Deficient iNOS in inflammatory bowel disease intestinal microvascular endothelial cells results in increased leukocyte adhesion

Abstract

Microvascular endothelial cells play a key role in inflammation by undergoing activation and recruiting circulating immune cells into tissues and foci of inflammation, an early and rate-limiting step in the inflammatory process. We have previously [Binion et al., Gastroenterology112:1898–1907, 1997] shown that human intestinal microvascular endothelial cells (HIMEC) isolated from surgically resected inflammatory bowel disease (IBD) patient tissue demonstrate significantly increased leukocyte binding in vitro compared to normal HIMEC. Our studies [Binion et al., Am. J. Physiol.275 (Gastrointest. Liver Physiol. 38):G592–G603, 1998] have also demonstrated that nitric oxide (NO) production by inducible nitric oxide synthase (iNOS) normally plays a key role in downregulating HIMEC activation and leukocyte adhesion. Using primary cultures of HIMEC derived from normal and IBD patient tissues, we sought to determine whether alterations in iNOS-derived NO production underlies leukocyte hyperadhesion in IBD. Both nonselective (N^G-monomethyl-l-arginine) and specific (N-Iminoethyl-l-lysine) inhibitors of iNOS significantly increased leukocyte binding by normal HIMEC activated with cytokines and lipopolysaccharide (LPS), but had no effect on leukocyte adhesion by similarly activated IBD HIMEC. When compared to normal HIMEC, IBD endothelial cells had significantly decreased levels of iNOS mRNA, protein, and NO production following activation. Addition of exogenous NO by co-culture with normal HIMEC or by pharmacologic delivery with the long-acting NO donor detaNONOate restored a normal leukocyte binding pattern in the IBD HIMEC. These data suggest that loss of iNOS expression is a feature of chronically inflamed microvascular endothelial cells, which leads to enhanced leukocyte binding, potentially contributing to chronic, destructive inflammation in IBD.

Introduction

It is now appreciated that the free radical messenger molecule nitric oxide (NO) has complex and diverse roles in the inflammatory process. Increased NO production is associated with various forms of gastrointestinal mucosal inflammation, including Barrett’s esophagus, Helicobacter pylori gastritis, human inflammatory bowel disease (IBD), and animal models of IBD [1], [2], [3], [4]. Inhibition of NO production has been demonstrated to improve experimental models of intestinal inflammation in some reports [5]. However, there is a growing body of evidence suggesting that NO can play a protective role in gastrointestinal inflammation, as evidenced by the exacerbation of experimental colitis in iNOS-deficient mice [6]. One mechanism of the anti-inflammatory effect of NO may be via its effects on endothelial cells lining the capillaries and venules of the microvasculature. Microvascular endothelial cells play a critical “gatekeeper” role in the inflammatory response through their ability to undergo activation and recruit circulating leukocytes into tissues and foci of inflammation. Nitric oxide produced by the vascular endothelium functions as a powerful downregulatory molecule, maintaining vascular homeostasis and inhibiting leukocyte binding to the vascular wall, an early and rate-limiting step in inflammation [7].

In order to better define immune-non-immune cell-cell interaction in the intestine during chronic inflammation in IBD, we have successfully isolated human intestinal microvascular endothelial cells (HIMEC) from normal, and chronically inflamed Crohn’s disease (CD) and ulcerative colitis (UC) intestinal tissue. The isolation of tissue and disease-specific microvascular endothelial lines from the human gastrointestinal tract, although technically challenging, is potentially very important as this allows for a direct analysis and comparison of vascular function in health and disease through in vitro modeling. Using passaged primary cultures of these tissue-specific microvascular endothelial cells, we have previously demonstrated that IBD HIMEC bind significantly more leukocytes following activation with cytokines and bacterial stimuli in vitro [8]. The increase in leukocyte binding is an acquired alteration, as IBD HIMEC isolated from uninvolved, noninflamed areas of intestine do not demonstrate the hyperadhesion associated with chronically inflamed IBD gut [9]. Following activation of control, normal HIMEC, NO produced via the high-output, inducible pathway (iNOS, NOS2) exerts an anti-inflammatory effect, downregulating leukocyte adhesion [10]. These actions could be attributed to iNOS specifically, since (i) iNOS expression was markedly increased, while that of the constitutive form of the enzyme, endothelial NOS (eNOS), was unaffected by stimulation, and (ii) selective inhibition of iNOS enzymatic activity increased leukocyte adhesion to the same degree as nonselective inhibition of all forms of NOS [10]. Since iNOS-derived NO inhibits leukocyte adhesion for activated HIMEC and IBD HIMEC have a consistently increased binding of leukocytes, we hypothesized that a loss of NO production from iNOS underlies the enhanced leukocyte binding in activated IBD HIMEC.