4+CD45RBhigh T cells. Collectively, systemic, but not intestinal, IL-7 is essential for the persistence of colitis, suggesting that therapeutic approaches targeting the systemic IL-7/IL-7R signaling pathway may be feasible in the treatment of inflammatory bowel diseases.     

gamma delta T cells in the human intestine express surface markers of activation and are preferentially located in the epithelium

We have investigated the expression of the alpha beta and the gamma delta T cell receptor (TCR) in the human intestine. By immunohistology we found that 39% of CD3+ intraepithelial lymphocytes (IEL) expressed the gamma delta TCR compared to 3% of CD3+ lamina propria lymphocytes (LPL). Cytofluorometric analysis of isolated cells revealed a significantly higher proportion of gamma delta T cells among CD3+ IEL compared to LPL and peripheral blood lymphocytes. This was due to an increase in both CD8+ (low density) and CD4-CD8- gamma delta T cells in IEL. Most alpha beta IEL expressed high-density CD8. About 30% of both IEL and LPL expressed CD25 (alpha-chain of the IL-2 receptor). HML-1 expression was detected on nearly all IEL and on 27% of LPL. CD25 and HML-1 were preferentially expressed on intestinal alpha beta and gamma delta T cells, respectively. Thus, human gamma delta T cells are located preferentially in the gut epithelium and are phenotypically different from alpha beta T cells, which constitute the majority of both LPL and IEL.     

Specific overexpression of IL-7 in the intestinal mucosa: the role in intestinal intraepithelial lymphocyte development

IL-7 plays a crucial role in controlling T cell development and homeostasis. Since IL-7 may be derived from extraintestinal sources, and exogenous IL-7 broadly affects lymphoid populations, the actions of epithelial cell (EC)-derived IL-7 are not fully understood. The effect of intestinal specific expression of IL-7 on intestinal mucosal lymphocytes was investigated by using an IL-7 transgenic mouse model. We generated an intestinal EC-specific overexpressing IL-7 transgenic mouse model (IL-7(vill)) and compared their phenotype and function to wild-type C57BL/6J mice. EC-derived IL-7 overexpression was found to be exclusively in the small and large intestine. Numbers and subtypes of mucosal lymphocytes, including intraepithelial lymphocytes (IEL) and lamina propria lymphocytes (LPL), significantly changed in IL-7(vill) mice. From a functional standpoint, IEL proliferation also significantly increased in IL-7(vill) mice. IEL cytokine expression significantly changed in both T cell receptor (TCR)-alphabeta(+) and TCR-gammadelta(+) IEL subpopulations, including a significant increase in IFN-gamma and TNF-alpha as well as an increase in keratinocyte growth factor expression. EC expression of CD103 (integrin alpha(E)beta(7)), the ligand of E-cadherin, markedly upregulated and may account for a mechanism of the massive expansion of IEL in transgenic mice. Systemic lymphoid populations did not change in transgenic mice. IL-7 overexpression by intestinal EC significantly affected IEL phenotype and function. These results offer insight into the role of IL-7 in IEL development and suggest a critical role of EC-derived expression of IL-7 in the phenotype and function of IEL.     

Induction of CD8+ regulatory T cells in the intestine by Heligmosomoides polygyrus infection

This study determined whether Heligmosomoides polygyrus induces intestinal regulatory T cells. Splenic T cells proliferate strongly when cultured with anti-CD3 and antigen-presenting cells (APC). Lamina propria T cells from mice with H. polygyrus mixed with normal splenic T cells from uninfected mice inhibited proliferation over 90%. Lamina propria T cells from mice without H. polygyrus only modestly affected T cell proliferation. The worm-induced regulatory T cell was CD8+ and required splenic T cell contact to inhibit proliferation. The regulation also was IL-10 independent, but TAP-dependent, suggesting that it requires major histocompatibility complex (MHC) class I interaction. Additional studies employed mice with transgenic T cells that did not express functional TGF-beta receptors. The lamina propria T regulator inhibited proliferation of these transgenic T cells nearly 100%, suggesting that TGF-beta signaling via the T cell was not required. CD8+ T cells were needed for worms to reverse piroxicam-induced colitis in Rag mice (T and B cell deficient) reconstituted with IL-10-/- T cells. Thus H. polygyrus induces a regulatory CD8+ lamina propria T cell that inhibits T cell proliferation and that appears to have a role in control of colitis.     

