Remission-inducing effect of anti-TNF monoclonal antibody in TNBS colitis: mechanisms beyond neutralization?

BACKGROUND: Tumor necrosis factor (TNF) plays an important role in the pathogenesis of several inflammatory diseases. Its expression is increased in inflamed mucosa of Crohn's disease patients and anti-TNF treatment improves mucosal inflammation. Besides neutralization, induction of apoptosis of monocytes/macrophages and T cells is thought to be an important mechanism of action of the anti-TNF monoclonal antibody therapy. The aim was to investigate the pathogenic role of TNF in hapten-induced colitis models and to study the relationship between apoptosis induction and disease remission. METHODS: In 2 murine colitis models (trinitrobenzene sulphonic acid, TNBS, and oxazolone colitis), mice were injected daily with anti-TNF monoclonal antibody (mAb). Macrophages were collected from lamina propria of TNBS colitis mice. 7AAD and anti-active-caspase-3 staining were used to study DNA degradation and intracellular caspase activation. A pan-caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK), was given to a subgroup of the colitis mice. RESULTS: Treatment with anti-TNF effectively reduced intestinal mucosal inflammation in TNBS colitis but not in oxazolone colitis. Effectiveness was evidenced by a more rapid recovery of body weight and reduced cell infiltration, and downregulation of proinflammatory cytokines interferon-gamma (IFN-gamma), TNF, and IL-18 at the mRNA level. Apoptosis was induced in lamina propria macrophages after treatment with anti-TNF, and it was abrogated through short-term pretreatment with Z-VAD-FMK. CONCLUSION: Anti-TNF downregulates proinflammatory cytokines and decreases cell infiltration in the bowel after TNBS application. The remission-inducing effect of anti-TNF may partly rely on apoptosis induction.     

Constitutive and cytokine induced expression of HLA molecules,secretory component,and intercellular adhesion molecule-1 is modulated by butyrate in the colonic epithelial cell line HT-29.

Normal colonic epithelial cells play an important part in the mucosal immune system and use butyrate, a bacterial fermentation product, as an important energy source. Butyrate deficiency has been associated with inflammatory bowel disease, diversion colitis, and pseudomembranous colitis. Butyrate effects on important molecules for epithelial immune functions were studied in a colonic epithelial cell line (HT-29): the constitutive and cytokine regulated expression of secretory component (poly-Ig receptor), HLA class I and II molecules, and intercellular adhesion molecule-1 (ICAM-1). Butyrate facilitated the constitutive expression of secretory component and HLA class I. Butyrate furthermore tended to enhance cytokine mediated stimulation of protein expression, although tumour necrosis factor alpha (TNF) and interleukin 4 (IL 4) responses on HLA class I and secretory component, respectively, were relatively inhibited by butyrate. Cytokine mediated accumulation in the various mRNAs usually increased even more in the presence of butyrate, with the exception of TNF response on HLA class I and secretory component mRNA concentrations. In conclusion, butyrate may substantially influence constitutive and cytokine mediated expression of molecules with immune functions in a complex and differentiated manner, and butyrate deficiencies, as seen in various clinical conditions, might influence mucosal immune responses.     

Germinated barley foodstuffs attenuate colonic mucosal damage and mucosal nuclear factor kappa B activity in a spontaneous colitis model

