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.

IntroductionClostridium difficile causes antibiotic-associated diarrhea and pseudomembranous colitis in humans (1). Pathogenic strains of C. difficile release 2 large exotoxins: toxin A (308 kDa) and toxin B (269 kDa). In animal models, only toxin A is enterotoxic and causes fluid secretion, mucosa edema, and villous disruption by inducing massive acute inflammation with neutrophil infiltration. We have shown that blocking neutrophil extravasation using an anti-CD18 antibody prevented toxin A-induced enteritis and mucosal damage (2).Toxin A and toxin B show 63% homology (3) and share similar domains. Their COOH-terminal portions carry repeating sequences that may be involved in receptor binding. The NH 2 -terminal portions carry an enzymatic domain, which hydrolyzes UDP-glucose and transfers the glucose moiety to a conserved threonine residue of the small GTP-binding proteins Rho, Cdc42, and Rac (4-6). The small GTP-binding protein Rap is glucosylated only by toxin A (7). These covalent modifications inactivate Rho proteins, which in turn induce cytoskeleton disaggregation and cell rounding. Whether Rho glucosylation is involved in toxin A enterotoxicity and in vitro cytokine production is not known. Toxin A or toxin B were shown to block Rho-regulated signaling pathways, including basophilic cell activation (8), receptor signaling to phospholipase D (9), NF-κB activation by bradykinin (10), and mitogen-activated protein-kinase (MAP kinase) activation (11,12). Blockage of the above signal transduction pathways by C. difficile toxins is consistent with the role of Rho proteins in signaling to MAP kinases and NF-κB (13-15). However, it appears at variance with the known in vivo and in vitro inflammatory actions of toxin A.In cells of the monocyte lineage, C. difficile toxins stimulate inflammatory cytokine release, including TNF-α, IL-1β, . In addition, we have reported recently that both toxins induce necrosis in human monocytes and in THP-1 human monocytic cells. This cell-death pathway is associated with potassium depletion, caspase activation, and maturation and release of preformed IL-1β (19). The mechanisms whereby these toxins activate and kill monocytes are not known.In this study, we investigated the role of MAP kinases in IL-8 production, IL-1β release, and necrosis induced by toxin A in monocytic cells. MAP kinases regulate cell responses to growth factors and stress stimuli and transmit signals from the cell surface to the nucleus via 3 distinct but related pathways. These culminate in the selective activation of extracellular signal-related kinases (ERK), p38, and c-Jun NH 2 -terminal kinase (JNK) Clostridium difficile toxin A causes acute neutrophil infiltration and intestinal mucosal injury. In cultured cells, toxin A inactivates Rho proteins by monoglucosylation. In monocytes, toxin A induces IL-8 production and necrosis by unknown mechanisms. We investigated the role of mitogen-activated protein (MAP) kinases in these events. In THP-1 monocytic cells, toxin A activated the 3 main MAP kinase cascad...

The gastric pathogen Helicobacter pylori is known to activate epithelial cell signaling pathways that regulate numerous inflammatory response genes. The aim of this study was to elucidate the pathway leading to extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in H. pylori-infected AGS gastric epithelial cells. We find that H. pylori, via activation of the epidermal growth factor (EGF) receptor activates the small GTP-binding protein Ras, which in turn, mediates ERK1/2 phosphorylation. cag؉ strains of H. pylori are able to induce greater EGF receptor activation than cag؊ strains, and studies with isogenic mutants indicate that an intact type IV bacterial secretion system is required for this effect. Blockade of EGF receptor activation using tyrphostin AG1478 prevents H. pylori-mediated Ras activation, inhibits ERK1/2 phosphorylation, and substantially decreases interleukin-8 gene expression and protein production. Investigations into the mechanism of EGF receptor activation, using heparin, a metalloproteinase inhibitor and neutralizing antibodies reveal that H. pylori transactivates the EGF receptor via activation of the endogenous ligand heparin-binding EGFlike growth factor. Transactivation of gastric epithelial cell EGF receptors may be instrumental in regulating both proliferative and inflammatory responses induced by cag؉ H. pylori infection.

Neurotensin (NT), a neuropeptide released in the gastrointestinal tract in response to several stimuli, is involved in the pathophysiology of colonic inflammation. However, the molecular mechanism(s) mediating this proinflammatory response remains unclear. We found that NCM460, non-transformed human colonocytes, express a functional high affinity NT receptor that mediates NT-induced Erk activation. By using NCM460 cells stably transfected with NTR1, we show that NTR1 activation leads to interleukin (IL)-8 secretion that is mediated via both NF-B-and Erk-dependent pathways. In addition, NT-stimulated NF-B activation is dependent on intracellular calcium release. NT-stimulated Erk activity requires Ras activation because overexpression of the dominant negative Ras mutant Ras-17N almost completely inhibits the Erk activation. Furthermore, NT directly stimulates Ras-GTP formation as shown by a Ras-GTP pull-down assay. By using reporter gene constructs containing targeted substitutions in the IL-8 promoter, we show that the NF-B, AP-1, and to a lesser degree the C/EBP sites in the IL-8 promoter region are required for IL-8 gene expression induced by NT. In summary, our results demonstrate that NT stimulates calcium-dependent NF-B and Ras-dependent Erk pathways that mediate the release of IL-8 from non-transformed human colonocytes. We speculate that these NT-related proinflammatory pathways are important in the pathophysiology of colonic inflammation.Neurotensin (NT), 1 a 13-amino acid neuropeptide originally isolated by Carraway and Leeman (1), is highly expressed in the gastrointestinal tract (2). In the ileal mucosa NT is synthesized and secreted by specific endocrine cells (3), in response to diverse stimuli (4). NT increases small bowel, colonic, and gastric motility and stimulates ileal, pancreatic, and biliary secretion (4, 5) as well as Cl Ϫ secretion from human colonic mucosa (6), indicating that this peptide may contribute to the pathophysiology of human diarrhea. NT also stimulates growth of the intestinal mucosa under physiological and pathological conditions and causes proliferation of intestinal epithelial cells in vivo and in vitro (7-11). Two G-protein-coupled receptors (GPCRs) have been described for NT, a high affinity (NTR1) and a low affinity (NTR2) receptor (12). Administration of the specific NTR1 antagonist SR 48692 to rats inhibits colonic mucin and prostaglandin E 2 secretion in response to immobilization stress (13), suggesting the importance of NTR1 in stress-mediated colonic responses. Our recent studies (14) demonstrate that NT is a proinflammatory peptide in the colon because blockage of the NT-NTR1 interaction with SR 48692 inhibited colonic secretion and inflammation mediated by Clostridium difficile toxin A. We also showed that, compared with normal colonic epithelial cells, there was a dramatic up-regulation of NTR1 during human colonic inflammation (15) as well as in Clostridium difficile toxin A-mediated colitis (14). NT exerts its proinflammatory effects by interacting with severa...