IL-7 exacerbates chronic colitis with expansion of memory IL-7Rhigh CD4+ mucosal T cells in mice

We have previously demonstrated that mucosal CD4(+) T cells expressing high levels of IL-7 receptor (IL-7R(high)) are pathogenic cells responsible for chronic colitis. Here we investigate whether IL-7 is directly involved in the expansion of IL-7R(high) memory CD4(+) mucosal T cells and the exacerbation of colitis. We first showed that CD4(+) lamina propria lymphocytes (LPLs) from wild-type, T cell receptor-alpha-deficient (TCR-alpha(-/-)), and recombinase-activating gene (RAG)-2(-/-)-transferred mice with or without colitis showed phenotypes of memory cells, but only CD4(+) LPLs from colitic mice showed IL-7R(high). In vitro stimulation by IL-7, but not by IL-15 and thymic stromal lymphopoietin, enhanced significant proliferative responses and survival of colitic CD4(+), but not normal CD4(+) LPLs. Importantly, in vivo administration of IL-7 mice accelerated the expansion of IL-7R(high) memory CD4(+) LPLs and thereby exacerbated chronic colitis in RAG-2(-/-) mice transferred with CD4(+) LPLs from colitic TCR-alpha(-/-) mice. Conversely, the administration of anti-IL-7R monoclonal antibody significantly inhibited the development of TCR-alpha(-/-) colitis with decreased expansion of CD4(+) LPLs. Collectively, the present data indicate that IL-7 is essential for the expansion of pathogenic memory CD4(+) T cells under pathological conditions. Therefore, therapeutic approaches targeting the IL-7R pathway may be feasible in the treatment of human inflammatory bowel disease.     

Essential involvement of IFN-gamma in Clostridium difficile toxin A-induced enteritis

Clostridium difficile has emerged as the important causative agent of antibiotics-associated pseudomembranous colitis; especially its toxin A is presumed to be responsible for the colitis. We examined the pathophysiological roles of IFN-gamma in toxin A-induced enteritis using IFN-gamma knockout (KO) mice. When toxin A of C. difficile was injected into the ileal loops of BALB/c wild-type (WT) mice, massive fluid secretion, disruption of intestinal epithelial structure, and massive neutrophil infiltration developed within 4 h after the injection. IFN-gamma protein was faintly detected in some CD3-positive lymphocytes in the lamina propria and submucosa of the ileum of untreated WT mice. On the contrary, at 2 and 4 h after toxin A injection, IFN-gamma protein was detected in infiltrating neutrophils and to a lesser degree in CD3-positive lymphocytes. In the ileum of WT mice, toxin A treatment markedly enhanced the gene expression of TNF-alpha, macrophage inflammatory protein-1alpha and -2, KC, and ICAM-1 >2 h after treatment. In contrast, the histopathological changes were marginal, without enhanced fluid secretion in the ileum of toxin A-treated IFN-gamma KO mice. Moreover, toxin A-induced gene expression of TNF-alpha, neutrophil chemotactic chemokines, and ICMA-1 was remarkably attenuated in IFN-gamma KO mice. Furthermore, pretreatment of WT mice with a neutralizing anti-IFN-gamma Ab prevented toxin A-induced enteritis. These observations indicate that IFN-gamma is the crucial mediator of toxin A-induced acute enteritis and suggest that IFN-gamma is an important molecular target for the control of C. difficile-associated pseudomembranous colitis.     

Novel function for intestinal intraepithelial lymphocytes. Murine CD3+, gamma/delta TCR+ T cells produce IFN-gamma and IL-5.

