Blocking lymphotoxin-beta receptor activation diminishes inflammation via reduced mucosal addressin cell adhesion molecule-1 (MAdCAM-1) expression and leucocyte margination in chronic DSS-induced colitis

Abstract

The lymphotoxin-beta receptor (LTbetaR) pathway is critical for maintenance of organized lymphoid structures and is involved in the development of colitis. To investigate the mechanisms by which LTbetaR activation contributes to the pathology of chronic inflammation we used a soluble LTbetaR-Ig fusion protein as a competitive inhibitor of LTbetaR activation in the mouse model of chronic colitis induced by oral administration of dextran sulphate sodium. Strong expression of LTbeta which constitutes part of the LTalpha(1)beta(2) ligand complex was detected in colonic tissue of mice with chronic colitis. Treatment with LTbetaR-Ig significantly attenuated the development and histological manifestations of the chronic inflammation and reduced the production of inflammatory cytokines such as TNF, IL-1beta, and IL-6. Moreover, LTbetaR-Ig treatment significantly down-regulated mucosal addressin cell adhesion molecule-1 (MAdCAM-1) expression, leading to reduced leucocyte rolling and sticking in postcapillary and collecting venules and reduced extravasation into the intestinal mucosa as quantified by in vivo fluorescence microscopy. Thus, LTbetaR pathway inhibition ameliorates DSS-induced experimental chronic colitis in mice by MAdCAM-1 down-regulation entailing reduced lymphocyte margination and extravasation into the inflamed mucosa. Therefore, a combined treatment with reagents blocking T cell-mediated perpetuation of chronic inflammation such as LTbetaR-Ig together with direct anti-inflammatory reagents such as TNF inhibitors could constitute a promising treatment strategy for chronic colitis.

Fig. 1

(a) The histological score was determined in mice (n = 6) with chronic colitis after treatment with either LTβR-Ig, anti-TNF or control IgG. Statistical significance was determined using the Mann–Whitney Rank Sum Test. (b) Histology of colon sections from the same mice as in (a).

Fig. 2

(a) The inflammatory infiltrate of granulocytes in colon tissue sections of mice (n = 6) with chronic colitis treated with either LTβR-Ig, anti-TNF, or control IgG was measured as MPO activity and statistical significance determined using the Mann–Whitney Rank Sum Test. (b) The length of the colons from the same mice as in (a) was measured and the statistic significance of the reduction in length was determined using the Mann–Whitney Rank Sum Test.

Fig. 3

Lymph node score was determined in mice (n = 6) with chronic colitis after treatment with either LTβR-Ig, anti-TNF, or control IgG. Statistical significance was determined using the Mann–Whitney-Rank Sum Test.

Fig. 4

Semiquantitative RT-PCR for mRNA determination of LTβ, and β-actin was performed from colon tissue of healthy mice or mice with chronic colitis (n = 5).

Fig. 5

Representative image of the distribution of MAdCAM-1 expression in the inflamed mucosa. Frozen colon sections of either (a) control IgG- or (b) LTβR-Ig-treated mice with chronic colitis were stained for MAdCAM-1. Representative pictures of at least 3 individual stainings (n = 6) are shown. (c) Expression of α₄β₇-integrin, the ligand for MAdCAM-1, on lymphocytes derived from the mesenteric lymph nodes of either control IgG- or LTβR-Ig-treated mice with chronic colitis. Representative data from at least 3 individual measurements are shown.

Fig. 6

In vivo microscopy of mucosal microcirculation in chronic colitis. The numbers (%) of rolling leucocytes or sticking leucocytes (per mm²) as well as pictures are given. Rolling and sticking events of lymphocytes (a) in submucosal collecting venules, (b) in mucosal post capillary venules and (c) sticking events and extravasation of lymphocytes in the mucosa.