William F. Stenson

Enhanced synthesis of leukotriene B4 by colonic mucosa in inflammatory bowel disease.

Leukotriene B4, an arachidonic acid metabolite, is a potent chemotactic agent, and is thought to be an important mediator of inflammation. To investigate the role of this compound as a mediator of inflammation in inflammatory bowel disease, arachidonic acid was incubated with ionophore and colonic mucosa from patients with inflammatory bowel disease and from normal subjects. Mucosa from patients with inflammatory bowel disease converted 2.17% of exogenous arachidonate to leukotriene B4; mucosa from normal subjects converted 0.37%. The production of leukotriene was blocked by sulfasalazine. To determine if inflammatory bowel mucosa contained endogenous leukotriene B4, lipid extracts were analyzed by high pressure liquid chromatography. Mucosa from patients with inflammatory bowel disease contained 254 ng of leukotriene B4 per gram and mucosa from normal subjects contained less than 5 ng of leukotriene B4 per gram. The presence of significant amounts of leukotriene B4 in colonic mucosa in patients with inflammatory bowel disease, combined with the known biologic effects of leukotriene B4, suggests that it may be an important mediator of inflammation in inflammatory bowel disease.

Cyclooxygenase 2 is induced in colonic epithelial cells in inflammatory bowel disease

BACKGROUND & AIMS Prostaglandins are synthesized by cyclooxygenases (COX)-1 and -2. The expression and cellular localization of COX-1 and COX-2 in normal human colon and inflammatory bowel disease (IBD) surgical resections were studied. METHODS COX-1 and COX-2 protein expression and cellular localization were assessed by Western blotting and immunohistochemistry. RESULTS COX-1 protein was expressed at equal levels in normal, Crohns disease, and ulcerative colitis colonic epithelial cells. COX-2 protein was not detected in normal epithelial cells but was detected in Crohns disease and ulcerative colitis epithelial cells. Immunohistochemistry of normal, Crohns colitis, and ulcerative colitis tissue showed equivalent COX-1 expression in epithelial cells in the lower half of the colonic crypts. COX-2 expression was absent from normal colon, whereas in Crohns colitis and ulcerative colitis, COX-2 was observed in apical epithelial cells and in lamina propria mononuclear cells. In Crohns ileitis, COX-2 was present in the villus epithelial cells. In ulcerative colitis, colonic epithelial cells expressing COX-2 also expressed inducible nitric oxide synthase. CONCLUSIONS COX-1 was localized in the crypt epithelium of the normal ileum and colon, and its expression was unchanged in IBD. COX-2 was undetectable in normal ileum or colon, but it was induced in apical epithelial cells of inflamed foci in IBD.

CYCLOOXYGENASE-2-DEPENDENT ARACHIDONIC ACID METABOLITES ARE ESSENTIAL MODULATORS OF THE INTESTINAL IMMUNE RESPONSE TO DIETARY ANTIGEN

Intestinal inflammatory diseases are mediated by dysregulated immune responses to undefined luminal antigens. Feeding hen egg-white lysozyme to mice expressing a transgenic T-cell receptor that recognizes hen egg-white lysozyme peptide 46–61 resulted in no intestinal pathology; however, simultaneous administration of cyclooxygenase-2 inhibitors and dietary hen egg-white lysozyme resulted in increased proliferation of lamina propria mononuclear cells and crypt epithelial cells, crypt expansion and villus blunting. Lamina propria mononuclear cells produce high levels of cyclooxygenase-2-dependent arachidonic acid metabolites, which act as immunomodulators in the immune response to dietary antigen. These findings establish that cyclooxygenase-2-dependent arachidonic acid metabolites are essential in the development and maintenance of intestinal immune homeostasis.

Metabolism of arachidonic acid in acetic acid colitis in rats. Similarity to human inflammatory bowel disease.

We recently reported that human inflammatory bowel disease mucosa contains large amounts of leukotriene B4, a potent chemotactic agent formed from arachidonic acid through the lipoxygenase pathway. To more fully evaluate the role of arachidonic acid metabolites in the mediation of intestinal inflammation, we studied arachidonate metabolism in an animal model: acetic acid colitis in the rat. Incubation of acetic acid colitis mucosa with arachidonic acid resulted in the production of leukotriene B4 and a series of monohydroxy fatty acids, all products of the lipoxygenase pathway, plus much smaller amounts of cyclooxygenase products including prostaglandin E2. All of these metabolities were made in significantly greater quantities by mucosa from acetic acid-treated rats than by controls. The pattern of arachidonate metabolism in acetic acid colitis was strikingly similar to that in human inflammatory bowel disease. Moreover, the concentration of leukotriene B4 in acetic acid-treated mucosa was almost identical to that in human inflammatory bowel disease mucosa and was 50 times greater than that in normal rat colonic mucosa. These data indicate that lipoxygenase products, including leukotriene B4, may be important mediators of intestinal inflammation in a wide variety of inflammatory conditions. Moreover, the similarities in the metabolism of arachidonate by human inflammatory bowel disease and by acetic acid colitis may allow the use of this model, and perhaps other animal models of intestinal inflammation, in the screening of potential therapeutic agents for inflammatory bowel disease.

