Development of chronic colitis is dependent on the cytokine MIF.

The cytokine macrophage-migration inhibitory factor (MIF) is secreted by a number of cell types upon induction by lipopolysaccharide (LPS). Because colitis is dependent on interplay between the mucosal immune system and intestinal bacteria, we investigated the role of MIF in experimental colitis. MIF-deficient mice failed to develop disease, but reconstitution of MIF-deficient mice with wild-type innate immune cells restored colitis. In addition, established colitis could be treated with anti-MIF immunoglobulins. Thus, murine colitis is dependent on continuous MIF production by the innate immune system. Because we found increased plasma MIF concentrations in patients with Crohn's disease, these data suggested that MIF is a new target for intervention in Crohn's disease.

Continuous stimulation by normal luminal bacteria is essential for the development and perpetuation of colitis in Tg(epsilon26) mice.

BACKGROUND & AIMS: Normal resident bacteria are required for development of colitis in several rodent models. We determined whether bacterial stimulation is necessary for both induction and perpetuation of mucosal inflammation and T-cell activation in Tg(epsilon26) mice, in which transplantation of wild-type bone marrow (BM-->Tg(epsilon26)) causes colitis under specific pathogen-free (SPF) conditions. METHODS: BM from (C57BL/6 X CBA/J) F1 mice was transplanted into germfree (GF) or SPF Tg(epsilon26) mice. Mesenteric lymph node (MLN) cells from these mice were then transferred into SPF or GF recipients. Colitis and activation of MLN cells were measured by histologic scores, membrane marker analysis, and intracellular cytokine staining. Cytokine secretion by MLN cells stimulated by anti-CD3 or by luminal or epithelial antigens was measured by ELISA. RESULTS: Colitis did not develop when BM was transferred into GF recipient mice (BM-->GF Tg(epsilon26)). T lymphocytes that secreted interferon gamma upon activation were present in the MLN of BM-->GF Tg(epsilon26) mice, albeit in lower frequency than in control BM-->SPF Tg(epsilon26) mice. Furthermore, transfer of MLN cells from BM-->SPF Tg(epsilon26) mice into SPF Tg(epsilon26) recipients induced active colitis, but not if the same cells were transferred into GF Tg(epsilon26) recipients. Although CD4 T cells were detected in the colonic mucosa of GF recipients, no inflammation was observed for at least 31 weeks. In a reciprocal experiment, MLN cells from BM-->GF Tg(epsilon26) mice without colitis transferred disease to SPF Tg(epsilon26) recipients within 2-4 weeks. CONCLUSIONS: Activated T cells are present in the mucosa of BM-->GF Tg(epsilon26) mice but are incapable of inducing disease unless colonic bacteria are present. Moreover, pathogenic T cells require the continuous presence of colonic bacteria to sustain colitis.

Chronic murine colitis is dependent on the CD154/CD40 pathway and can be attenuated by anti-CD154 administration.

BACKGROUND & AIMS: Experimental colitis in most animal models is caused by dysregulation of T lymphocytes that display a T helper 1 (Th1) phenotype. CD154 (CD40L/gp39), a member of the tumor necrosis factor (TNF) family, is up-regulated on T cells on activation and has been shown to play a key role in the induction of a Th1 response. We investigated whether chronic experimental colitis is dependent on the CD154/CD40 pathway and whether disease can be prevented by anti-CD154 antibody treatment. METHODS: Two models of chronic colitis were used: CD45Rb(hi) cell transfer into recombination activation gene-deficient (Rag(-/-)) mice and bone marrow transplant of tgepsilon26 animals. In both models, mice were reconstituted with cells from CD154-deficient animals. In another series of experiments, wild-type CD45Rb(hi) T cell-reconstituted recipients were treated with anti-CD154, either from the start of the experiment or after onset of disease. RESULTS: T cells deficient in CD154 induced a milder clinical disease, less weight loss, and fewer histologic signs of colitis than wild-type cells. The level of interleukin 12 in the serum of CD154-deficient T-cell recipients was 5-fold less than that of wild-type cell recipients. Nevertheless, no signs of deviation from a Th1 phenotype were observed. Treatment with anti-CD154 antibodies substantially impaired disease development, even when started after the onset of colitis. CONCLUSIONS: The CD154/CD40 pathway plays a critical role in Th1-induced chronic experimental colitis. Blocking CD154, even after the onset of disease, ameliorates colitis but does not induce a T helper 2 (Th2) phenotype.

Activation of natural killer T cells by alpha-galactosylceramide in the presence of CD1d provides protection against colitis in mice.

