- Email: [email protected]
EICOSANOIDS AND INFLAMMATORY BOWEL DISEASE
NSAIDS, EICOSANOIDS, AND THE GASTROENTERIC TRACT
0889-8553/96 $0.00
+ .20
EICOSANOIDS AND INFLAMMATORY BOWEL DISEASE Vincent W. Yang, MD, PhD
Inflammatory bowel disease (IBD) is a chronic, debilitating illness of the gastrointestinal tract afflicting individuals usually between the ages of 16 and 40. The two primary forms of idiopathic intestinal inflammation, ulcerative colitis and Crohn's disease, cause significant morbidity in the young adult population. This article reviews aspects of the inflammatory response in IBD that involve metabolism of arachidonic acid. For an overview of the pathogenesis and clinical features of the disease, readers are referred to the excellent review by P o d ~ l s k y .51~ ~ , PATHOGENESIS .OF INFLAMMATORY BOWEL DISEASE
Although the exact cause of IBD remains unknown, the pathogenesis of intestinal inflammation has been postulated to include two stages. In the first stage, an initial insulting event, precipitated by an unknown factor (or factors), occurs in the intestinal mucosa and leads to tissue damage. These insults may be derived from luminal stimuli such as dietary antigens or persistently infectious microbial agents. An intrinsic defect in the mucosal barrier may also be present that permits access for the offending agents. Alternatively the regulation of the mucosal immune system may be inherently abnormal, leading to an exaggerated
From the Departments of Medicine and Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland
GASTROENTEROLOGY CLINICS OF NORTH AMERICA VOLUME 25 * NUMBER 2 * JUNE 1996
317
318
YANG
response to luminal factors or to a defective feedback system that normally down-regulates the mucosal response to luminal factors (Fig. 1). The second stage of the disease involves an amplification of the inflammatory response. Several types of cells, including lymphocytes, neutrophils, macrophages, and mast cells, are attracted to the site of the initial injury. Products of these immune and inflammatory cells include a wide variety of mediators, such as cytokines, eicosanoids, free radicals, and components of the complement pathway. Amplification of the inflammatory response is important in the pathogenesis of IBD for two reasons. First, amplification of the inflammatory response, not the initiating event, causes the tissue destruction observed in IBD. Second, drugs used to treat IBD are designed for their ability either to modulate effects of the soluble mediators or to attenuate the cells from which these mediators are derived. Because the initiating event of IBD remains unknown, pharmacologic advances in the treatment of the disease are
Stimulus
Inflammatory Response Antigen Nonspecific
Immune Response Antigen Specific
Sustained Inflammatory Mucosal Destruction
1
IBD
Figure 1, Potential pathogenic mechanisms of inflammatory bowel disease (IBD). The lumen of the intestine is confronted by a variety of substances (e.g., dietary antigens and microbial agents) that elicit an antigen-specific immune response by the mucosal immune system or an antigenic-nonspecific inflammatory response by the epithelium. This process is normally balanced by feedback mechanisms that down-regulate the immune and inflammatory responses, leading to elimination of stimuli that breach the mucosal barrier. In the presence of disease, one or several fundamental alterations in the mucosal defense mechanisms may contribute to sustained and enhanced inflammation. Alternatively, regulation of immune and inflammatory responses may be intrinsically abnormal, with either exaggerated activations to normal stimuli (solid arrows) or defective feedback mechanisms for down-regulation (dotted arrows). These alterations result in sustained tissue destruction that leads to IBD. (Adapted from Podolsky DK: Inflammatory bowel disease. N Engl J Med 325932, 1991; 0 1991 with permission of the Massachusetts Medical Society.)
EICOSANOIDS AND INFLAMMATORY BOWEL DISEASE
319
more likely to result from an intervention of the inflammatory response than from the treatment of the cause of IBD. ARACHIDONIC ACID METABOLISM AND THE INFLAMMATORY RESPONSE Arachidonic acid (AA), a 20-carbon unsaturated fatty acid, is the source of a class of soluble inflammatory mediators called eicosanoids. AA is available from extracellular sources or can be released from within the cell. The phospholipid fraction in the cell constitutes the major source of AA. At least three enzymatic pathways exist for AA release from cellular phospholipids (Fig. 2): (1) direct action by phospholipase A, (PLA,); (2) combined action of phospholipase C and diacylglycerol (DAG) lipase or of phospholipase C, DAG kinase, and PLA,; and (3) combined action of phospholipase D and PLA,. Evidence suggests that an AA-specific PLA, is responsible for AA release from the membrane, which leads to eicosanoid formation. Further metabolism of AA by cellular components is mediated by one of three enzymatic pathways: the cyclooxygenase, lipoxygenase, and monooxygenase pathways, each giving rise to a distinct array of products. This section reviews the formation of eicosanoids by the cyclooxygenase and lipoxygenase pathways. In addition, the biologic effects of these lipid mediators are reviewed. For a detailed summary of AA metabolism, readers are referred to several excellent review articles.12,19, 22,30, 31 Prostaglandins and the Cyclooxygenase Pathway Cyclooxygenase (prostaglandin endoperoxide synthase or prostaglandin synthase) catalyzes the conversion of AA to prostaglandin endoperoxides, which are the precursors to a series of biologic compounds that include thromboxanes, prostacyclin, and several prostaglandins, including PGD,, PGE,, and PGF,,. Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit cyclooxygenase activity. In particular, aspirin irreversibly inactivates the enzyme by acetylation, which accounts for the therapeutic effect of this drug. To date, two different isoforms of cyclooxygenase have been identified: prostaglandin synthase-1 (PGS-1) and prostaglandin synthase-2 (PGS-2).The regulation of these two isozymes and their respective genes ~ ) . is constitutively are quite different (reviewed in H e r ~ c h m a n ~PGS-1 expressed, whereas PGS-2 is highly sensitive to extracellular stimuli. PGS-2 messenger RNA (mRNA) is rapidly induced in cells treated with serum, growth factors, forskolin, or phorbol ester.34,36 Moreover, the induction of the PGS-2 gene in response to stimulation is inhibited by the concomitant treatment of cells with glucocorticoids, whereas the PGS-1 gene is not inhibited by g l ~ ~ o ~ o r t i ~ 43 o iEvidence d s . ~ ~ ~suggests that PGS-1 has a housekeeping function, producing prostaglandins neces-
320
YANG
Phospholipid
I
Phospholipase A2
/
AA
Phospholipase D
Phosphonpase C
1
Phosphatidic Acid
I
Diacylglycerol I
DAG Lipase DAG Kinase
f
AA
+
Phospholipase A2
\
AA Phosphatidic Acid
I
+
Phospholipase A2 AA Figure 2. Enzymatic pathways for the formation of arachidonic acid (AA). Three predominant mechanisms for generating AA from membrane phospholipid have been elucidated in mammalian cells: (1) phospholipaseA, acting chiefly on phosphatidylcholineand phosphatidylethanolamine; (2) phospholipase C, acting principally on phosphatidylinositol to yield diacylglycerol (DAG), which in turn is a substrate for DAG lipase or the sequential actions by a kinase and a specific phospholipase A;, (3) phospholipase D, acting principally on phosphatidylcholine, combined with the subsequent action of another phospholipase A., (From Holtzman MJ: Arachidonic acid metabolism: Implications of biological chemistry for lung function and disease. Am Rev Respir Dis 143:193, 1991; with permission.)
sary for normal cellular processes, whereas PGS-2 is responsible for the inflammatory and mitogenic responses, producing prostaglandins involved in inflammation and growth regulation. Studies have identified certain NSAIDs that exhibit differential inhibitory effects on the activity of the two PGS isozyrnes.l3,68 These studies may lead to the eventual identification of NSAIDs that reduce inflammation but spare those prostaglandins required for normal cell physiology, thus reducing the untoward side effects associated with most NSAIDs. The physiologic effects of the various PGS products vary, depending on their sites of action and on the cells from which the products are derived. Thromboxane A, (TXA,), the predominant product of AA metabolism of platelets, is a potent platelet aggregating agent as well as a
EICOSANOIDS AND INFLAMMATORY BOWEL DISEASE
321
vasoconstrictor and bronchoconstrictor. In contrast, prostacyclin (PGI,), derived primarily from endothelial cells, is a potent inhibitor of platelet aggregation and, in some vascular systems, serves as a vasodilator and antagonizes the formation of TXA,. The prostaglandin synthases D, E, and F are responsible for the conversion of PGH, to prostaglandins D, E, and F. PGD, is formed from mast cells on antigen binding to IgE receptors and is a potent bronchoconstrictor, as is PGF,,. PGE, is a vasodilator and bronchodilator. Human intestinal epithelial cells have been shown to produce both PGE, and PGF,, in response to inflammatory stimuli, and these mediators act to increase chloride secretion.55 Importantly, intestinal epithelial cells interface with the luminal environment from which inflammatory stimuli are potentially originated. Leukotrienes and the 5-Lipoxygenase Pathway An alternate pathway of AA oxidation is mediated by the lipoxygenases. There are three major lipoxygenases: 5, 12, and 15, named for their ability to add a molecular oxygen at a specific carbon of AA.76The products of 5-lipoxygenase include leukotrienes, which constitute an important class of inflammatory mediators. This section reviews the biochemistry of the 5-lipoxygenase pathway and its relationship to the inflammatory response. The biochemical pathway leading to leukotriene formation has been 60, 61, 5-Lipoxygenase catalyzes the formation of well characteri~ed.~~, LTA, and 5(S)-hydroxy-6,8,11,14-eicosatetraenoicacid (5-HETE) from AA through the intermediary molecule of 5(S)-hydroperoxy-6,8,11,14eicosatetraenoic acid (5-HPETE) (Fig. 3). LTA, can be metabolized by two specific enzymes to produce biologically active compounds. The first enzyme, leukotriene A, hydrolase, catalyzes the hydrolysis of LTA, to LTB,. The second enzyme, leukotriene C, synthase, catalyzes the conjugation of LTA, to the tripeptide glutathione, leading to the formation of LTC,. Further metabolism of LTC, occurs when its glutamyl residue is cleaved by a membrane-bound gamma-glutamyl transferase to produce LTD,, which is further metabolized to LTE, by the action of a membrane-bound dipeptidase.