Myd88-dependent positioning of Ptgs2-expressing stromal cells maintains colonic epithelial proliferation during injury

We identified cellular and molecular mechanisms within the stem cell niche that control the activity of colonic epithelial progenitors (ColEPs) during injury. Here, we show that while WT mice maintained ColEP proliferation in the rectum following injury with dextran sodium sulfate, similarly treated Myd88(-/-) (TLR signaling-deficient) and prostaglandin-endoperoxide synthase 2(-/-) (Ptgs2(-/-)) mice exhibited a profound inhibition of epithelial proliferation and cellular organization within rectal crypts. Exogenous addition of 16,16-dimethyl PGE(2) (dmPGE(2)) rescued the effects of this injury in both knockout mouse strains, indicating that Myd88 signaling is upstream of Ptgs2 and PGE(2). In WT and Myd88(-/-) mice, Ptgs2 was expressed in scattered mesenchymal cells. Surprisingly, Ptgs2 expression was not regulated by injury. Rather, in WT mice, the combination of injury and Myd88 signaling led to the repositioning of a subset of the Ptgs2-expressing stromal cells from the mesenchyme surrounding the middle and upper crypts to an area surrounding the crypt base adjacent to ColEPs. These findings demonstrate that Myd88 and prostaglandin signaling pathways interact to preserve epithelial proliferation during injury using what we believe to be a previously undescribed mechanism requiring proper cellular mobilization within the crypt niche.

Sulfasalazine inhibits the synthesis of chemotactic lipids by neutrophils.

Neutrophils metabolize arachidonic acid through the liposygenase pathway to 5-hydroxy-6,8,11,14-eicosatetrenoic acid (5-HETE) and 5,12-dihydroxy-6,8,10,14-eicosatraenoic acid (5,12 diHETE). 5-HETE and 5,12diHETE are potent chemotactic agents and are thought to have important roles in the inflammatory response. In this study we demonstrate the sulfasalazine, at concentrations found in the stool of patients being treated for ulcerative colitis, blocks the synthesis of both 5-HETE and 5,12 diHETE by human neutrophils. A sulfasalazine metabolite, 5-aminosalicylate, also blocks the synthesis of 5,12 diHETE.

Crypt stem cell survival in the mouse intestinal epithelium is regulated by prostaglandins synthesized through cyclooxygenase-1.

Prostaglandins (PGs) are important mediators of epithelial integrity and function in the gastrointestinal tract. Relatively little is known, however, about the mechanism by which PGs affect stem cells in the intestine during normal epithelial turnover, or during wound repair. PGs are synthesized from arachidonate by either of two cyclooxygenases, cyclooxygenase-1 (Cox-1) or cyclooxygenase-2 (Cox-2), which are present in a wide variety of mamalian cells. Cox-1 is thought to be a constitutively expressed enzyme, and the expression of Cox-2 is inducible by cytokines or other stimuli in a variety of cell types. We investigated the role of PGs in mouse intestinal stem cell survival and proliferation following radiation injury. The number of surviving crypt stem cells was determined 3.5 d after irradiation by the microcolony assay. Radiation injury induced a dose-dependent decrease in the number of surviving crypts. Indomethacin, an inhibitor of Cox-1 and Cox-2, further reduced the number of surviving crypts in irradiated mice. The indomethacin dose response for inhibition of PGE2 production and reduction of crypt survival were similar. DimethylPGE2 reversed the indomethacin-induced decrease in crypt survival. Selective Cox-2 inhibitors had no effect on crypt survival. PGE2, Cox-1 mRNA, and Cox-1 protein levels all increase in the 3 d after irradiation. Immunohistochemistry for Cox-1 demonstrated localization in epithelial cells of the crypt in the unirradiated mouse, and in the regenerating crypt epithelium in the irradiated mouse. We conclude that radiation injury results in increased Cox-1 levels in crypt stem cells and their progeny, and that PGE2 produced through Cox-1 promotes crypt stem cell survival and proliferation.