BACKGROUND & AIMS: CD1d is a major histocompatibility complex class I-like molecule that presents glycolipid antigens to a subset of natural killer (NK)1.1(+) T cells. These NK T cells exhibit important immunoregulatory functions in several autoimmune disease models. METHODS: To investigate whether CD1d and NK T cells have a similar role in intestinal inflammation, the effects of the glycolipid, alpha-galactosylceramide (alpha-GalCer), on dextran sodium sulfate (DSS)-induced colitis were examined. Wild-type (WT), CD1d(-/-), and RAG(-/-) mice were examined for their response to either alpha-GalCer or the control analogue, alpha-mannosylceramide (alpha-ManCer). RESULTS: WT mice, but not CD1d(-/-) and RAG(-/-) mice, receiving alpha-GalCer had a significant improvement in DSS-induced colitis based on body weight, bleeding, diarrhea, and survival when compared with those receiving alpha-ManCer. Elimination of NK T cells through antibody-mediated depletion resulted in a reduction of the effect of alpha-GalCer. Furthermore, adoptive transfer of NK T cells preactivated by alpha-GalCer, but not alpha-ManCer, resulted in diminished colitis. Using a fluorescent-labeled analogue of alpha-GalCer, confocal microscopy localized alpha-GalCer to the colonic surface epithelium of WT but not CD1d(-/-) mice, indicating alpha-GalCer binds CD1d in the intestinal epithelium and may be functionally active at this site. CONCLUSIONS: These results show an important functional role for NK T cells, activated by alpha-GalCer in a CD1d-restricted manner, in regulating intestinal inflammation.

High- and low-affinity single-peptide/MHC ligands have distinct effects on the development of mucosal CD8alphaalpha and CD8alphabeta T lymphocytes.

In this study, we compared the influence of two peptides on the selection of CD8alphaalpha and CD8alphabeta intraepithelial lymphocytes (IELs) of the intestine, which develop by a unique and partially thymus-independent process. Mice were used in which all T cells carried one transgenic T cell antigen receptor (TCR) (F5), and in which only well defined transgenic peptides were presented by H-2Db. The first peptide, for which the F5 TCR has a high affinity, derives from the influenza virus nucleoprotein (NP68). The second peptide, NP34, is an antagonistic variant of NP68 and is recognized by the F5 TCR with low affinity. To avoid presentation of endogenous peptides or production of T cells carrying alternative TCRs, F5 TCR transgenic mice were generated that were deficient for Tap-1 and Rag-1. In these mice, no CD3(+)CD8(+) cells were found in lymph nodes, spleen, or intestine. Introduction of transgenes encoding either NP34 or NP68 along with an endoplasmic reticulum signal sequence enabled Tap-1-independent expression of each peptide in these mice. Positive selection of F5TCR+CD8(+) thymocytes was not rescued by these transgenic peptides. However, the high-affinity NP68 peptide induced maturation of CD8alphaalpha IEL, whereas the low-affinity NP34 peptide stimulated development of both CD8alphabeta and CD8alphaalpha IEL, but in smaller numbers. When both peptides were present, CD8alphabeta T cells failed to develop and the number of CD8alphaalpha IELs was lower than in mice carrying the NP68 transgene alone. These data demonstrate that single ligands with a high or low affinity for TCR are capable of inducing or inhibiting the maturation of alternative subsets of IELs.

Development of colonic adenocarcinomas in a mouse model of ulcerative colitis.

Mice deficient in both interleukin-2 and beta 2-microglobulin expression (Beta 2mullnull x IL-2null mice) develop an inflammatory disease of the colon resembling ulcerative colitis. To examine long-term complications of disease in these mice, a group of 34 Beta 2mnull x IL-2null mice was monitored for 6-12 months. Development of clinical disease was assessed by wasting, general appearance, and diarrhea. Further analysis included histologic examination of the distal colon for colitis, staining of CD4+ T cells for surface activation markers, and cytoplasmic staining of CD4+ T cells for IFN-gamma and TNF-alpha. These older Beta 2mnull x IL-2null mice had activated CD4+ T cells as assessed by surface markers on flow cytometry. Cytoplasmic staining revealed IFN-gamma production, but not TNF-alpha production by CD4+ T cells. The majority of these older Beta 2mnull x IL-2null mice continued to have colitis on histology. However, they lived much longer and had less wasting in comparison to IL-2null mice. At necropsy, 11 (32%) of 34 of the Beta 2mnull x IL-2null mice had tumors in the proximal half of the colon. Histologic examination confirmed these tumors to be adenocarcinomas. These mice may be useful as a model for studying carcinogenesis in chronic colitis.

Consequences of Fas-ligand and perforin expression by colon T cells in a mouse model of inflammatory bowel disease.

