Tissue origin of peptide-responsive eicosanoid production in rabbit intestine.

Different layers of rabbit large and small intestine display different peptide sensitivity and different profiles of eicosanoid release. Isolated perfused mesenteric pedicle alone, with muscularis/submucosa or with muscularis and mucosa from normal small bowel, normal colon, or inflamed colon were stimulated with bradykinin (BK) or n-formyl-methionyl-leucyl-phenylalanine (fMLP). Released prostaglandin (PG)E2, thromboxane (Tx)B2, and leukotriene (LT)B4 were assayed using extensively validated radioimmunoassays. In rabbit colon, PGE2 arises primarily from the mesentery, while in small intestine the muscularis/mucosa releases 70-80% of the total PGE2. BK releases no significant thromboxane from healthy colon, although both muscularis/submucosa and mucosa respond in inflamed colon. In contrast, fMLP stimulates thromboxane from muscularis/submucosa and mucosa of even healthy colon, while release is greatly potentiated in inflammation. Lipoxygenase in the colon is regulated differently than cyclooxygenase; it is not stimulated by BK in either healthy or inflamed colon. fMLP releases equal amounts of LTB4 from healthy and inflamed colon, but release was primarily from healthy colonic mucosa, whereas it was distributed throughout mesenteric pedicle, muscularis, and mucosa in inflamed colon. The ability of normal colonic mucosa to release proinflammatory LTB4 in response to a chemotactic factor (fMLP) produced by enteric bacteria suggests a possible role for these compounds as a stimulus for inflammation in some patients with inflammatory bowel disease.

Chemotactic peptide-induced acute colitis in rabbits.

Bacterial chemotactic peptides from the intestinal lumen could potentially induce inflammation if they reached the mucosa. We tested several peptides chemotactic for different inflammatory cells, as well as a nonchemotactic peptide, bradykinin, for their ability to induce colitis in vivo in rabbits. These peptides were also assessed for their ability to stimulate release of the eicosanoids leukotrienes B4 and C4 and prostaglandin E2 from normal rabbit colons perfused ex vivo. Intracolonic administration of n-formyl-methionyl-leucyl-phenylalanine (chemotactic for neutrophils); its methyl ester (chemotactic for monocytes), and alanyl-glycyl-seryl-glutamic acid (chemotactic for eosinophils) all produced colitis (assessed grossly and histologically) within 4 days. Bradykinin did not induce colitis although it did release prostaglandin E2. n-Formyl-methionyl-leucyl-phenylalanine methyl ester induced the greatest degree of colitis in vivo and released prostaglandin E2 and leukotrienes ex vivo. n-Formyl-methionyl-leucyl-phenylalanine and alanyl-glycyl-seryl-glutamic acid induced comparable degrees of inflammation, but alanyl-glycyl-seryl-glutamic acid produced no eicosanoid release while n-formyl-methionyl-leucyl-phenylalanine released both prostaglandin E2 and leukotriene B4 and leukotriene C4 products from normal ex vivo perfused colons. Thus alanyl-glycyl-seryl-glutamic acid produces colitis independent of proinflammatory eicosanoids while eicosanoid release could contribute to colitis produced by n-formyl-methionyl-leucyl-phenylalanine methyl ester. This experimental model of colitis may reflect one possible etiology of inflammatory bowel disease in humans, when bacterial chemotactic peptides breach mucosal defenses in susceptible individuals.

Chemotactic peptides. Mechanisms, functions, and possible role in inflammatory bowel disease.

An important component of host defenses is the ability of inflammatory cells to detect and respond to minute concentrations of chemoattractant substances. Chemotactic peptides elaborated by both bacteria and leukocytes are the focus of this review. These peptides induce directed migration of inflammatory cells towards their targets, and stimulate biological functions including degranulation, release of oxygen radicals, phagocytosis, and eicosanoid production. Among the released eicosanoids, leukotriene B4 potentiates the leukocyte response. As with other chemotactic factors, these functions are regulated partially through differential coupling to high- and low-affinity receptors and via calcium as the second messenger. Some chemotactic peptides are elaborated by normal colonic luminal bacteria. Recent evidence demonstrates that these peptides can produce mucosal inflammation in vivo. A possible mechanism for this effect involves abnormal colonic permeability in susceptible individuals that allows bacterial chemotactic peptides access to the mucosa where they may induce inflammation. Remaining questions include the mechanism by which the mucosal barrier is breached and the role of leukotrienes in the potentiation of colonic inflammation.

Bradykinin and FMLP stimulate prostanoid production by adult rabbit colonocytes in culture.

Normal colonocytes in culture produce prostaglandins both constitutively and in response to inflammatory stimuli. These highly purified proliferative cell populations were isolated from normal adult rabbit proximal and distal colon. Basal prostaglandin production ranged from 3.4 to 11.7 ng.15 min-1 x 10(6) cells-1. Cultures were incubated at 37 degrees C in the presence or absence of bradykinin or N-formyl-methionine-leucine-phenylalanine (FMLP) over concentrations from 10(-9) to 10(-5) M. In both distal and proximal colonocytes bradykinin stimulated a dose-dependent increase in prostaglandin E2 (PGE2) and 6-keto-PGF1 alpha, the stable metabolite of prostacyclin; production peaked at 10(-6) M. Proximal colonocytes responded to FMLP with a bell-shaped curve, with maximal stimulation of both PGE2 and 6-keto-PGF1 alpha occurring at 10(-8) M. Distal colonocytes responded variably to FMLP. Arachidonic acid also stimulated prostanoid production in a concentration-dependent manner, with maximal stimulation occurring at 100 microM. The full synthetic profile of prostanoid production was determined by labeling with [14C]arachidonic acid and by analyzing metabolites using radiochromatography on reverse-phase high-pressure liquid chromatography. Only PGE2 and 6-keto-PGF1 alpha were detected. A similar profile of labeled metabolites occurred when colonocytes were prelabeled with [14C]arachidonic acid and stimulated with bradykinin or FMLP. The degradative capacity of the colonocytes appeared very low. Colonocyte production of protective prostaglandins in response to luminal or other inflammatory stimuli may serve as a mucosal defense mechanism. Prostanoids so produced may also modulate the functions of colonocytes, surrounding cells, or both.