5-Lipoxygenase and 5-Lipoxygenase-Activating Protein Because 5-lipoxygenase plays a central role in the biosynthesis of leukotrienes, considerable effort has been devoted to the characterization of this enzyme (reviewed by Ford-Hutchinson and colleague^^^). It is a cytosolic enzyme that requires Ca2 and adenosine triphosphate (ATP) for maximal activity.59The human enzyme has been purified to homogeneity from peripheral blood leukocytes and is a 78-kilodalton (kd) protein. When neutrophils are activated by the calcium ionophore A23187, 5-lipoxygenase, normally cytosolic, is translocated to a membrane fraction, where it gains access to its substrate, AA.58 These observations +
322
YANG
Arachidonic Acid
5i-HrTEJ
5-llpoxygenase FLAP
5-HETE
Leukotrlene A4
Leukotriene A4 Hydrolase
Leukotriene C4 Synthase
l + l
Leukotriene B4
1
Receptor Activation
c
Leukotriene C4
i
Gamma-glutamyl Transferase
Leukotrlene D4
I
Dipeptidase
Leukotriene E4
c
Receptor Activation
Figure 3. Enzymatic pathways of leukotriene biosynthesis. Key enzymes (italics) lead to the formation of various metabolites.
suggest the presence of a defined regulatory process for the activation of 5-lipoxygenase within the cell. The key to understanding 5-lipoxygenase activation came from the study of a specific inhibitor of leukotriene biosynthesis, MK-886.26This compound has no effect on the synthesis of cyclooxygenase, 12-lipoxygenase, or 15-lipoxygenase-derivedproducts of AA. Surprisingly, MK886 inhibits only the synthesis of leukotrienes in intact human neutrophils stimulated with calcium ionophore but has no effect on the 5lipoxygenase enzyme itself, either in broken cell preparations or in purified enzyme preparations.26,57 These findings suggest that additional cellular components are required for the cellular activation of 5-lipoxygenase. A protein was subsequently identified and shown to be the target of MK-886.@This 18-kDa protein, called 5-lipoxygenase-activating
EICOSANOIDS AND INFLAMMATORY BOWEL DISEASE
323
protein (FLAP), was purified to homogeneity, and its full-length cDNA cloned and ~equenced.'~ Only when both 5-lipoxygenase and FLAP are expressed in the same cell can cellular leukotriene synthesis be observed following ionophore challenge, and this synthesis is inhibited by MK886.15 FLAP, a membrane protein, is also involved in the translocation of 5-lipoxygenase from the cytosol to the membrane following exposure to ionophore, resulting in the activation of 5-lipoxygenase. This translocation, together with the accompanying leukotriene synthesis, is inhibited by MK-886. Evidence suggests that FLAP is an AA-binding protein that facilitates the transfer of AA to activated 5-lipo~ygenase.~~ The current hypothesis on the mechanism of action of MK-886 is that its binding site on FLAP coincides with the AA-binding site and that the interaction of MK-886 and FLAP impedes the proper delivery of AA to 5-lipoxygena ~ e Figure . ~ ~ 4 illustrates the complex pathway of activation of the 5lipoxygenase/FLAP system and the mechanism by which MK-886 inhibits LT formation. Another interesting observation depicted in Figure 4 is the subcellular localization of 5-lipoxygenase and FLAP. In activated leukocytes, both 5-lipoxygenase and FLAP are localized to the lumen of the nuclear envelope and endoplasmic reticulum.75The distribution of FLAP in resting cells is identical to that in activated cells, but 5-lipoxygenase is not detected in any specific cellular compartment. Hence, in unstimulated cells, FLAP is present in the membrane, and following stimulation 5-lipoxygenase moves to the same membrane site as FLAP. Biologic Effects of Leukotrienes The biologic effects of leukotrienes are numerous. Although all leukotrienes are primarily products of leukocytes, including neutrophils, mast cells, and macrophages, LTB4 alone acts as a calcium ionophore, leading to further stimulation of 5-lipoxygenase activity.62In addition, LTB, is a potent chemotactic substance for leukocytes22and has been shown to influence lymphocyte activity.20In contrast, the peptidyl derivatives of leukotrienes (LTC,, LTD4, and LTE,) primarily affect smooth muscle contractility and other cells with contractile capacity. For example, on a molar basis, LTC, and LTD, are 1000 times more potent than histamine in inducing constriction of the airway.17Many other actions of leukotrienes have been described and can be found in the review articles by Samuelsson and colleagues,61 Lewis and colleagues,4° and Henderson.29 Although leukotrienes are primarily produced by the inflammatory cells, whether intestinal epithelial cells possess the ability to synthesize leukotrienes has only recently been explored. Immunohistochemical examination of the porcine ileum with a polyclonal 5-lipoxygenase antibody indicates no immunoreactivity in the e p i t h e l i ~ m In . ~ ~contrast, isolated rat colonic crypt epithelial cells can produce leukotrienes.10 Furthermore, several epithelial cell lines derived from human small and large intestines are also capable of synthesizing leuk~trienes.~,67 The
324
YANG
+Cell Activation
+MK-886
fiT
+Cell Activation
UlFLAP * 5 - L 0 A
oCPLA2
* MK-886
B
Figure 4. A model for the cellular activation of 5-lipoxygenase (5-LO) and proposed mechanism of action of the leukotriene inhibitor, MK-886. A, Activation of a cell surface receptor by a given stimulus leads to the mobilization of intracellular Ca2+,which results in the translocation of the cytosolic phospholipaseA2 (cPLA,) to the plasma membrane where it liberates AA from membrane phospholipids. Free AA is then available to an activated 5LO, which in response to the increase in intracellular Ca2+ is translocated to the nuclear membrane where FLAP resides. Evidence suggests that FLAP is an AA-binding protein, which presumably facilitates the transfer of this substrate to the activated 5-LO to produce leukotrienes. 8,The interaction of MK-886 with FLAP interferes with the proper translocation of 5-LO to the membrane in response to a stimulus, thus resulting in the inhibition of leukotriene synthesis. (From Ford-HutchinsonAW, Gresser M, Young RN: 5-Lipoxygenase. Annu Rev Biochem 63:413, 1994; 0 1994 with permission of Annual Reviews, Inc.)
EICOSANOIDS AND INFLAMMATORY BOWEL DISEASE
325
human colonic epithelial cell line, HT29-C1,32for example, contains the mRNA for both 5-lipoxygenase and FLAP and possesses the capacity to synthesize LTB, and 5-HETE under certain conditions? Thus, the intestinal epithelium can produce limited quantities of leukotrienes, which may be implicated in intestinal cell physiology and potentially in intestinal inflammation. EICOSANOIDS AND INFLAMMATORY BOWEL DISEASE
As a result of the amplification of the inflammatory process, soluble mediators such as eicosanoids play a significant role in the pathogenesis of IBD. Numerous studies have demonstrated a relationship between elevated eicosanoid levels in the inflamed intestine and disease severity (reviewed in Donowitz,16 Rask-Madsen:'j and S t e n ~ o n ~This ~ ) . section summarizes some of the changes in AA metabolism observed in IBD. The ability of the human colon to produce both cyclooxygenase and I* When homogenates lipoxygenase metabolites has been dem~nstrated.~, of human colonic mucosa are incubated with radiolabeled AA, the predominant cyclooxygenase products include PGE2, PGF2,, PGD2, and TxB2. 12-HETE and 15-HETE are the major lipoxygenase products formed. LTB., and the peptidyl derivatives of leukotrienes are also present but in lesser amounts than the prostaglandins. In active IBD, high levels of prostaglandins are found in the rectal mucosa and urine of patients with active disease.@The levels of prostaglandins decline in IBD patients treated with either corticosteroids or sulfasalazine. By contrast, in those with inactive IBD, the levels of prostaglandins are not significantly different from those of normal control subjects. The pathologic effects of elevated prostaglandin levels in IBD are more difficult to determine, however. Although prostaglandin levels in mucosal specimens of IBD patients decline when they are treated with NSAIDs, there is no clinical improvement.2sIn fact, some evidence indicates that NSAIDs actually increase the severity of IBD. These findings suggest that prostaglandins may not be the important mediators of the inflammation observed in IBD and that the mechanism of action of corticosteroids and sulfasalazine may not be related to the inhibition of prostaglandin synthesis. The pathologic role of the lipoxygenase products in IBD is better documented. Several studies demonstrate that colonic mucosa from patients with active IBD synthesize significantly higher amounts of lipoxygenase products, including LTB, and 5-HETE, compared with normal control tissue.6,7, 65 Likewise, LTB, levels are much higher in dialysates from dialysis bags placed in the rectums of patients with ulcerative colitis than in controls.38Also, increased synthesis of LTB, and the peptidyl leukotrienes is observed in rectal biopsy specimens from IBD patients that are stimulated with the calcium ionophore, A23187.49Similarly, in various animal models of colitis, the synthesis of leukotrienes