Alterations of the Immune System in Ulcerative Colitis and Crohn's Disease

Publisher Summary Ulcerative colitis involves only the colon; Crohns disease can involve either the colon, or the small bowel, or both. In ulcerative colitis, there are mucosal ulcers and infiltration of the mucosa and submucosa with neutrophils, macrophages, and lymphocytes. In Crohns disease, the inflammatory infiltrate frequently contains granulomas and extends through all layers of the bowel wall rather than being confined to the mucosa and submucosa as in ulcerative colitis. Both diseases have been viewed as “chronic” inflammatory diseases because of their prolonged clinical courses and because their inflammatory infiltrates contain lymphocytes and macrophages, a histologic picture that is characteristic of chronic inflammation. Although the etiologic agents are unknown, recent studies have given some insights into the mechanism for the amplification of the inflammatory response that result in the histologic and clinical changes characteristic of these diseases. It is common thought that a response to a lumenal antigen would result in more uniform inflammation, as is seen in gluten-sensitive enteropa The limitation to the intestine may reflect a defect in the regulation of the mucosal immune system but not the general immune system. The immune response seen in IBD is determined by the characteristics of the intestinal immune system, which is a unique functional immmunologic compartment in comparison with peripheral blood MNC.

Chemotactic activity in inflammatory bowel disease. Role of leukotriene B4.

An important histologic feature of inflammatory bowel disease (IBD) is infiltration of the colonic mucosa with neutrophils. To investigate the nature of the chemotactic agents responsible for this infiltration, colonic mucosa from three normals and nine patients with inflammatory bowel disease (seven ulcerative colitis, two Crohns colitis) was assayed for chemotactic activity for human neutrophilsin vitro in a Boyden chamber. There was more (>10-fold more) chemotactic activity in homogenates of inflammatory bowel disease mucosa than in homogenates of normal colonic mucosa. Analysis of the chemotactic activity in the inflammatory bowel disease mucosa revealed that most was lipid extractable. Moreover, when the lipid extract was fractionated by reverse-phase high-pressure liquid chromatography, the only fraction with significant chemotactic activity was the fraction that coeluted with leukotriene B4. The chemotactic response to IBD mucosa was blocked by anti-LTB4 antisera. The amount of chemotactic activity in lipid extracts of different inflammatory bowel disease specimens correlated well with the concentration of leukotriene B4 measured by UV absorbance (250 ng/g of mucosa). These data suggest that leukotriene B4 is an important stimulus to neutrophil chemotaxis in inflammatory bowel disease and, thus, may play a major role in the amplification of the inflammatory response in this condition.

Prostaglandin E2 reduces radiation-induced epithelial apoptosis through a mechanism involving AKT activation and bax translocation

Prostaglandin E2 (PGE2) synthesis modulates the response to radiation injury in the mouse intestinal epithelium through effects on crypt survival and apoptosis; however, the downstream signaling events have not been elucidated. WT mice receiving 16,16-dimethyl PGE2 (dmPGE2) had fewer apoptotic cells per crypt than untreated mice. Apoptosis in Bax(-/-) mice receiving 12 Gy was approximately 50% less than in WT mice, and the ability of dmPGE2 to attenuate apoptosis was lost in Bax(-/-) mice. Positional analysis revealed that apoptosis in the Bax(-/-) mice was diminished only in the bax-expressing cells of the lower crypts and that in WT mice, dmPGE2 decreased apoptosis only in the bax-expressing cells. The HCT-116 intestinal cell line and Bax(-/-) HCT-116 recapitulated the apoptotic response of the mouse small intestine with regard to irradiation and dmPGE2. Irradiation of HCT-116 cells resulted in phosphorylation of AKT that was enhanced by dmPGE2 through transactivation of the EGFR. Inhibition of AKT phosphorylation prevented the reduction of apoptosis by dmPGE2 following radiation. Transfection of HCT-116 cells with a constitutively active AKT reduced apoptosis in irradiated cells to the same extent as in nontransfected cells treated with dmPGE2. Treatment with dmPGE2 did not alter bax or bcl-x expression but suppressed bax translocation to the mitochondrial membrane. Our in vivo studies indicate that there are bax-dependent and bax-independent radiation-induced apoptosis in the intestine but that only the bax-dependent apoptosis is reduced by dmPGE2. The in vitro studies indicate that dmPGE2, most likely by signaling through the E prostaglandin receptor EP2, reduces radiation-induced apoptosis through transactivation of the EGFR and enhanced activation of AKT and that this results in reduced bax translocation to the mitochondria.