BACKGROUND: Germinated barley foodstuffs (GBF), which are derived from brewer's spent grain and are a highly safe food substance, increased butyrate production in the lower intestine and prevented mucosal damage and bloody diarrhoea in an acute experimental colitis model. As human histocompatibility leucocyte antigen (HLA)-B27 transgenic rats develop spontaneous and chronic intestinal inflammation resembling ulcerative colitis, we investigated the mechanisms underlying the preventive effects of GBF against a spontaneous and chronic colitis model. Specifically, the production of bacterial butyrate and the regulation of proinflammatory cytokine production were examined. METHODS: A GBF diet and a cellulose (CE) diet were fed to HLA-B27 transgenic rats for 13 weeks. The presence of faecal occult blood, colonic mucosal protein, DNA and RNA content, colonic myeloperoxidase activity, nuclear factor kappa B (NFkappaB) DNA binding activity, the depth of the crypts and serum inflammatory parameters were then evaluated. Butyrate production in the caecal contents was also determined. RESULTS: Feeding GBF significantly increased bacterial butyrate production and simultaneously attenuated the presence of faecal occult blood and colonic mucosal hyperplasia. Colonic mucosal NFkappaB-DNA binding activity and the production of interleukin-8 were also suppressed by the butyrate produced from GBF. CONCLUSIONS: Germinated barley foodstuffs feeding promotes bacterial butyrate production and attenuated inflammation in both spontaneous and chronic colitis in HLA-B27 transgenic rats.     

cDNA array analysis of cytokines, chemokines, and receptors involved in the development of TNBS-induced colitis: homeostatic role of VIP

Crohn's disease (CD) is a chronic inflammatory pathology of the intestine, characterized by diarrhea and weight loss. A healing effect of vasoactive intestinal peptide (VIP) in the murine model of CD based on 2,4,6-trinitrobencene sulfonic acid (TNBS) administration has been previously shown. The aim of this work was to analyze the expression of several mediators related to the inflammatory cascade in colitic and VIP-treated animals. With this aim, mice received either only TNBS or TNBS and VIP treatment on alternate days. cDNA microarray analysis and real-time polymerase chain reaction were performed on total mRNA from colon to study the expression of a battery of proinflammatory molecules such as the enzyme COX-2, the chemokines CX3CL1, CXCL12, CXCL13, CXCL14, CCR5, and CXCR2, and the cytokines interleukin (IL)-1beta, IL-12, IL-18, IL-10, interferon-gamma, and IL-4. TNBS administration induced the expression of all the proinflammatory mediators studied, whereas VIP treatment reduced their levels, increasing the anti-inflammatory IL-10 and the TH2 cytokine IL-4, explaining its beneficial action through inhibition of the inflammatory/TH1 response. These data describe not only the relation of several proinflammatory mediators to the development of TNBS colitis, reporting their time-course, but also show the beneficial action of VIP in this model through complete blockage of the inflammatory cascade and recovery of the colon homeostasis, providing a potential new alternative for CD therapy.     

Variable Impact of CD39 in Experimental Murine Colitis

Background

Dysregulation of immune responses in inflammatory bowel diseases (IBD) results in intestinal inflammation and vascular injury while exacerbating systemic disease. CD39 is an ectonucleotidase, expressed by T regulatory cells and dendritic cells, that hydrolyzes extracellular nucleotides to modify those cellular immune responses implicated in IBD. Genetic polymorphisms of CD39 have been linked to Crohn??s disease while gene deletion in mice exacerbates dextran sodium sulphate-induced colitis.

Aim

The aim of this study was to test how global deletion of CD39 in mice impacts other models of experimental colitis.

Methods

Colitis was induced in CD39-null and -wt mice, using trinitrobenzene sulfonic acid (TNBS, 125 mg/kg) administered intrarectally. Oxazolone colitis (1.5% oxazolone in 50% alcohol) was induced in comparable groups. Morphology, clinical and molecular parameters, and FACS analyses of lamina propria mononuclear cells (LPMC) were examined in CD39-null mice. CD39 expression was analyzed in human IBD biopsies.

Results

Paradoxically, TNBS colitis in CD39-null mice was characterized by improved survival, favorable clinical scores, and decreased MPO activity, when compared to wt mice (P < 0.05). LPMC from TNBS colitis contained significantly increased amounts of T-cells (CD3⁺ and CD4⁺) and TNF-?? mRNA expression were increased over those in CD39 null mice (P < 0.05). In contrast, oxazolone treated CD39-null and wt mice had comparable outcomes. In both ulcerative colitis and Crohn??s disease, CD39 is present at high levels in intestinal tissue biopsies.