326
YANG
in the inflamed mucosa is elevated.53* 64, 78 Inhibition of leukotriene synthesis by specific drugs decreases inflammation and accelerates healing in a rat model of IBD.” In particular, corticosteroids and sulfasalazine, two drugs known to be effective in the treatment of IBD, decrease colonic leukotriene formation.’, 37, 38 These findings indicate the importance of the lipoxygenase products of AA metabolism in the pathogenesis of IBD. The exact role played by the lipoxygenase products in IBD remains unclear. LTB,, as noted, is a potent chemotactic and aggregating substance for neutrophils and mononuclear cells.2oWhen homogenates of colonic mucosa are assayed for the chemotactic activity for human neutrophils, IBD patients have a 10-fold greater activity than normal control^.^' When the homogenates are fractionated to identify the substance responsible for the increased chemotactic activity, the fraction with significant activity coelutes with LTB,. Furthermore, this chemotactic response is abolished by anti-LTB, antisera. These studies indicate that LTB, within the IBD mucosa is responsible for the promulgation of the chemotactic reaction and the subsequent recruitment of circulating neutrophils to the inflamed mucosa. Thus, the enhanced synthesis of LTB, may account, in part, for the amplification of the inflammatory response in IBD. Aside from the chemotactic and proinflammatory natures of LTB,, products of the lipoxygenase and cyclooxygenase pathways may contribute directly to the diarrhea seen in IBD patients. Various AA metabolites may alter different aspects of intestinal fluid regulation, including absorption and secretion of solutes and intestinal motility. PGE, decreases active sodium and chloride absorption and increases chloride secretion in both the small intestine and the TxA, has also been shown to cause colonic secretion. Products of the lipoxygenase pathway alter active intestinal electrolyte transport as well, although this effect is not uniform among all products. For example, 5-HETE and 5-HPETE cause chloride secretion in rabbit distal colon at low concentrations but not in rabbit ileum.45In contrast, LTB, and LTC, do not alter either colonic or ileal electrolyte transport. The effects of lipoxygenase products on intestinal motility have not been clearly demonstrated, although the myotrophic effects of several leukotrienes may potentially influence motility, leading to diarrheal formation. TREATING INFLAMMATORY BOWEL DISEASE WITH DRUGS TARGETED TOWARD ARACHIDONIC ACID METABOLISM
Based on the preceding discussion, a number of AA metabolites seem important in the amplification of the inflammatory process observed in IBD. Therefore, drugs interfering with the metabolism of AA and leading to the inhibition of eicosanoid formation are potential therapeutic agents for IBD. One of the many functions of corticosteroids, commonly used in IBD, is the stabilization of membrane phospholipase
EICOSANOIDS AND INFLAMMATORY BOWEL DISEASE
327
A, activity, making the substrate AA less available for leukotriene synthesis (see Fig. 2). Sulfasalazine, another frequently used drug in IBD treatment, is a second example. Sulfasalazine contains two moieties: sulfapyridine, a sulfonamide, and 5-aminosalicylate (5-ASA).Sulfasalazine inhibits 5-lipoxygenase activity and lowers leukotriene levels in IBD patients.4s,70 The 5-ASA moiety of sulfasalazine is considered the active component required for 5-lipoxygenase inhibition. 5-ASA is now available as both an oral and a topical agent for IBD treatment.71,74
5-Lipoxygenase Inhibitors
Pharmacologic evaluation of 5-lipoxygenase inhibition by many new drugs for the treatment of IBD and other inflammatory disorders is These drugs can be divided into two major under active classes, depending on their mode of action: (1) direct inhibitors of 5lipoxygenase activity and (2) inhibitors of FLAP activity. Zileuton (A64077), a benzothiophene hydroxyurea, is an example of a direct, orally active 5-lipoxygenase i n h i b i t ~ rIt. ~compares favorably with sulfasalazine in attenuating the lesions seen in experimentally induced colitis in ratsn In initial clinical trials, oral zileuton significantly reduces LTB, levels in rectal dialysates from patients with active ulcerative colitis39and improves the symptom and sigmoidoscopy scores in these patients when compared with placebo control.8These patients have not had significant side effects. A long-term controlled, double-blind, and dose-range trial of the efficacy and safety of zileuton is currently being conducted. Newer, longer-acting 5-lipoxygenase inhibitors with improved bioavailability are also currently under investigation in experimental animal^.^
5-Lipoxygenase-Activating Protein Inhibitors
In contrast to zileuton, MK-886 (L-663,536) inhibits 5-lipoxygenase activity by interacting with FLAP (see Fig. 4). This drug is a potent inhibitor of leukotriene biosynthesis in intact human polymorphonuclear (PMN) leukocytes. MK-886 appears effective in controlling the inflammatory response produced in several experimentally induced inflammatory animal models.26This drug has excellent bioavailability, prolonged duration of action, and lack of toxicity in initial animal studies.26In a rat model of chronic colitis, MK-886 reduced colonic LTB, synthesis and accelerated healing.7zIn a human trial, MK-886 significantly reduced calcium ionophore-stimulated LTB, biosynthesis ex vivo in healthy male subjects receiving the drug and was well tolerated.'l Moreover, MK-886 blocks allergen-induced airway responses in atopic patients challenged with histamine.25Other potent FLAP inhibitors have been
328
YANG
Potential Treatment Strategies of Inflammatory Bowel Disease That Target the Leukotriene Pathway of Arachidonic Acid Metabolism In addition to inhibiting leukotriene formation by targeting 5-lipoxygenase, many other pharmacologic strategies for interfering with AA metabolism can be exploited. Figure 5 illustrates several potential points of interference with the leukotriene pathway of AA metabolism that would be explored for therapeutic purposes. Corticosteroids stabilize membrane phospholipids and inhibit phospholipase A, activity, resulting in less AA available for eicosanoid formation. Other specific inhibitors of phospholipase A, that lead to decreased AA release from membrane phospholipids are also potential candidates. N-3 fatty acids, such as eicosapentaenoic acid (EPA), a component of fish oil lipids, may lower normal stores of AA, compete with AA for oxygenation, and potentially alter substrate milieu to influence AA uptake and availability. Dietary supplementation with EPA markedly attenuates leukocyte generation of leukotrienes and the late asthmatic response to allergens? Clinical trials of IBD patients with dietary EPA supplementation is feasible. Lastly, many compounds that are leukotriene analogs, thus competitive inhibitors of leukotriene receptors, have been developed. These agents can be designed to target individual leukotriene receptors, therefore blocking only the response of the individual cell type con-
n-3 Fatty Acids
Membrane Phospholipids
'
I*
PLA2
57
Corticosteroids
PLA2 Inhibitors
Arachidonic Acid 5-LO
5-LO and FLAP lnhibitors
Leukotrienes
1
Membrane Receptors
Leukotriene Receptor Antagonists
Cells
Figure 5. Pharmacologic strategies for the inhibition of AA metabolism. Potential sites of inhibition of the leukotriene pathway of AA metabolism are categorized. Examples include (1) dietary therapy with n-3 fatty acids such as EPA; (2) phospholipase A2 (PLA,) inhibition by corticosteroids or specific PLA, inhibitors; (3) 5-LO/FLAP inhibition by inhibitors such as sulfasalazine, zileuton, and MK-886; and (4) direct leukotriene receptor antagonists. (From Holtzman MJ: Arachidonic acid metabolism: Implications of biological chemistry for lung function and disease. Am Rev Respir Dis 143:198, 1991; with permission.)