Eicosanoid production by a differentiated canine colonic epithelial cell line, VNCC.

BACKGROUND/AIMS: The lack of pure, proliferative, but not transformed intestinal epithelial cells has impeded progress in understanding their role in chronic intestinal inflammation. To clarify that role, the present study characterized the epithelial cell line VNCC, derived from normal adult dog distal colon. METHODS: Cells were cultured on plastic and permeable supports for analysis of eicosanoid production (by radioimmunoassay and high-performance liquid chromatography) and transport characteristics (by Ussing chamber short-circuit determinations). RESULTS: In culture, VNCC formed confluent monolayers and domes, suggesting formation of tight junctions and active solute absorption. When cultured on permeable supports, VNCC developed modest, but variable, transepithelial resistances (563 +/- 94 omega/cm2) with a spontaneous short-circuit current of 5.0 +/- 0.4 microA/cm2. Forskolin caused a prolonged increase in the short-circuit current, inhibited by amiloride but not bumetanide, suggesting that VNCC display 5'-cyclic adenosine monophosphate-stimulated sodium absorption. VNCC incubated with arachidonic acid released a variety of eicosanoids including 6-keto-prostaglandin (PG)F1 alpha, PGE2, thromboxane B2, and PGF2 alpha, but no hydroxyarachidonate metabolites. Bradykinin stimulated VNCC eicosanoid release. CONCLUSIONS: The ability of VNCC to divide and differentiate in culture, to form polarized monolayers capable of active sodium absorption, and to respond to inflammatory mediators with eicosanoid release makes them a unique tool for the study of the interactions of inflammation on colonocyte function.

Chemotactic peptide stimulation of leukotrienes from healthy and inflamed rabbit colons.

Prostaglandins, thromboxanes and leukotrienes are increased in human and experimental colitis. To evaluate the biosynthesis of these eicosanoids, colon inflammation was induced in rabbits by formalin enema followed by i.v. immune complexes, and the distal colon was perfused ex vivo. Bradykinin increased synthesis of prostaglandin E2 and thromboxane B2 more from colitis than from control colons (both P less than .001) but had no effect on leukotriene synthesis. The inflammatory cell agonist N-formylmethionyl-leucyl-phenylalanine (30 ng) also induced greater synthesis of prostaglandin E2 (70 +/- 13 vs. 14 +/- 6) and thromboxane B2 (84 +/- 22 vs. 20 +/- 11) from colitis than from control colons (P less than .01), but leukotriene B4 (416 +/- 68 vs. 438 +/- 128 ng/5 min) and leukotriene C4 (171 +/- 50 vs. 203 +/- 25 ng/5 min) synthesis were greatly augmented in both colitis and control colons. In vitro incubations demonstrated similar dose-dependent stimulation of leukotriene B4 by N-formylmethionyl-leucyl-phenylalanine in both colitis and control colons. These studies demonstrate that healthy colon tissue as well as colitis tissue can produce proinflammatory leukotrienes in response to bacterial peptides. Leukotriene production may contribute to the induction or mediation of colon inflammation.

Depression of lymphocyte traffic in sheep by anaesthesia and associated changes in efferent-lymph PGE2 and antibody levels.

General anaesthesia of sheep with ketamine and xylazine has been found to produce a profound and prolonged depression in lymphocyte traffic through primary peripheral lymph nodes, as mirrored in the output of lymphocytes into efferent lymph. In this study, the depression has been found to be associated with a marked and sustained elevation in prostaglandin E2 (PGE2) levels in efferent lymph. The degree and duration of lymphocyte output depression was found to be modulated (diminished), both in degree and duration, by study-node drainage-area stimulation from prior surgery, inflammation or bacterial immunization. Even when the anaesthesia-associated lymphocyte-output depression was modulated by drainage-area inflammation, the period of lymphocyte-output depression was correlated still with elevated levels of PGE2 in efferent lymph. When drainage-area stimulation was produced by bacterial immunization (killed Salmonella muenchen), the anaesthesia-associated depression in lymphocyte output into efferent lymph (small as well as blast) was accompanied by a depression in antibody output into efferent lymph.

Effects of cyclooxygenase and lipoxygenase inhibition on eicosanoids and healing of acetic acid colitis in rats.

Both cyclooxygenase products, such as prostaglandin (PG) E2, and lipoxygenase products, such as leukotriene (LT) B4, are increased in colitis and have potent proinflammatory actions. We studied effects of specific inhibitors of cyclooxygenase and 5-lipoxygenase on the healing of acetic acid colitis in rats. Acetic acid colitis was induced 24 hr before enzyme inhibition began. Four days after induction of colitis, the area of gross colonic mucosal damage was determined by image analysis. Eicosanoid content in the intestinal lumen was quantitated by radioimmunoassay following chromatographic purification. Under these conditions, indomethacin significantly retarded the healing of colonic lesions and inhibited PGE2 by > 90% compared to placebo-treated colitis rats. AA861 had no effect on the healing of lesions, although > 75% inhibition of leukotriene synthesis was demonstrated. These results suggest that inhibition of endogenous colonic prostaglandins can impair healing mechanisms in acute colitis even after inflammation has developed. In contrast, inhibition of leukotriene synthesis did not affect healing.