Intestinal intraepithelial lymphocytes (IEL) from mice are greater than 80% CD3+ T cells and could be separated into four subsets according to expression of CD4 and CD8. In our studies designed to assess the functions of IEL, namely, cytokine production, it was important to initially characterize the various subsets of T cells that reside in IEL. The major subset was CD4-, CD8+ (75% of CD3+ T cells), which contained approximately 45 to 65% gamma/delta TCR+ and 35 to 45% alpha/beta TCR+ T cells. Approximately 7.5% of IEL T cells were CD4-, CD8- (double negative) and gamma/delta+ population. On the other hand, CD4+, CD8+ (double positive) and CD4+, CD8- fractions represented 10% and 7.5% of CD3+ T cells, respectively, which were all alpha/beta TCR+. Inasmuch as CD3+, CD4-, CD8+ T cells are a major subset of IEL which contain both gamma/delta TCR or alpha/beta TCR-bearing cells, the present study was focused on the capability of this subset of IEL T cells to produce the cytokines IFN-gamma and IL-5. Both gamma/delta TCR+ and alpha/beta TCR+ IEL spontaneously produced IFN-gamma and IL-5, although higher frequencies of cytokine spot-forming cells were associated with the alpha/beta TCR+ subset. Approximately 30% of CD8+, gamma/delta TCR+ cells produced both cytokines, whereas approximately 90% of alpha/beta TCR+ T cells produced either IFN-gamma or IL-5. Both gamma/delta TCR+ and alpha/beta TCR+ IEL possessed large quantities of cytokine-specific mRNA, clearly showing that these IEL were programmed for cytokine production. When IEL were activated with anti-gamma/delta or anti-CD8 antibodies, higher numbers of IFN-gamma and IL-5 spot-forming cells were noted. The present study has provided direct evidence that a major function of IEL involves cytokine production, and this is the first evidence that gamma/delta TCR+ cells in IEL possess the capability of producing both IL-5 and IFN-gamma.     

Analysis of the functional capabilities of CD3+CD4-CD8- and CD3+CD4+CD8+ human T cell clones

The functional capabilities of human peripheral blood CD3+CD4-CD8- and CD3+CD4+CD8+ T cell clones were examined. The clones were generated by culturing purified populations of CD3+CD4-CD8- and CD3+CD4+CD8+ T cells at limiting dilution (0.3 cell/well) in the presence of PHA, rIL-2, and irradiated PBMC as feeders. Twelve CD3+CD4-CD8- and 5 CD3+CD4+CD8+ clones were generated. Clonality was documented by analyzing TCR gamma- and beta-chain rearrangement patterns. All CD3+CD4-CD8- clones were stained by the TCR-delta 1 mAb that identifies a framework epitope of the TCR delta-chain, but not by mAb WT31 that identifies the TCR-alpha beta on mature T cells. In contrast, the CD3+CD4+CD8+ clones were all stained by WT31 and not by TCR-delta 1. All 17 clones were screened for various functional activities. Each secreted IL-2, IFN-gamma, and lymphotoxin/TNF-like factors when stimulated with immobilized mAb to CD3 (64.1), albeit in varying quantities. These clones secreted far less IL-2 and IFN-gamma than CD3+CD4+CD8- or CD3+CD4-CD8+ alpha beta expressing clones, but comparable amounts of lymphotoxin/TNF. All clones also functioned as MHC-unrestricted cytotoxic cells. This activity was comparable to that mediated by the CD3+CD4+CD8- or CD3+CD4-CD8+ alpha beta clones. Nine of 12 CD3+CD4-CD8- and 4 of 5 CD3+CD4+CD8+ clones were able to support B cell differentiation when activated by immobilized anti-CD3, but usually not as effectively as the CD3+CD4+CD8- or CD3+CD4-CD8+ alpha beta clones. The differences in the functional capabilities of the various clones could not be accounted for by alterations in the signaling capacity of the CD3 molecular complex as mAb to CD3 induced comparable increases in intracellular free calcium in each clone examined. When clones were stimulated with PWM, each suppressed B cell differentiation supported by mitomycin C-treated fresh CD4+ T lymphocytes. Suppression was dependent on the number of clone cells added to culture, but could be observed with as few as 12,500 cells per microtiter well. Phenotypic analysis of the clones revealed that all expressed CD29, CD11b, and the NKH1 surface Ag. These results demonstrate that the CD3+CD4-CD8- and CD3+CD4+CD8+ T cell clones exhibit many of the functional characteristics of mature T cells, although they produce IL-2 and IFN-gamma and provide help for B cell differentiation less effectively than CD3+CD4+CD8- and CD3+CD4-CD8+ alpha beta T cell clones.     