EICOSANOIDS AND INFLAMMATORY BOWEL DISEASE
329
taining that specific receptor. Examples of specific leukotrienes receptor antagonists currently being tested in animals or humans include the LTB, receptor antagonist, SC-41930,24and the LTD,/LTE, receptor antagonist, SR 2640.47 SUMMARY
IBD is a disease of unknown cause that involves an amplification of the inflammatory response in the intestinal mucosa. Although not the only offending agents leading to the disease, eicosanoids, the collective group of AA metabolites, play a significant role in the pathogenesis of IBD. This article reviewed the biochemical pathways of eicosanoid formation and the clinical relevance of eicosanoids to IBD. Potential strategies designed to interfere with various aspects of AA metabolism were also outlined. Further clinical trials of newer compounds may soon prove them effective in the management of IBD. References 1. Allgayer H, Stenson WF: A comparison of effects of sulfasalazine and its metabolites on the metabolism of endogenous vs. exogenous arachidonic acid. Immunopharmacology 15:39, 1988 2. Arm JP, Horton CE, Spur BW, et al: The effects of dietary supplementation with fish oil lipids on airway response to inhaled allergen in bronchial asthma. Am Rev Respir Dis 139:1395, 1989 3. Batt DG: 5-Lipoxygenase inhibitors and their anti-inflammatory activities. Prog Med Chem 291, 1992 4. Bell RL, Bouska.J, Young PR, et al: The properties of A-69412: A small hydrophilic 5lipoxygenase inhibitor. Agents Actions 38:178, 1993 5. Bell RL, Young PR, Albert D, et al: The discovery and development of zileuton: An orally active 5-lipoxygenase inhibitor. Int J Immunopharmacol 14:505, 1992 6. Beyer C, Maier KE, Klotz U: Enhanced synthesis of hydroxyeicosatetraenoic acids by colonic mucosa in chronic inflammatory bowel disease. In MacDermott RP (ed): Inflammatory Bowel Disease: Current Status and Future Approach. New York, Elsevier Science Publishers, 1988, p 291-296 7. Boughton-Smith NK, Hawkey CJ, Whittle BJR Biosynthesis of lipoxygenase and cyclooxygenase products from [14C]-arachidonicacid by human colonic mucosa. Gut 24:1176, 1983 8. Collawn C, Rubin P, Perez N, et al: Phase I1 study of the safety and efficacy of a 5lipoxygenase inhibitor in patients with ulcerative colitis. Am J Gastroenterol 87:342, 1992 9. Cortese JF, Spannhake EW, Eisinger W, et al: The 5-lipoxygenase pathway in cultured human intestinal epithelial cells. Prostaglandins 49:155, 1995 10. Craven PA, DeRubertis FR Profiles of eicosanoid production by superficial and proliferative colonic epithelial cells and sub-epithelial colonic tissue. Prostaglandins 32:387, 1986 11. Depre M, Friedman B, Tanaka W, et al: Biochemical activity, pharmacokinetics, and tolerability of MK-886, a leukotriene biosynthesis inhibitor, in humans. Clin Pharmacol Ther 53:602, 1993 12. DeWitt D L Prostaglandin endoperoxide synthase: Regulation of enzyme expression. Biochim Biophys Acta 1083:121, 1991
330
YANG
13. DeWitt DL, Meade EA, Smith WL: PGH synthase isoenzyme selectivity: The potential for safer nonsteroidal antiinflammatory drugs. Am J Med 95(suppl2A):40S, 1993 14. Dias VC, Wallace JL, Parsons HG: Modulation of cellular phospholipid fatty acids and leukotriene B, synthesis in the human intestinal cell (CaCo-2). Gut 33:622, 1991 15. Dixon RAF, Diehl RE, Opas E, et al: Requirement of a 5-lipoxygenase-activatingprotein for leukotriene synthesis. Nature 343282, 1990 16. Donowitz M: Arachidonic acid metabolites and their role in inflammatory bowel disease: An update requiring addition of a pathway. Gastroenterology 88:580, 1985 17. Drazen JM, Austen KF: Leukotrienes and airway responses. Am Rev Respir Dis 136:985, 1987 18. Dreyling KW, Hoppe U, Peskar BA, et al: Leukotriene synthesis by human gastrointestinal tissues. Biochim Biophys Acta 878:184, 1986 19. Feuerstein G, Hallenbeck JM: Leukotrienes in health and disease. FASEB J 1:186, 1987 20. Ford-Hutchinson A W Leukotriene B, in inflammation. Crit Rev Immunol l O : l , 1990 21. Ford-Hutchinson AW: Regulation of leukotriene biosynthesis. Cancer Metastasis Rev 13257, 1994 22. Ford-Hutchinson AW, Bray MA, Doig MV, et al: Leukotriene B,, a potent chernokinetic and aggregating substance released from polymorphonuclear leukocytes. Nature 286264, 1980 23. Ford-Hutchinson AW, Gresser M, Young RN: 5-Lipoxygenase. Annu Rev Biochem 63:383, 1994 24. Fretland DJ, Widornski D, Tsai BS, et al: Effect of the leukotriene B, receptor antagonist SC-41930 on colonic inflammation in rat, guinea pig and rabbit. J Pharrnacol Exp Ther 255572, 1990 25. Friedman BA, Be1 EH, Buntinx A, et al: Oral leukotriene inhibitor (MK-886) blocks allergen-induced airway responses. Am Rev Respir Dis 147839, 1993 26. Gillard J, Ford-Hutchinson AW, Chan C, et a1 L-663,536 (MK-886), a novel, orally active leukotriene biosynthesis inhibitor. Can J Physiol Pharmacol 67456, 1989 27. Goetzl EJ, Pickett WC: The human PMN leukocyte chemotactic activity of complex hydroxy-eicosatetraenoic acids (HETEs). J Immunol 1251789, 1980 28. Gould SR, Brash AR, Conolly ME, et al: Studies of prostaglandins and sulphasalazine in ulcerative colitis. Prostaglandins Med 6:165, 1981 29. Henderson W R The role of leukotrienes in inflammation. Ann Intern Med 121:684, 1994 30. Herschman HR: Regulation of prostaglandin synthase-1 and prostaglandin synthase2. Cancer Metastasis Rev 13241, 1994 31. Holtzman MJ: Arachidonic acid metabolism: Implications of biological chemistry for lung function and disease. Am Rev Respir Dis 143:188, 1991 32. Huet C, Sahuquillo-Merino C, Coudrier E, et al: Absorptive and mucus-secreting subclones isolated from a multipotent intestinal cell line (HT-29) provide new models for cell polarity and terminal differentiation. J Cell Biol 105:345, 1987 33. Komatsu N, Natsui K, Ueda N, et al: Immunohistochemical study on arachidonate 5lipoxygenase in porcine leukocytes and other tissues. J Histochem Cytochem 39:655, 1991 34. Kujubu DA, Fletcher BS, Vamum BC, et al: TIS10, a phorbol ester tumor promoterinducible mRNA from Swiss 3T3 cells, encodes a novel prostaglandin synthase/ cyclooxygenase homologue. J Biol Chem 266:12866, 1991 35. Kujubu DA, Herschman HR: Dexamethasone inhibits mitogen induction of the TISlO prostaglandin synthase/cyclooxygenase gene. J Biol Chem 2677991, 1992 36. Kujubu DA, Reddy ST, Fletcher BS, et al: Expression of the protein product of the prostaglandin synthase-Z/TISlO gene in mitogen-stimulated Swiss 3T3 cells. J Biol Chem 2685425, 1993 37. Lauritsen K, Laursen LS, Bukhave K, et al: In vivo effects of orally administered prednisolone on prostaglandin and leucotriene production in ulcerative colitis. Gut 281095, 1987 38. Lauritsen K, Laursen LS, Bukhave K, et al: Longterm olsalazine treatment: Pharmacokinetics, tolerance and effects on local eicosanoid formation in ulcerative colitis and Crohn’s colitis. Gut 29:974, 1988
EICOSANOIDS AND INFLAMMATORY BOWEL DISEASE
331
39. Laursen LS, Naesdal J, Bukhave K, et al: Selective 5-lipoxygenase inhibition in ulcerative colitis. Lancet 335:683, 1992 40. Lewis RA, Austen KF, Soberman RJ: Leukotrienes and other products of the 5lipoxygenase pathway: Biochemistry and relation to pathobiology in human diseases. N Engl J Med 323:645,1990 41. Lobos EA, Sharon P, Stenson WF: Chemotactic activity in inflammatory bowel disease: Role of leukotriene B,. Dig Dis Sci 32:1380, 1987 42. Mancini JA, Abramovitz M, Cox ME, et a1 5-Lipoxygenase-activating protein is an arachidonate binding protein. FEBS Lett 318:277, 1993 43. Masferrer JL, Seibert K, Zweifel 8, et al: Endogenous glucocorticoids regulate an inducible cyclooxygenase enzyme. J Biol Chem 89:3917, 1992 Vickers PJ, et al: Identification and isolation of a membrane 44. Miller DK, Gillard JW, protein necessary for leukotriene production. Nature 343:278, 1990 45. Musch MW, Miller RJ, Field M, et al: Stimulation of colonic secretion by lipoxygenase metabolites of arachidonic acid. Science 2171255, 1982 46. Musser JH, Kreft A F 5-Lipoxygenase: Properties, pharmacology, and the quinolinyl (bridged)aryl class of inhibitors. J Med Chem 35:2501, 1992 47. Nielsen OH, Ahnfeit-Ronne I, Thomsen MK, et al: Effect of the leukotriene LTD,/ LTE, antagonist, SR 2640, in ulcerative colitis: An open clinical study. Prostaglandins Leukotrienes Essent Fatty Acids 42181, 1991 48. Nielsen OH, Bukhave K, Elmgreen J, et a1 Inhibition of 5-lipoxygenase pathway of arachidonic acid metabolism in human neutrophils by sulfasalazine and 5-aminosalicylic acid. Dig Dis Sci 6:577, 1987 49. Peskar BM, Dreyling KW, Peskar BA, et al: Enhanced formation of sulfidopeptideleukotrienes in ulcerative colitis and Crohn’s disease: Inhibition by sulfasalazine and 5-aminosalicylic acid. Agents Actions 18381, 1986 50. Podolsky DK Inflammatory bowel disease (first of two parts). N Engl J Med 325:928, 1991 51. Podolsky DK Inflammatory bowel disease (second of two parts). N Engl J Med 325:1008, 1991 52. Prasit P, Belley M, Blouin M, et al: A new class of leukotriene biosynthesis inhibitor: The development of MK-0591. J Lipid Mediat 6:239, 1993 53 Rachmilewitz D, Simon PL, Schwartz LW, et al: Inflammatory mediators of experimental colitis in rats. Gastroenterology 97326, 1989 54. Racusen LC, Binder HI: Effects of prostaglandin on ion transport across isolated colonic mucosa. Dig Dis Sci 25:900, 1680 55. Rask-Madsen J: Eicosanoids and their role in the pathogenesis of diarrhoea1 diseases. Clin Gastroenterol 15:545, 1986 56. Rask-Madsen J: The role of eicosanoids in the gastrointestinal tract. Scand J Gastroenterol 127(suppl):7, 1987 57. Rouzer CA, Ford-Hutchinson AW, Morton HE, et al: MK-886, a potent and specific leukotriene biosynthesis inhibitor blocks and reverses the membrane association of 5lipoxygenase in ionophore-challenged leukocytes. J Biol Chem 265:1436, 1990 58. Rouzer CA, Kargman S Translocation of 5-lipoxygenase to the membrane in human leukocytes challenged with ionophore A23187. J Biol Chem 263:10980, 1988 59. Rouzer CA, Samuelsson B: On the nature of the 5-lipoxygenase reaction in human leukocytes: Enzyme purification and requirement for multiple stimulatory factors. Proc Natl Acad Sci USA 82:6040, 1985 60. Samuelsson B: Leukotrienes: Mediators of immediate hypersensitivity reactions and inflammation. Science 220568, 1983 61. Samuelsson B, Dahlen S, Lindgren JA, et al: Leukotrienes and lipoxins: Structures, biosynthesis, and biological effects. Science 2371171, 1987 62. Serhan CN, Fridovich J, Goetzl EJ, et al: Leukotriene B, and phosphatidic acid are calcium ionophores: Studies employing arsenazo 111 in liposomes. J Biol Chem 2574746, 1982 63. Sharon P, Ligumsky M, Rachmilewitz D, et al: Role of prostaglandins in ulcerative colitis: Enhanced production during active disease and inhibition by sulfasalazine. Gastroenterology 75638, 1978
