Neutrophil transmigration in inflammatory bowel disease is associated with differential expression of epithelial intercellular junction proteins

Abstract

Inflammatory bowel disease (IBD) consisting of ulcerative colitis (UC) and Crohn's (CD) typically displays a waxing and waning course punctuated by disease flares that are characterized by transepithelial migration of neutrophils (PMN) and altered barrier function. Since epithelial barrier function is primarily regulated by the apical most intercellular junction referred to as the tight junction (TJ), our aim was to examine expression of TJ and adherens junction (AJ) proteins in relation to PMN infiltration in mucosal tissue samples from patients with active IBD. Expression of epithelial intercellular TJ proteins (occludin, ZO-1, claudin-1, and JAM) and subjacent AJ (beta-catenin and E-cadherin) proteins were examined by immunoflourescence/confocal microscopy, immunohistochemistry, and Western blotting. Colonic mucosa from patients with UC revealed dramatic, global down-regulation of the key TJ transmembrane protein occludin in regions of actively transmigrating PMN and in quiescent areas in the biopsy samples. Significant decreases in occludin expression were observed at the protein and mRNA levels by Western and Northern blotting. In contrast, expression of other TJ and AJ proteins such as ZO-1, claudin-1, JAM, beta-catenin, and E-cadherin were down-regulated only in epithelial cells immediately adjacent to transmigrating PMN. Analysis of inflamed mucosa from Crohn's disease patients mirrored the results obtained with UC patients. No change in TJ and AJ protein expression was observed in colonic epithelium from patients with collagenous colitis or lymphocytic colitis that are respectively characterized by a thickened subepithelial collagen plate and increased intraepithelial lymphocytes. These results suggest that occludin expression is diminished in IBD by mechanisms distinct from those regulating expression of other intercellular junction proteins. We speculate that down-regulation of epithelial occludin may play a role in enhanced paracellular permeability and PMN transmigration that is observed in active inflammatory bowel disease.

Figure 1.

Differential down-regulation of TJ proteins occludin and ZO-1 in chronic active IBD. Hematoxylin and eosin staining of colonic mucosa from control non-IBD (a, b) and UC (c, d) patients was used to evaluate the histology of tissue sections. Classic features of crypt architectural distortion during mild inflammation (c) and PMN-transmigration with crypt abscesses during active inflammation (d) consistent with ulcerative colitis were noted. To analyze expression of TJ proteins in epithelial cells relative to active inflammation, mucosal tissue sections were double labeled for occludin or ZO-1 (green) and CD11b/CD18 as a marker for PMN (red). Labeled sections were analyzed by confocal microscopy. Occludin was identified in TJs of normal control surface (e) and crypt (f) epithelial cells and was markedly down-regulated to absent in tissue sections from UC patients. Decreased intensity of occludin staining in epithelial cells was observed in regions of active inflammation (h) and in areas not exposed to transmigrating PMN (g). In contrast to occludin, ZO-1 expression in colonic tissues from normal (i, j) and IBD (k, l) patients is down-regulated only in epithelial cells exposed to transmigrating PMN (l). Arrows: tight junctions; arrowhead: PMN; Lumen, lumen; Epi, epithelial cell. Scale bar, 15 μm.

Figure 2.

A: Immunoblot analysis of TJ and AJ protein expression in tissues from patients with chronic active IBD. Mucosal colonic tissue from control and IBD patients was analyzed for TJ and AJ protein expression by performing Western blots. Equal protein was loaded in each lane as determined by protein analysis and Western blots for cytokeratin. Data are representative of results obtained from five control patients and five patients with active IBD. Densitometry results from these patients were presented as mean ± SEM. B: Transcriptional down-regulation of occludin in chronic active UC. RNA was isolated from tissue of normal controls and patients with chronic active UC and transferred to a nitrocellulose membrane. Hybridization with a radioactive labeled probe for full length occludin reveals a signal that is markedly diminished in patients with active UC. Data are representative for results obtained from three control patients and two patients with chronic active UC.

Figure 3.

Decreased expression of TJ and AJ proteins in IBD colonic mucosa is restricted to epithelium with actively transmigrating PMN. Immunohistochemistry was used to map the distribution of TJ and AJ proteins, JAM (a, b, c), claudin-1 (d, e, f), E-cadherin (g, h, i), and β-catenin (j, k, l) in normal and actively inflammed IBD colonic mucosa. JAM and claudin-1 in normal mucosa are expressed in focal concentrations at the apical region of the lateral membranes of epithelial cells consistent with its localization in TJs. Additional pool of these proteins are identified in the lateral membrane subjacent to the TJ in the AJ. The AJ proteins, β-catenin and E-cadherin, are abundantly expressed in lateral membranes of normal epithelial cells. In active UC, expression of all four proteins was diminished exclusively in epithelial cells adjacent to actively transmigrating PMN (arrowheads). However, no change in expression or distribution of these TJ and AJ proteins was observed in epithelial cells not exposed to transmigrating PMN. Arrows, TJ, AJ; arrowhead, PMN. Scale bar, 15 μm