L-selectin, alpha 4 beta 1, and alpha 4 beta 7 integrins participate in CD4+ T cell recruitment to chronically inflamed small intestine

CD4+ T cells are essential for development and perpetuation of Crohn's disease, a chronic immune-mediated condition that affects primarily the small intestine. Using novel models of Crohn's disease-like ileitis (i.e., SAMP1/YitFc and CD4+ T cell transfer models), we have begun to understand the adhesive pathways that mediate lymphocyte trafficking to the chronically inflamed small bowel. Expansion of the CD4/beta7+ population and increased mucosal addressin cell adhesion molecule-1 (MAdCAM-1) expression were observed within the intestinal lamina propria with disease progression. However, Ab blockade of the beta7 integrin, the alpha4beta7 heterodimer, MAdCAM-1, or L-selectin did not attenuate inflammation. Blockade of two pathways (L-selectin and MAdCAM-1 or alpha4 integrins) was required to improve ileitis. Further analyses showed that 55 +/- 7% of the mesenteric lymph node alpha4beta7+CD4 expressed L-selectin. These L-selectin+ T cells were the main producers of TNF-alpha and the predominant ileitis-inducing subpopulation. Mechanistically, combined blockade of L-selectin and MAdCAM-1 depleted the intestinal lamina propria of CD4+ T cells that aberrantly coexpressed alpha4beta7 and alpha4beta1 integrins, markedly decreasing local production of TNF-alpha and IFN-gamma. Thus, pathogenic CD4+ T cells not only use the physiologic alpha4beta7/MAdCAM-1 pathway, but alternatively engage alpha4beta1 and L-selectin to recirculate to the chronically inflamed small intestine.     

Infection with a helminth parasite prevents experimental colitis via a macrophage-mediated mechanism

The propensity of a range of parasitic helminths to stimulate a Th2 or regulatory cell-biased response has been proposed to reduce the severity of experimental inflammatory bowel disease. We examined whether infection with Schistosoma mansoni, a trematode parasite, altered the susceptibility of mice to colitis induced by dextran sodium sulfate (DSS). Mice infected with schistosome worms were refractory to DSS-induced colitis. Egg-laying schistosome infections or injection of eggs did not render mice resistant to colitis induced by DSS. Schistosome worm infections prevent colitis by a novel mechanism dependent on macrophages, and not by simple modulation of Th2 responses, or via induction of regulatory CD4+ or CD25+ cells, IL-10, or TGF-beta. Infected mice had marked infiltration of macrophages (F4/80+CD11b+CD11c(-)) into the colon lamina propria and protection from DSS-induced colitis was shown to be macrophage dependent. Resistance from colitis was not due to alternatively activated macrophages. Transfer of colon lamina propria F4/80+ macrophages isolated from worm-infected mice induced significant protection from colitis in recipient mice treated with DSS. Therefore, we propose a new mechanism whereby a parasitic worm suppresses DSS-induced colitis via a novel colon-infiltrating macrophage population.     

Mice with a selective deletion of the CC chemokine receptors 5 or 2 are protected from dextran sodium sulfate-mediated colitis: lack of CC chemokine receptor 5 expression results in a NK1.1+ lymphocyte-associated Th2-type immune response in the intestine

The chemokine receptors CCR2 and CCR5 and their respective ligands regulate leukocyte chemotaxis and activation. To determine the role of these chemokine receptors in the regulation of the intestinal immune response, we induced colitis in CCR2- and CCR5-deficient mice by continuous oral administration of dextran sodium sulfate (DSS). Both CCR2- and CCR5-deficient mice were susceptible to DSS-induced intestinal inflammation. The lack of CCR2 or CCR5 did not reduce the DSS-induced migration of macrophages into the colonic lamina propria. However, both CCR5-deficient mice and, to a lesser degree, CCR2-deficient mice were protected from DSS-induced intestinal adhesions and mucosal ulcerations. CCR5-deficient mice were characterized by a greater relative infiltration of CD4+ and NK1.1+ lymphocyte in the colonic lamina propria when compared to wild-type and CCR2-deficient mice. In CCR5-deficient mice, mucosal mRNA expression of IL-4, IL-5, and IL-10 was increased, whereas that of IFN-gamma was decreased, corresponding to a Th2 pattern of T cell activation. In CCR2-deficient mice, the infiltration of Th2-type T cells in the lamina propria was absent, but increased levels of IL-10 and decreased levels of IFN-gamma may have down regulated mucosal inflammation. Our data indicate that CCR5 may be critical for the promotion of intestinal Th1-type immune responses in mice.     