-
332
YANG
64. Sharon P, Stenson WF: Metabolism of arachidonic acid in acetic acid colitis in rats: Similarity to human inflammatory bowel disease. Gastroenterology 8855, 1985 65. Sharon P, Stenson W F Enhanced synthesis of leukotriene B4 by colonic mucosa in inflammatory bowel disease. Gastroenterology 86453, 1984 66. Sigal E The molecular biology of mammalian arachidonic acid metabolism. Am J Physiol 260:L13, 1991 67. Siolander A, Schippert A, Hammarstrom S A human epithelial cell line, intestine 407, can produce ^<-hydroxyeicosatetraenoicacid and leikotriene 8,. Prostaglandins 4585, 1993 68. Smith WL, Meade EA, DeWitt D L Pharmacology of prostaglandin endoperoxide synthase isozymes-1 and -2. Ann NY Acad Sci 714:136, 1994 69. Stenson WF: Role of eicosanoids as mediators of inflammation in inflammatory bowel disease. Scand J Gastroenterol 172(suppl):13, 1990 70. Stenson WF, Lobos EA: Sulfasalazine inhibits the synthesis of chemotactic lipids by neutrophils. J Clin Invest 69:494, 1982 71. Travis SPL, Jewel1 DP: Salicylates for ulcerative colitis-their mode of action. Pharmacol Ther 63:135, 1994 72. Wallace JL, Keenan CM: An orally active inhibitor of leukotriene synthesis accelerates healing in a rat model of colitis. Am J Physiol258:G527, 1990 73. Wallace JL, MacNaughton WK, Morris GP, et a 1 Inhibition of leukotriene synthesis markedly accelerates healing in a rat model of inflammatory bowel disease. Gastroenterology 9699, 1989 74. Williams C N Overview of 5-ASA in therapy of inflammatory bowel disease. Can J Gastroenterol8:379, 1994 75. Woods JW, Evans JF, Ethier D, et a1 5-Lipoxygenase and 5-lipoxygenase-activating protein are localized in the nuclear envelope of activated human leukocytes. J Exp Med 1781935,1993 76. Yamamoto S Mammalian lipoxygenases: Molecular structures and functions. Biochim Biophys Acta 1128117, 1992 77. Zingarelli B, Squadrito F, Graziani P, et al: Effects of zileuton, a new 5-lipoxygenase inhibitor, in experimentally induced colitis in rats. Agents Actions 39:150, 1993 78. Zipser RD, Nast CC, Lee M, et al: In vivo production of leukotriene B, and leukotriene C, in rabbit colitis: Relationship to inflammation. Gastroenterology 9233, 1987 Address reprint requests to Vincent W. Yang, MD, PhD Ross 918 The Johns Hopkins University School of Medicine 720 Rutland Avenue Baltimore, MD 21205
0889-8553/96 $0.00
+ .20
EICOSANOIDS AND INFLAMMATORY BOWEL DISEASE Vincent W. Yang, MD, PhD
Inflammatory bowel disease (IBD) is a chronic, debilitating illness of the gastrointestinal tract afflicting individuals usually between the ages of 16 and 40. The two primary forms of idiopathic intestinal inflammation, ulcerative colitis and Crohn's disease, cause significant morbidity in the young adult population. This article reviews aspects of the inflammatory response in IBD that involve metabolism of arachidonic acid. For an overview of the pathogenesis and clinical features of the disease, readers are referred to the excellent review by P o d ~ l s k y .51~ ~ , PATHOGENESIS .OF INFLAMMATORY BOWEL DISEASE
Although the exact cause of IBD remains unknown, the pathogenesis of intestinal inflammation has been postulated to include two stages. In the first stage, an initial insulting event, precipitated by an unknown factor (or factors), occurs in the intestinal mucosa and leads to tissue damage. These insults may be derived from luminal stimuli such as dietary antigens or persistently infectious microbial agents. An intrinsic defect in the mucosal barrier may also be present that permits access for the offending agents. Alternatively the regulation of the mucosal immune system may be inherently abnormal, leading to an exaggerated
From the Departments of Medicine and Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland
GASTROENTEROLOGY CLINICS OF NORTH AMERICA VOLUME 25 * NUMBER 2 * JUNE 1996
317
318
YANG
response to luminal factors or to a defective feedback system that normally down-regulates the mucosal response to luminal factors (Fig. 1). The second stage of the disease involves an amplification of the inflammatory response. Several types of cells, including lymphocytes, neutrophils, macrophages, and mast cells, are attracted to the site of the initial injury. Products of these immune and inflammatory cells include a wide variety of mediators, such as cytokines, eicosanoids, free radicals, and components of the complement pathway. Amplification of the inflammatory response is important in the pathogenesis of IBD for two reasons. First, amplification of the inflammatory response, not the initiating event, causes the tissue destruction observed in IBD. Second, drugs used to treat IBD are designed for their ability either to modulate effects of the soluble mediators or to attenuate the cells from which these mediators are derived. Because the initiating event of IBD remains unknown, pharmacologic advances in the treatment of the disease are
Stimulus
Inflammatory Response Antigen Nonspecific
Immune Response Antigen Specific
Sustained Inflammatory Mucosal Destruction
1
IBD
Figure 1, Potential pathogenic mechanisms of inflammatory bowel disease (IBD). The lumen of the intestine is confronted by a variety of substances (e.g., dietary antigens and microbial agents) that elicit an antigen-specific immune response by the mucosal immune system or an antigenic-nonspecific inflammatory response by the epithelium. This process is normally balanced by feedback mechanisms that down-regulate the immune and inflammatory responses, leading to elimination of stimuli that breach the mucosal barrier. In the presence of disease, one or several fundamental alterations in the mucosal defense mechanisms may contribute to sustained and enhanced inflammation. Alternatively, regulation of immune and inflammatory responses may be intrinsically abnormal, with either exaggerated activations to normal stimuli (solid arrows) or defective feedback mechanisms for down-regulation (dotted arrows). These alterations result in sustained tissue destruction that leads to IBD. (Adapted from Podolsky DK: Inflammatory bowel disease. N Engl J Med 325932, 1991; 0 1991 with permission of the Massachusetts Medical Society.)
EICOSANOIDS AND INFLAMMATORY BOWEL DISEASE
319
more likely to result from an intervention of the inflammatory response than from the treatment of the cause of IBD. ARACHIDONIC ACID METABOLISM AND THE INFLAMMATORY RESPONSE Arachidonic acid (AA), a 20-carbon unsaturated fatty acid, is the source of a class of soluble inflammatory mediators called eicosanoids. AA is available from extracellular sources or can be released from within the cell. The phospholipid fraction in the cell constitutes the major source of AA. At least three enzymatic pathways exist for AA release from cellular phospholipids (Fig. 2): (1) direct action by phospholipase A, (PLA,); (2) combined action of phospholipase C and diacylglycerol (DAG) lipase or of phospholipase C, DAG kinase, and PLA,; and (3) combined action of phospholipase D and PLA,. Evidence suggests that an AA-specific PLA, is responsible for AA release from the membrane, which leads to eicosanoid formation. Further metabolism of AA by cellular components is mediated by one of three enzymatic pathways: the cyclooxygenase, lipoxygenase, and monooxygenase pathways, each giving rise to a distinct array of products. This section reviews the formation of eicosanoids by the cyclooxygenase and lipoxygenase pathways. In addition, the biologic effects of these lipid mediators are reviewed. For a detailed summary of AA metabolism, readers are referred to several excellent review articles.12,19, 22,30, 31 Prostaglandins and the Cyclooxygenase Pathway Cyclooxygenase (prostaglandin endoperoxide synthase or prostaglandin synthase) catalyzes the conversion of AA to prostaglandin endoperoxides, which are the precursors to a series of biologic compounds that include thromboxanes, prostacyclin, and several prostaglandins, including PGD,, PGE,, and PGF,,. Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit cyclooxygenase activity. In particular, aspirin irreversibly inactivates the enzyme by acetylation, which accounts for the therapeutic effect of this drug. To date, two different isoforms of cyclooxygenase have been identified: prostaglandin synthase-1 (PGS-1) and prostaglandin synthase-2 (PGS-2).The regulation of these two isozymes and their respective genes ~ ) . is constitutively are quite different (reviewed in H e r ~ c h m a n ~PGS-1 expressed, whereas PGS-2 is highly sensitive to extracellular stimuli. PGS-2 messenger RNA (mRNA) is rapidly induced in cells treated with serum, growth factors, forskolin, or phorbol ester.34,36 Moreover, the induction of the PGS-2 gene in response to stimulation is inhibited by the concomitant treatment of cells with glucocorticoids, whereas the PGS-1 gene is not inhibited by g l ~ ~ o ~ o r t i ~ 43 o iEvidence d s . ~ ~ ~suggests that PGS-1 has a housekeeping function, producing prostaglandins neces-
320
YANG
Phospholipid
I
Phospholipase A2
/
AA
Phospholipase D
Phosphonpase C
1
Phosphatidic Acid
I
Diacylglycerol I
DAG Lipase DAG Kinase
f
AA
+
Phospholipase A2
\
AA Phosphatidic Acid
I
+
Phospholipase A2 AA Figure 2. Enzymatic pathways for the formation of arachidonic acid (AA). Three predominant mechanisms for generating AA from membrane phospholipid have been elucidated in mammalian cells: (1) phospholipaseA, acting chiefly on phosphatidylcholineand phosphatidylethanolamine; (2) phospholipase C, acting principally on phosphatidylinositol to yield diacylglycerol (DAG), which in turn is a substrate for DAG lipase or the sequential actions by a kinase and a specific phospholipase A;, (3) phospholipase D, acting principally on phosphatidylcholine, combined with the subsequent action of another phospholipase A., (From Holtzman MJ: Arachidonic acid metabolism: Implications of biological chemistry for lung function and disease. Am Rev Respir Dis 143:193, 1991; with permission.)