Conclusions

TNBS colitis was attenuated in CD39-null mice whereas oxazolone-induced colitis was not impacted. Impaired adaptive cellular immune reactivity in the CD39-null environment appears protective in hapten-mediated Th1-type colitis. CD39 is expressed at high levels in clinical IBD tissues.     

Amelioration of 2,4,6-trinitrobenzene sulphonic acid induced colitis in angiotensinogen gene knockout mice

BACKGROUND: A number of recent studies have demonstrated a protective effect of renin-angiotensin system (RAS) antagonism against immune mediated diseases such as myocarditis, chronic allograft rejection, and antiglomerular basement membrane nephritis. To our knowledge, there has been no report on the immunological contribution of the RAS in colonic tissue. AIMS: We evaluated the direct effect of angiotensin II (AII) on the pathogenesis of immune mediated colitis using angiotensinogen deficient homozygous (Atg-/-) mice. SUBJECTS: 2,4,6-Trinitrobenzene sulphonic acid (TNBS) colitis was induced in Atg-/- and wild-type (Atg+/+) mice. METHODS: Levels of proinflammatory cytokines in the colon were determined by enzyme linked immunosorbent assay. Histological analysis was performed simultaneously. RESULTS: Although Atg-/- mice developed colitis, the degree was much milder than that in Atg+/+ mice (p<0.05). Colonic cytokine analysis showed that the production of proinflammatory cytokines (interleukin (IL)-1beta, interferon gamma (IFN-gamma)) was impaired in Atg-/- mice. Furthermore, expression of cytokines such as IL-4 and IL-10 in the colon was predominant in Atg-/- compared with Atg+/+ mice after TNBS instillation (p<0.005, p<0.01, respectively). Similarly, subcutaneous infusion of losartan suppressed colitis (p<0.05) and the production of proinflammatory cytokines (IL-1beta, IFN-gamma). These results indicate that the RAS is directly involved in the pathogenesis of TNBS colitis through regulation of proinflammatory and anti-inflammatory cytokines in the colon. CONCLUSIONS: This study revealed that the RAS is involved in the immune system in the colon. Antagonism of the RAS is a potential prophylactic strategy for the treatment of human inflammatory bowel disease.     

The in vitro anti-inflammatory effects of recombinant anti-CD25 immunotoxin on lamina propria T cells of patients with inflammatory bowel disease are not sufficient to cure experimental colitis in mice

BACKGROUND AND AIMS: In chronic inflammatory bowel disease (IBD) such as Crohn's disease and ulcerative colitis an aberrant mucosal immune regulation is observed accompanied by upregulation of proinflammatory cytokines. Lamina propria T cells of inflamed mucosa have an activated phenotype characterized by increased expression of surface markers such as CD25. We therefore determined the anti-inflammatory effect of a recombinant immunotoxin consisting of an anti-CD25 single chain variable fragment (scFv) fused to a deletion mutant of Pseudomonas exotoxin A [RFT5(scFv)ETA'] on isolated lamina propria lymphocytes of patients with IBD and in the murine model of trinitrobenzene sulfonic acid (TNBS) induced colitis. PATIENTS AND/METHODS: Lamina propria lymphocytes of 25 patients with IBD and 19 control patients were stimulated in absence or presence of RFT5(scFv)ETA'. Interferon-gamma production was determined in the supernatant by ELISA and the induction of apoptosis by flow cytometry after propidium iodide staining. BALB/c mice received TNBS intrarectally and were treated with RFT5(scFv)ETA'. RESULTS: In vitro the administration of RFT5(scFv)ETA' significantly reduced interferon-gamma production and increased apoptosis in lamina propria lymphocytes isolated of inflamed mucosa. However, this contrainflammatory regulation did not result in gain of weight or increased life span in experimental colitis in vivo. CONCLUSION: In addition to the downregulation of the proinflammatory cytokine in vitro, RFT5(scFv)ETA' induced neither a direct nor a bystander effect in an in vivo model of colitis. Therefore our data do not support potential therapeutic implications of targeting CD25 by RFT5(scFv)ETA' in chronic IBD.