Bacterial-reactive T regulatory cells inhibit pathogenic immune responses to the enteric flora

We showed previously that cecal bacterial Ag (CBA)-specific CD4(+) T cells induce colitis when transferred into SCID mice. The purpose of this study was to generate and characterize CBA-specific regulatory T cells in C3H/HeJBir (Bir) mice. CD4(+) T cells were stimulated with CBA-pulsed APC in the presence of IL-10 every 10-14 days. After four or more cycles, these T cells produced high levels of IL-10, low levels of IL-4 and IFN-gamma, and no IL-2, consistent with the phenotype of T regulatory-1 (Tr1) cells. Bir Tr1 cells proliferated poorly, but their proliferation was dependent on CD28-B7 interactions and was MHC class II-restricted. Transfer of Bir Tr1 cells into SCID mice did not result in colitis, and cotransfer of Bir Tr1 T cells with pathogenic Bir CD4(+) Th1 cells prevented colitis. Bir Tr1 cells inhibited proliferation and IFN-gamma production of a CBA-specific Th1 cell line in vitro. Such inhibition was partly due to IL-10 and TGFbeta1, but cognate interactions with either APCs or Th1 cells were also involved. Normal intestinal lamina propria CD4(+) T cells had Tr1-like activity when stimulated with CBA-pulsed APCs. We conclude that CD4(+) T cells with the properties of Tr1 cells are present in the intestinal lamina propria and hypothesize that these cells maintain intestinal immune homeostasis to the enteric flora.     

Treatment of T cell-dependent experimental colitis in SCID mice by local administration of an adenovirus expressing IL-18 antisense mRNA.

Recent studies have shown that IL-18, a pleiotropic cytokine that augments IFN-gamma production, is produced by intestinal epithelial cells and lamina propria cells from patients with Crohn's disease. In this study, we show that IL-18 is strongly expressed by intestinal epithelial cells in a murine model of Crohn's disease induced by transfer of CD62L+ CD4+ T cells into SCID mice. To specifically down-regulate IL-18 expression in this model, we constructed an E1/E3-deleted adenovirus expressing IL-18 antisense mRNA, denoted Ad-asIL-18, and demonstrated the capacity of such a vector to down-regulate IL-18 expression in colon-derived DLD-1 cells and RAW264.7 macrophages. Local administration of the Ad-asIL-18 vector to SCID mice with established colitis led to transduction of epithelial cells and caused a significant suppression of colitis activity, as assessed by a newly developed endoscopic analysis system for colitis. Furthermore, treatment with Ad-asIL-18 induced a significant suppression of histologic colitis activity and caused suppression of mucosal IFN-gamma production, whereas IFN-gamma production by spleen T cells was unaffected. Taken together, these data indicate an important role for IL-18 in the effector phase of a T cell-dependent murine model of colitis and suggest that strategies targeting IL-18 expression may be used for the treatment of patients with Crohn's disease.     

Commensal gut flora drives the expansion of proinflammatory CD4 T cells in the colonic lamina propria under normal and inflammatory conditions

We tested in B6 mice whether the local expansion of CD4 T cells producing proinflammatory cytokines including IL-17 (Th17 cells) in the colonic lamina propria (cLP) depends on the commensal microflora. High numbers of CD4 Th17 cells were found in the lamina propria of the ileum and colon but not the duodenum, jejunum, mesenteric lymph nodes, spleen, or liver of specific pathogen-free (SPF) mice. The microflora is required for the accumulation of cytokine (IL-17, IFN-gamma, TNF-alpha, IL-10)-producing CD4 T cells in the cLP because only low numbers of cytokine-producing cLP CD4 T cells were found in syngeneic (age- and sex-matched) germfree mice. The fraction of cLP Th17 cells was higher in (type I and type II) IFN- but not IL-4- or IL-12p40-deficient SPF congenics. cLP CD4 Th17 cells produce IL-17 but not IFN-gamma, TNF-alpha, IL-4, or IL-10. cLP CD4 Th17 cells accumulate locally in colitis induced by adoptive transfer of IFN-gamma+/+ or IFN-gamma-/- CD4 T cells into congenic SPF (but not germfree) RAG-/- hosts. In this colitis model, cLP CD4 T cells that "spontaneously" produce IL-17 progressively increase in number in the inflamed cLP, and increasing serum IL-17 levels appear as the disease progresses. Commensal bacteria-driven, local expansion of cLP CD4 Th17 cells may contribute to the pathogenesis of this inflammatory bowel disease.