sary for normal cellular processes, whereas PGS-2 is responsible for the inflammatory and mitogenic responses, producing prostaglandins involved in inflammation and growth regulation. Studies have identified certain NSAIDs that exhibit differential inhibitory effects on the activity of the two PGS isozyrnes.l3,68 These studies may lead to the eventual identification of NSAIDs that reduce inflammation but spare those prostaglandins required for normal cell physiology, thus reducing the untoward side effects associated with most NSAIDs. The physiologic effects of the various PGS products vary, depending on their sites of action and on the cells from which the products are derived. Thromboxane A, (TXA,), the predominant product of AA metabolism of platelets, is a potent platelet aggregating agent as well as a
EICOSANOIDS AND INFLAMMATORY BOWEL DISEASE
321
vasoconstrictor and bronchoconstrictor. In contrast, prostacyclin (PGI,), derived primarily from endothelial cells, is a potent inhibitor of platelet aggregation and, in some vascular systems, serves as a vasodilator and antagonizes the formation of TXA,. The prostaglandin synthases D, E, and F are responsible for the conversion of PGH, to prostaglandins D, E, and F. PGD, is formed from mast cells on antigen binding to IgE receptors and is a potent bronchoconstrictor, as is PGF,,. PGE, is a vasodilator and bronchodilator. Human intestinal epithelial cells have been shown to produce both PGE, and PGF,, in response to inflammatory stimuli, and these mediators act to increase chloride secretion.55 Importantly, intestinal epithelial cells interface with the luminal environment from which inflammatory stimuli are potentially originated. Leukotrienes and the 5-Lipoxygenase Pathway An alternate pathway of AA oxidation is mediated by the lipoxygenases. There are three major lipoxygenases: 5, 12, and 15, named for their ability to add a molecular oxygen at a specific carbon of AA.76The products of 5-lipoxygenase include leukotrienes, which constitute an important class of inflammatory mediators. This section reviews the biochemistry of the 5-lipoxygenase pathway and its relationship to the inflammatory response. The biochemical pathway leading to leukotriene formation has been 60, 61, 5-Lipoxygenase catalyzes the formation of well characteri~ed.~~, LTA, and 5(S)-hydroxy-6,8,11,14-eicosatetraenoicacid (5-HETE) from AA through the intermediary molecule of 5(S)-hydroperoxy-6,8,11,14eicosatetraenoic acid (5-HPETE) (Fig. 3). LTA, can be metabolized by two specific enzymes to produce biologically active compounds. The first enzyme, leukotriene A, hydrolase, catalyzes the hydrolysis of LTA, to LTB,. The second enzyme, leukotriene C, synthase, catalyzes the conjugation of LTA, to the tripeptide glutathione, leading to the formation of LTC,. Further metabolism of LTC, occurs when its glutamyl residue is cleaved by a membrane-bound gamma-glutamyl transferase to produce LTD,, which is further metabolized to LTE, by the action of a membrane-bound dipeptidase.
5-Lipoxygenase and 5-Lipoxygenase-Activating Protein Because 5-lipoxygenase plays a central role in the biosynthesis of leukotrienes, considerable effort has been devoted to the characterization of this enzyme (reviewed by Ford-Hutchinson and colleague^^^). It is a cytosolic enzyme that requires Ca2 and adenosine triphosphate (ATP) for maximal activity.59The human enzyme has been purified to homogeneity from peripheral blood leukocytes and is a 78-kilodalton (kd) protein. When neutrophils are activated by the calcium ionophore A23187, 5-lipoxygenase, normally cytosolic, is translocated to a membrane fraction, where it gains access to its substrate, AA.58 These observations +
322
YANG
Arachidonic Acid
5i-HrTEJ
5-llpoxygenase FLAP
5-HETE
Leukotrlene A4
Leukotriene A4 Hydrolase
Leukotriene C4 Synthase
l + l
Leukotriene B4
1
Receptor Activation
c
Leukotriene C4
i
Gamma-glutamyl Transferase
Leukotrlene D4
I
Dipeptidase
Leukotriene E4
c
Receptor Activation
Figure 3. Enzymatic pathways of leukotriene biosynthesis. Key enzymes (italics) lead to the formation of various metabolites.
suggest the presence of a defined regulatory process for the activation of 5-lipoxygenase within the cell. The key to understanding 5-lipoxygenase activation came from the study of a specific inhibitor of leukotriene biosynthesis, MK-886.26This compound has no effect on the synthesis of cyclooxygenase, 12-lipoxygenase, or 15-lipoxygenase-derivedproducts of AA. Surprisingly, MK886 inhibits only the synthesis of leukotrienes in intact human neutrophils stimulated with calcium ionophore but has no effect on the 5lipoxygenase enzyme itself, either in broken cell preparations or in purified enzyme preparations.26,57 These findings suggest that additional cellular components are required for the cellular activation of 5-lipoxygenase. A protein was subsequently identified and shown to be the target of MK-886.@This 18-kDa protein, called 5-lipoxygenase-activating
EICOSANOIDS AND INFLAMMATORY BOWEL DISEASE
323
protein (FLAP), was purified to homogeneity, and its full-length cDNA cloned and ~equenced.'~ Only when both 5-lipoxygenase and FLAP are expressed in the same cell can cellular leukotriene synthesis be observed following ionophore challenge, and this synthesis is inhibited by MK886.15 FLAP, a membrane protein, is also involved in the translocation of 5-lipoxygenase from the cytosol to the membrane following exposure to ionophore, resulting in the activation of 5-lipoxygenase. This translocation, together with the accompanying leukotriene synthesis, is inhibited by MK-886. Evidence suggests that FLAP is an AA-binding protein that facilitates the transfer of AA to activated 5-lipo~ygenase.~~ The current hypothesis on the mechanism of action of MK-886 is that its binding site on FLAP coincides with the AA-binding site and that the interaction of MK-886 and FLAP impedes the proper delivery of AA to 5-lipoxygena ~ e Figure . ~ ~ 4 illustrates the complex pathway of activation of the 5lipoxygenase/FLAP system and the mechanism by which MK-886 inhibits LT formation. Another interesting observation depicted in Figure 4 is the subcellular localization of 5-lipoxygenase and FLAP. In activated leukocytes, both 5-lipoxygenase and FLAP are localized to the lumen of the nuclear envelope and endoplasmic reticulum.75The distribution of FLAP in resting cells is identical to that in activated cells, but 5-lipoxygenase is not detected in any specific cellular compartment. Hence, in unstimulated cells, FLAP is present in the membrane, and following stimulation 5-lipoxygenase moves to the same membrane site as FLAP. Biologic Effects of Leukotrienes The biologic effects of leukotrienes are numerous. Although all leukotrienes are primarily products of leukocytes, including neutrophils, mast cells, and macrophages, LTB4 alone acts as a calcium ionophore, leading to further stimulation of 5-lipoxygenase activity.62In addition, LTB, is a potent chemotactic substance for leukocytes22and has been shown to influence lymphocyte activity.20In contrast, the peptidyl derivatives of leukotrienes (LTC,, LTD4, and LTE,) primarily affect smooth muscle contractility and other cells with contractile capacity. For example, on a molar basis, LTC, and LTD, are 1000 times more potent than histamine in inducing constriction of the airway.17Many other actions of leukotrienes have been described and can be found in the review articles by Samuelsson and colleagues,61 Lewis and colleagues,4° and Henderson.29 Although leukotrienes are primarily produced by the inflammatory cells, whether intestinal epithelial cells possess the ability to synthesize leukotrienes has only recently been explored. Immunohistochemical examination of the porcine ileum with a polyclonal 5-lipoxygenase antibody indicates no immunoreactivity in the e p i t h e l i ~ m In . ~ ~contrast, isolated rat colonic crypt epithelial cells can produce leukotrienes.10 Furthermore, several epithelial cell lines derived from human small and large intestines are also capable of synthesizing leuk~trienes.~,67 The
324
YANG
+Cell Activation
+MK-886
fiT
+Cell Activation
UlFLAP * 5 - L 0 A
oCPLA2
* MK-886
B
Figure 4. A model for the cellular activation of 5-lipoxygenase (5-LO) and proposed mechanism of action of the leukotriene inhibitor, MK-886. A, Activation of a cell surface receptor by a given stimulus leads to the mobilization of intracellular Ca2+,which results in the translocation of the cytosolic phospholipaseA2 (cPLA,) to the plasma membrane where it liberates AA from membrane phospholipids. Free AA is then available to an activated 5LO, which in response to the increase in intracellular Ca2+ is translocated to the nuclear membrane where FLAP resides. Evidence suggests that FLAP is an AA-binding protein, which presumably facilitates the transfer of this substrate to the activated 5-LO to produce leukotrienes. 8,The interaction of MK-886 with FLAP interferes with the proper translocation of 5-LO to the membrane in response to a stimulus, thus resulting in the inhibition of leukotriene synthesis. (From Ford-HutchinsonAW, Gresser M, Young RN: 5-Lipoxygenase. Annu Rev Biochem 63:413, 1994; 0 1994 with permission of Annual Reviews, Inc.)
