K. Menzel

In vivo treatment with the herbal phenylethanoid acteoside ameliorates intestinal inflammation in dextran sulphate sodium-induced colitis.

Recently we demonstrated that in inflammatory bowel disease (IBD) macrophage‐oxidative burst activity is increased and NADPH oxidase mRNA is induced. The herbal phenylethanoid acteoside isolated from Plantago lanceolata L. was shown to exhibit anti‐oxidative potential. Using the dextran sulphate sodium (DSS)‐induced colitis model, in this study we have assessed whether systemic application of acteoside affects colitis. Colitis was induced by DSS in Balb/c mice. Treatment with acteoside (120, 600 µg/mouse/day) was performed intraperitoneally. The colon lengths were determined. Colonic tissue was scored histologically (max. score 8) by a blinded investigator. T cells isolated from mesenteric lymph nodes (MLN) were stimulated with anti‐CD3 antibody in the presence of interleukin (IL)‐2 (final concentration 10 U/ml). After incubation for 24 h, IL‐1β, IL‐6, IL‐12 tumour necrosis factor (TNF)‐α and interferon (IFN)‐γ levels in supernatants were analysed by the beadlyte® cytokine detection system. Histological scoring of colonic tissue revealed that application of acteoside was followed by a significantly improved histological score. In acute colitis the histological score was 3·2 with acteoside versus 5·2 with phosphate‐buffered saline (PBS) (P < 0·02). In chronic colitis both 120 µg (3·3 versus 5·2) or 600 µg acteoside (3·0 versus 5·2) significantly ameliorated colitis (both P < 0·02). Stimulated MLN from mice with chronic DSS‐induced colitis treated with acteoside showed a significant down‐regulation of IFN‐γ secretion (195 pg/ml with 600 µg acteoside versus 612 pg/ml with PBS, P < 0·02). Inhibition of oxidative burst activity with acteoside reduced mucosal tissue damage in DSS colitis and could be a therapeutic alternative for IBD treatment. Further studies of this agent are warranted.

Cathepsins B, L and D in inflammatory bowel disease macrophages and potential therapeutic effects of cathepsin inhibition in vivo.

The cathepsins D (CTSD), B (CTSB) and L (CTSL) are important for the intracellular degradation of proteins. Increased cathepsin expression is associated with inflammatory diseases. We have shown previously an induction of CTSD expression in intestinal macrophages (IMAC) in inflamed mucosa of patients with inflammatory bowel disease (IBD). Here we investigated the regulation of CTSB and CTSL in IMAC during IBD and effects of CTSD and CTSB/CTSL inhibition in vivo. Human IMAC were isolated from normal and inflamed mucosa. Reverse transcription–polymerase chain reaction (RT–PCR) was performed for CTSB and CTSL mRNA. Immunostaining was used to confirm PCR results. Cathepsin inhibition was investigated in the dextran–sulphate–sodium (DSS) colitis model in mice with application of pepstatin A (CTSD inhibitor), CA‐074 (CTSB inhibitor) and Z‐Phe‐Tyr‐aldehyde (CTSL inhibitor). CTSL mRNA was significantly up‐regulated in IMAC isolated from IBD mucosa. Up‐regulated protein expression was found mainly in areas of mucosal damage by immunostaining. Inhibition of CTSD in mouse DSS colitis was followed by an amelioration of the disease. Inhibitor‐treated mice showed a significant lower histological score (HS) and less colon reduction in comparison to controls. Similarly, simultaneous inhibition of CTSB/CTSL was followed by a significant amelioration of colitis. Expression of tissue‐degrading cathepsins is increased in IMAC in IBD. Inhibition of CTSD as well as CTSB/CTSL is followed by an amelioration of experimental colitis. The prevention of mucosal damage by cathepsin inhibition could represent a new approach for the therapy of IBD.

(GT)n Dinucleotide repeat polymorphism of haem oxygenase-1 promotor region is not associated with inflammatory bowel disease risk or disease course

Haem oxygenase‐1 (HO‐1) up‐regulation was suggested to reduce mucosal tissue damage in inflammatory bowel disease (IBD) and an up‐regulation of HO‐1 expression in patients with Crohns disease (CD) and ulcerative colitis (UC) was demonstrated. A HO‐1 gene promoter microsatellite (GT)n dinucleotide repeat polymorphism was associated with regulation of HO‐1 in response to inflammatory stimuli. We therefore hypothesized that IBD patients might segregate into phenotypes with high or low HO‐1 inducibility. Ethylenediamine tetraacetic acid blood samples were obtained from 179 CD patients, 110 UC patients and 56 control patients without inflammation. Genomic DNA was purified and the 5′‐flanking region of the HO‐1 gene containing the (GT)n dinucleotide repeat was amplified. Polymerase chain reaction (PCR) products were purified and the length of the PCR fragments was analysed. The number of (GT)n repeats in the population studied ranged from 13 to 42. The distribution of the allele frequencies was comparable in patients and controls for both the short and the long alleles. The frequencies of short‐, middle‐ and long‐sized alleles were not changed among the groups studied. No correlation was found between IBD and microsatellite instability detected in five individals. Our data indicate that (GT)n dinucleotide repeats of the HO‐1 promotor region have no significance for the pathophysiology and disease course of IBD.

W1215 MDP Stimulates Memory T-Cell Recruitment Via NOD2 Connecting Bacterial Sensing and Adaptive Immunity

BACKGROUND: Macrophage inflammatory protein (Mip)-3α is a CC chemokine attracting memory T lymphocytes and is induced during differentiation of blood monocytes into intestinal macrophages (IMACs) in the intestinal mucosa. It is further released via NFκB activation upon stimulation with proinflammatory cytokines, such as TNF. Bacterial translocation across the epithelial barrier is crucial during the pathogenesis of Crohns disease (CD). NOD2 is an intracellular sensor of bacterial muramyl dipeptide (MDP) and is able to activate the NF-κB pathway. NOD2 variants have been shown to be the most important susceptibility factor for the development of CD. We have tested the ability of MDP to induce Mip-3α expression and subsequent recruitment of memory T lymphocytes. METHODS: Cells were transfected with vector (pcDNA) to express wtNOD2, SNP13 or empty vector. Cells were stimulated with 0/100/500/1000/2000 ng/ml MDP for 18 hours. Supernatants were collected and Mip-3α was quantified by ELISA and real time-PCR. Functional assays on migration of memory (CD45R0+) T cells were performed in a three dimensional In Vitro differentiation model for IMACs: the multicellular spheroid (MCS) coculture model. Neutralizing antibodies to human Mip-3α (polyclonal, rabbit, acris-antibodies; final concentrations, 15, 1.5, and 0.15 mg/ml) were used for blocking studies. RESULTS: Taqman analysis revealed significantly elevated Mip-3αmRNA expression in HEK293 cells transfected with pcDNA to express NOD2 after stimulation with MDP. Increased Mip-3α secretion was demonstrated by ELISA. MDP induced Mip-3α mRNA and protein secretion in a dose dependent manner. A functional deficit was found after transfection with pcDNA to express SNP13. During the differentiation of monocytes into IMACs Mip-3α was induced in the MCS-In Vitro model during 7 days of coculture. Mip-3α+ IMACs attracted CD45R0+ T cells to migrate into the MCS. CONCLUSION: Mip-3α is induced during differentiation of IMACs. There is evidence for a NOD2 mediated induction of Mip-3α expression followed by subsequent memory T-cell recruitment connecting bacterial sensingwithmemory T-cell functions.