Original contributionDeficient iNOS in inflammatory bowel disease intestinal microvascular endothelial cells results in increased leukocyte adhesion
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.
Section snippets
Isolation and culture of mucosal microvascular endothelial cells
HIMEC isolation was performed using a technique adapted from dermal microvascular endothelium, as we have previously described [8]. In brief, surgical specimens were rinsed and full thickness samples of intestinal tissue were obtained. Mucosal strips were dissected and washed to remove debris and contaminating bacteria, minced, and digested in a type II collagenase solution (Worthington Biochemical Corp., Freehold, NJ, USA). Mechanical compression was used to express clusters of microvascular
Results
Since NO downregulates leukocyte adhesion, we reasoned that if NO production in activated HIMEC from IBD is diminished, then NOS inhibitors would not affect their ability to adhere leukocytes. A nonselective inhibitor of NO production, l-NMMA, was first used to assess the effect of total NOS inhibition on leukocyte binding in HIMEC isolated from the chronically inflamed mucosa from patients with medically unresponsive ulcerative colitis or Crohn’s disease. Although NOS inhibition with l-NMMA is
Discussion
The results of our study suggest that in IBD, intestinal blood vessels subjected to chronic inflammation in vivo acquire a fundamental alteration in their ability to generate NO via iNOS. This observation has important implications, since NO production by the vascular endothelium appears to be a critical factor in downregulating leukocyte adhesion [7], a key regulatory step in the inflammatory process. In the context of chronic inflammation, the loss of endothelial NO production via iNOS may
Acknowledgements
This work was supported in part by the National Institutes of Health Grants K08 DK02417 (D.G.B.), R03 DK56234 (D.G.B.), K08 DK02469 (K.T.W.), R01 CA67497 (K.T.W.), R01 DK53620 (K.T.W.), the Medical College of Wisconsin, Digestive Disease Center (D.G.B.), the University of Maryland (K.T.W.), and the Crohn’s and Colitis Foundation of America (D.G.B. and K.T.W.). We would like to thank Dr. Bellur Seetharam, Division of Gastroenterology and Hepatology, The Medical College of Wisconsin, for his
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