BACKGROUND & AIMS: We describe a type of colitis that develops after transplantation of nonallogeneic wt bone marrow cells into T cell- and natural killer cell-deficient Tg26 mice (BM-->Tg26). In these animals, severe wasting and inflammation of the colon correlates with the expansion of mucosal T lymphocytes that displays cytotoxic activity. The aims of this study were to determine the relative contribution of perforin and Fas ligand (Fas-L) expression to the cytotoxic action of these T cells and to examine the influence of each pathway in this model of colitis. METHODS: Colonic T cells were tested for their ability to mediate Fas- and perforin-dependent killing in redirected cytotoxicity assays. Bone marrow cells from donor mice lacking either Fas-L (gld mice) or perforin (PFPnull mice) or both molecules were used to reconstitute Tg26 mice. RESULTS: Colon cytotoxic T lymphocyte displayed both Fas- and perforin-dependent killing. Deficiency in perforin, but not Fas-L, resulted in reduced incidence of wasting and, to a lesser extent, severe colitis in BM-->Tg26 animals. CONCLUSIONS: Colon T cells from BM-->Tg26 mice express both perforin and Fas-L. Although neither pathway is critical in the development of colitis, perforin does have a measurable influence on disease in the BM-->Tg26 colitis model.

T cell-mediated pathology in two models of experimental colitis depends predominantly on the interleukin 12/Signal transducer and activator of transcription (Stat)-4 pathway, but is not conditional on interferon gamma expression by T cells.

The requirements for interleukin (IL)-12/signal transducer and activator of transcription (Stat)-4 signaling and induction of T cell-specific interferon (IFN)-gamma expression in the development of T helper cell (Th)1-type pathology were examined in two different models of experimental colitis. In each model, abnormal reconstitution of the T cell compartment in immunodeficient mice by adoptive cell transfer leads to a wasting syndrome and inflammation of the colon, induced by IFN-gamma and tumor necrosis factor (TNF)-alpha-producing T cells. We show here that treatment with anti-IL-12 antibodies in one of the models, or reconstitution with T cells from Stat-4-deficient (Stat-4(null)) mice in both models resulted in a milder disease in the majority of recipient animals, compared with those that were left untreated or that had been reconstituted with wt cells. Protected mice in each group also harbored lower frequencies of IFN-gamma-producing T cells than did diseased mice, suggesting that effects on wasting and colitis resulted from the attenuation of IFN-gamma expression by T cells. To test whether the development of pathogenic T cells in the two colitis models was directly dependent on T cell-specific IFN-gamma expression, IFN-gammanull donors were used for T cell reconstitution in each system. Surprisingly, large numbers of IFN-gammanull-reconstituted mice developed wasting and colitis, which in many cases was of comparable severity to that seen in animals reconstituted with wt cells. Furthermore, T cells from these animals expressed TNF-alpha, demonstrating that they had retained the ability to produce another proinflammatory cytokine. Taken together, these results demonstrate that in some forms of chronic experimental colitis the development of pathogenic T cells is influenced predominantly, though not exclusively, by IL-12 via the actions of Stat-4 proteins. Furthermore, our data suggest that in the models of colitis studied here the effects of IL-12/Stat-4 or other Th1 promoting pathways are not limited to the induction of IFN-gamma gene expression in T lymphocytes.

Interleukin 8 released after acute myocardial infarction is mainly bound to erythrocytes.

OBJECTIVE: To determine whether rapid clearance of interleukin 8 (IL-8) from plasma through binding to the erythrocyte chemokine receptor may be responsible for failure to detect IL-8 consistently after acute myocardial infarction. DESIGN: Plasma concentrations of IL-8 were measured at frequent intervals in 43 consecutive patients. In 21 of these, erythrocyte bound IL-8 concentrations were also measured. The influence of infarct size, type of treatment, and the presence of early successful reperfusion on IL-8 release was assessed. RESULTS: Peak IL-8 concentrations in plasma were raised in 31 of the 43 patients (68%). Median plasma IL-8 concentrations were 16.0 pg/ml (range 2.4 to 225.0 pg/ml) six hours after the onset of chest pain. Twelve hours after the onset of symptoms, plasma IL-8 concentrations had already returned to normal in 27 patients. In contrast, in 18 of 21 patients (86%), erythrocyte bound IL-8 concentrations were raised at between 6 and 30 hours, with a median peak value of 59.8 pg/ml (range 19 to 148 pg/ml). No correlation between peak creatine kinase MB and peak IL-8 (plasma or erythrocyte bound) was observed. There was a significant difference in peak plasma IL-8 concentrations between patients who underwent direct PTCA (19.4 pg/ml) and those who received conservative treatment (9.9 pg/ml; p = 0.0206), but no correlation with the presence of early successful reperfusion. CONCLUSIONS: IL-8 is released in plasma after acute myocardial infarction and subsequently binds to red blood cells, resulting in only a transient rise of plasma IL-8 and a more prolonged increase of erythrocyte bound IL-8.