EICOSANOIDS AND INFLAMMATORY BOWEL DISEASE
325
human colonic epithelial cell line, HT29-C1,32for example, contains the mRNA for both 5-lipoxygenase and FLAP and possesses the capacity to synthesize LTB, and 5-HETE under certain conditions? Thus, the intestinal epithelium can produce limited quantities of leukotrienes, which may be implicated in intestinal cell physiology and potentially in intestinal inflammation. EICOSANOIDS AND INFLAMMATORY BOWEL DISEASE
As a result of the amplification of the inflammatory process, soluble mediators such as eicosanoids play a significant role in the pathogenesis of IBD. Numerous studies have demonstrated a relationship between elevated eicosanoid levels in the inflamed intestine and disease severity (reviewed in Donowitz,16 Rask-Madsen:'j and S t e n ~ o n ~This ~ ) . section summarizes some of the changes in AA metabolism observed in IBD. The ability of the human colon to produce both cyclooxygenase and I* When homogenates lipoxygenase metabolites has been dem~nstrated.~, of human colonic mucosa are incubated with radiolabeled AA, the predominant cyclooxygenase products include PGE2, PGF2,, PGD2, and TxB2. 12-HETE and 15-HETE are the major lipoxygenase products formed. LTB., and the peptidyl derivatives of leukotrienes are also present but in lesser amounts than the prostaglandins. In active IBD, high levels of prostaglandins are found in the rectal mucosa and urine of patients with active disease.@The levels of prostaglandins decline in IBD patients treated with either corticosteroids or sulfasalazine. By contrast, in those with inactive IBD, the levels of prostaglandins are not significantly different from those of normal control subjects. The pathologic effects of elevated prostaglandin levels in IBD are more difficult to determine, however. Although prostaglandin levels in mucosal specimens of IBD patients decline when they are treated with NSAIDs, there is no clinical improvement.2sIn fact, some evidence indicates that NSAIDs actually increase the severity of IBD. These findings suggest that prostaglandins may not be the important mediators of the inflammation observed in IBD and that the mechanism of action of corticosteroids and sulfasalazine may not be related to the inhibition of prostaglandin synthesis. The pathologic role of the lipoxygenase products in IBD is better documented. Several studies demonstrate that colonic mucosa from patients with active IBD synthesize significantly higher amounts of lipoxygenase products, including LTB, and 5-HETE, compared with normal control tissue.6,7, 65 Likewise, LTB, levels are much higher in dialysates from dialysis bags placed in the rectums of patients with ulcerative colitis than in controls.38Also, increased synthesis of LTB, and the peptidyl leukotrienes is observed in rectal biopsy specimens from IBD patients that are stimulated with the calcium ionophore, A23187.49Similarly, in various animal models of colitis, the synthesis of leukotrienes
326
YANG
in the inflamed mucosa is elevated.53* 64, 78 Inhibition of leukotriene synthesis by specific drugs decreases inflammation and accelerates healing in a rat model of IBD.” In particular, corticosteroids and sulfasalazine, two drugs known to be effective in the treatment of IBD, decrease colonic leukotriene formation.’, 37, 38 These findings indicate the importance of the lipoxygenase products of AA metabolism in the pathogenesis of IBD. The exact role played by the lipoxygenase products in IBD remains unclear. LTB,, as noted, is a potent chemotactic and aggregating substance for neutrophils and mononuclear cells.2oWhen homogenates of colonic mucosa are assayed for the chemotactic activity for human neutrophils, IBD patients have a 10-fold greater activity than normal control^.^' When the homogenates are fractionated to identify the substance responsible for the increased chemotactic activity, the fraction with significant activity coelutes with LTB,. Furthermore, this chemotactic response is abolished by anti-LTB, antisera. These studies indicate that LTB, within the IBD mucosa is responsible for the promulgation of the chemotactic reaction and the subsequent recruitment of circulating neutrophils to the inflamed mucosa. Thus, the enhanced synthesis of LTB, may account, in part, for the amplification of the inflammatory response in IBD. Aside from the chemotactic and proinflammatory natures of LTB,, products of the lipoxygenase and cyclooxygenase pathways may contribute directly to the diarrhea seen in IBD patients. Various AA metabolites may alter different aspects of intestinal fluid regulation, including absorption and secretion of solutes and intestinal motility. PGE, decreases active sodium and chloride absorption and increases chloride secretion in both the small intestine and the TxA, has also been shown to cause colonic secretion. Products of the lipoxygenase pathway alter active intestinal electrolyte transport as well, although this effect is not uniform among all products. For example, 5-HETE and 5-HPETE cause chloride secretion in rabbit distal colon at low concentrations but not in rabbit ileum.45In contrast, LTB, and LTC, do not alter either colonic or ileal electrolyte transport. The effects of lipoxygenase products on intestinal motility have not been clearly demonstrated, although the myotrophic effects of several leukotrienes may potentially influence motility, leading to diarrheal formation. TREATING INFLAMMATORY BOWEL DISEASE WITH DRUGS TARGETED TOWARD ARACHIDONIC ACID METABOLISM
Based on the preceding discussion, a number of AA metabolites seem important in the amplification of the inflammatory process observed in IBD. Therefore, drugs interfering with the metabolism of AA and leading to the inhibition of eicosanoid formation are potential therapeutic agents for IBD. One of the many functions of corticosteroids, commonly used in IBD, is the stabilization of membrane phospholipase
EICOSANOIDS AND INFLAMMATORY BOWEL DISEASE
327
A, activity, making the substrate AA less available for leukotriene synthesis (see Fig. 2). Sulfasalazine, another frequently used drug in IBD treatment, is a second example. Sulfasalazine contains two moieties: sulfapyridine, a sulfonamide, and 5-aminosalicylate (5-ASA).Sulfasalazine inhibits 5-lipoxygenase activity and lowers leukotriene levels in IBD patients.4s,70 The 5-ASA moiety of sulfasalazine is considered the active component required for 5-lipoxygenase inhibition. 5-ASA is now available as both an oral and a topical agent for IBD treatment.71,74
5-Lipoxygenase Inhibitors
Pharmacologic evaluation of 5-lipoxygenase inhibition by many new drugs for the treatment of IBD and other inflammatory disorders is These drugs can be divided into two major under active classes, depending on their mode of action: (1) direct inhibitors of 5lipoxygenase activity and (2) inhibitors of FLAP activity. Zileuton (A64077), a benzothiophene hydroxyurea, is an example of a direct, orally active 5-lipoxygenase i n h i b i t ~ rIt. ~compares favorably with sulfasalazine in attenuating the lesions seen in experimentally induced colitis in ratsn In initial clinical trials, oral zileuton significantly reduces LTB, levels in rectal dialysates from patients with active ulcerative colitis39and improves the symptom and sigmoidoscopy scores in these patients when compared with placebo control.8These patients have not had significant side effects. A long-term controlled, double-blind, and dose-range trial of the efficacy and safety of zileuton is currently being conducted. Newer, longer-acting 5-lipoxygenase inhibitors with improved bioavailability are also currently under investigation in experimental animal^.^
5-Lipoxygenase-Activating Protein Inhibitors
In contrast to zileuton, MK-886 (L-663,536) inhibits 5-lipoxygenase activity by interacting with FLAP (see Fig. 4). This drug is a potent inhibitor of leukotriene biosynthesis in intact human polymorphonuclear (PMN) leukocytes. MK-886 appears effective in controlling the inflammatory response produced in several experimentally induced inflammatory animal models.26This drug has excellent bioavailability, prolonged duration of action, and lack of toxicity in initial animal studies.26In a rat model of chronic colitis, MK-886 reduced colonic LTB, synthesis and accelerated healing.7zIn a human trial, MK-886 significantly reduced calcium ionophore-stimulated LTB, biosynthesis ex vivo in healthy male subjects receiving the drug and was well tolerated.'l Moreover, MK-886 blocks allergen-induced airway responses in atopic patients challenged with histamine.25Other potent FLAP inhibitors have been
328
YANG
Potential Treatment Strategies of Inflammatory Bowel Disease That Target the Leukotriene Pathway of Arachidonic Acid Metabolism In addition to inhibiting leukotriene formation by targeting 5-lipoxygenase, many other pharmacologic strategies for interfering with AA metabolism can be exploited. Figure 5 illustrates several potential points of interference with the leukotriene pathway of AA metabolism that would be explored for therapeutic purposes. Corticosteroids stabilize membrane phospholipids and inhibit phospholipase A, activity, resulting in less AA available for eicosanoid formation. Other specific inhibitors of phospholipase A, that lead to decreased AA release from membrane phospholipids are also potential candidates. N-3 fatty acids, such as eicosapentaenoic acid (EPA), a component of fish oil lipids, may lower normal stores of AA, compete with AA for oxygenation, and potentially alter substrate milieu to influence AA uptake and availability. Dietary supplementation with EPA markedly attenuates leukocyte generation of leukotrienes and the late asthmatic response to allergens? Clinical trials of IBD patients with dietary EPA supplementation is feasible. Lastly, many compounds that are leukotriene analogs, thus competitive inhibitors of leukotriene receptors, have been developed. These agents can be designed to target individual leukotriene receptors, therefore blocking only the response of the individual cell type con-
n-3 Fatty Acids
Membrane Phospholipids
'
I*
PLA2
57
Corticosteroids
PLA2 Inhibitors
Arachidonic Acid 5-LO
5-LO and FLAP lnhibitors
Leukotrienes
1
Membrane Receptors
Leukotriene Receptor Antagonists
Cells
Figure 5. Pharmacologic strategies for the inhibition of AA metabolism. Potential sites of inhibition of the leukotriene pathway of AA metabolism are categorized. Examples include (1) dietary therapy with n-3 fatty acids such as EPA; (2) phospholipase A2 (PLA,) inhibition by corticosteroids or specific PLA, inhibitors; (3) 5-LO/FLAP inhibition by inhibitors such as sulfasalazine, zileuton, and MK-886; and (4) direct leukotriene receptor antagonists. (From Holtzman MJ: Arachidonic acid metabolism: Implications of biological chemistry for lung function and disease. Am Rev Respir Dis 143:198, 1991; with permission.)
EICOSANOIDS AND INFLAMMATORY BOWEL DISEASE
329
taining that specific receptor. Examples of specific leukotrienes receptor antagonists currently being tested in animals or humans include the LTB, receptor antagonist, SC-41930,24and the LTD,/LTE, receptor antagonist, SR 2640.47 SUMMARY
IBD is a disease of unknown cause that involves an amplification of the inflammatory response in the intestinal mucosa. Although not the only offending agents leading to the disease, eicosanoids, the collective group of AA metabolites, play a significant role in the pathogenesis of IBD. This article reviewed the biochemical pathways of eicosanoid formation and the clinical relevance of eicosanoids to IBD. Potential strategies designed to interfere with various aspects of AA metabolism were also outlined. Further clinical trials of newer compounds may soon prove them effective in the management of IBD. References 1. Allgayer H, Stenson WF: A comparison of effects of sulfasalazine and its metabolites on the metabolism of endogenous vs. exogenous arachidonic acid. Immunopharmacology 15:39, 1988 2. Arm JP, Horton CE, Spur BW, et al: The effects of dietary supplementation with fish oil lipids on airway response to inhaled allergen in bronchial asthma. Am Rev Respir Dis 139:1395, 1989 3. Batt DG: 5-Lipoxygenase inhibitors and their anti-inflammatory activities. Prog Med Chem 291, 1992 4. Bell RL, Bouska.J, Young PR, et al: The properties of A-69412: A small hydrophilic 5lipoxygenase inhibitor. Agents Actions 38:178, 1993 5. Bell RL, Young PR, Albert D, et al: The discovery and development of zileuton: An orally active 5-lipoxygenase inhibitor. Int J Immunopharmacol 14:505, 1992 6. Beyer C, Maier KE, Klotz U: Enhanced synthesis of hydroxyeicosatetraenoic acids by colonic mucosa in chronic inflammatory bowel disease. In MacDermott RP (ed): Inflammatory Bowel Disease: Current Status and Future Approach. New York, Elsevier Science Publishers, 1988, p 291-296 7. Boughton-Smith NK, Hawkey CJ, Whittle BJR Biosynthesis of lipoxygenase and cyclooxygenase products from [14C]-arachidonicacid by human colonic mucosa. Gut 24:1176, 1983 8. Collawn C, Rubin P, Perez N, et al: Phase I1 study of the safety and efficacy of a 5lipoxygenase inhibitor in patients with ulcerative colitis. Am J Gastroenterol 87:342, 1992 9. Cortese JF, Spannhake EW, Eisinger W, et al: The 5-lipoxygenase pathway in cultured human intestinal epithelial cells. Prostaglandins 49:155, 1995 10. Craven PA, DeRubertis FR Profiles of eicosanoid production by superficial and proliferative colonic epithelial cells and sub-epithelial colonic tissue. Prostaglandins 32:387, 1986 11. Depre M, Friedman B, Tanaka W, et al: Biochemical activity, pharmacokinetics, and tolerability of MK-886, a leukotriene biosynthesis inhibitor, in humans. Clin Pharmacol Ther 53:602, 1993 12. DeWitt D L Prostaglandin endoperoxide synthase: Regulation of enzyme expression. Biochim Biophys Acta 1083:121, 1991
330
YANG
13. DeWitt DL, Meade EA, Smith WL: PGH synthase isoenzyme selectivity: The potential for safer nonsteroidal antiinflammatory drugs. Am J Med 95(suppl2A):40S, 1993 14. Dias VC, Wallace JL, Parsons HG: Modulation of cellular phospholipid fatty acids and leukotriene B, synthesis in the human intestinal cell (CaCo-2). Gut 33:622, 1991 15. Dixon RAF, Diehl RE, Opas E, et al: Requirement of a 5-lipoxygenase-activatingprotein for leukotriene synthesis. Nature 343282, 1990 16. Donowitz M: Arachidonic acid metabolites and their role in inflammatory bowel disease: An update requiring addition of a pathway. Gastroenterology 88:580, 1985 17. Drazen JM, Austen KF: Leukotrienes and airway responses. Am Rev Respir Dis 136:985, 1987 18. Dreyling KW, Hoppe U, Peskar BA, et al: Leukotriene synthesis by human gastrointestinal tissues. Biochim Biophys Acta 878:184, 1986 19. Feuerstein G, Hallenbeck JM: Leukotrienes in health and disease. FASEB J 1:186, 1987 20. Ford-Hutchinson A W Leukotriene B, in inflammation. Crit Rev Immunol l O : l , 1990 21. Ford-Hutchinson AW: Regulation of leukotriene biosynthesis. Cancer Metastasis Rev 13257, 1994 22. Ford-Hutchinson AW, Bray MA, Doig MV, et al: Leukotriene B,, a potent chernokinetic and aggregating substance released from polymorphonuclear leukocytes. Nature 286264, 1980 23. Ford-Hutchinson AW, Gresser M, Young RN: 5-Lipoxygenase. Annu Rev Biochem 63:383, 1994 24. Fretland DJ, Widornski D, Tsai BS, et al: Effect of the leukotriene B, receptor antagonist SC-41930 on colonic inflammation in rat, guinea pig and rabbit. J Pharrnacol Exp Ther 255572, 1990 25. Friedman BA, Be1 EH, Buntinx A, et al: Oral leukotriene inhibitor (MK-886) blocks allergen-induced airway responses. Am Rev Respir Dis 147839, 1993 26. Gillard J, Ford-Hutchinson AW, Chan C, et a1 L-663,536 (MK-886), a novel, orally active leukotriene biosynthesis inhibitor. Can J Physiol Pharmacol 67456, 1989 27. Goetzl EJ, Pickett WC: The human PMN leukocyte chemotactic activity of complex hydroxy-eicosatetraenoic acids (HETEs). J Immunol 1251789, 1980 28. Gould SR, Brash AR, Conolly ME, et al: Studies of prostaglandins and sulphasalazine in ulcerative colitis. Prostaglandins Med 6:165, 1981 29. Henderson W R The role of leukotrienes in inflammation. Ann Intern Med 121:684, 1994 30. Herschman HR: Regulation of prostaglandin synthase-1 and prostaglandin synthase2. Cancer Metastasis Rev 13241, 1994 31. Holtzman MJ: Arachidonic acid metabolism: Implications of biological chemistry for lung function and disease. Am Rev Respir Dis 143:188, 1991 32. Huet C, Sahuquillo-Merino C, Coudrier E, et al: Absorptive and mucus-secreting subclones isolated from a multipotent intestinal cell line (HT-29) provide new models for cell polarity and terminal differentiation. J Cell Biol 105:345, 1987 33. Komatsu N, Natsui K, Ueda N, et al: Immunohistochemical study on arachidonate 5lipoxygenase in porcine leukocytes and other tissues. J Histochem Cytochem 39:655, 1991 34. Kujubu DA, Fletcher BS, Vamum BC, et al: TIS10, a phorbol ester tumor promoterinducible mRNA from Swiss 3T3 cells, encodes a novel prostaglandin synthase/ cyclooxygenase homologue. J Biol Chem 266:12866, 1991 35. Kujubu DA, Herschman HR: Dexamethasone inhibits mitogen induction of the TISlO prostaglandin synthase/cyclooxygenase gene. J Biol Chem 2677991, 1992 36. Kujubu DA, Reddy ST, Fletcher BS, et al: Expression of the protein product of the prostaglandin synthase-Z/TISlO gene in mitogen-stimulated Swiss 3T3 cells. J Biol Chem 2685425, 1993 37. Lauritsen K, Laursen LS, Bukhave K, et al: In vivo effects of orally administered prednisolone on prostaglandin and leucotriene production in ulcerative colitis. Gut 281095, 1987 38. Lauritsen K, Laursen LS, Bukhave K, et al: Longterm olsalazine treatment: Pharmacokinetics, tolerance and effects on local eicosanoid formation in ulcerative colitis and Crohn’s colitis. Gut 29:974, 1988
EICOSANOIDS AND INFLAMMATORY BOWEL DISEASE
331
39. Laursen LS, Naesdal J, Bukhave K, et al: Selective 5-lipoxygenase inhibition in ulcerative colitis. Lancet 335:683, 1992 40. Lewis RA, Austen KF, Soberman RJ: Leukotrienes and other products of the 5lipoxygenase pathway: Biochemistry and relation to pathobiology in human diseases. N Engl J Med 323:645,1990 41. Lobos EA, Sharon P, Stenson WF: Chemotactic activity in inflammatory bowel disease: Role of leukotriene B,. Dig Dis Sci 32:1380, 1987 42. Mancini JA, Abramovitz M, Cox ME, et a1 5-Lipoxygenase-activating protein is an arachidonate binding protein. FEBS Lett 318:277, 1993 43. Masferrer JL, Seibert K, Zweifel 8, et al: Endogenous glucocorticoids regulate an inducible cyclooxygenase enzyme. J Biol Chem 89:3917, 1992 Vickers PJ, et al: Identification and isolation of a membrane 44. Miller DK, Gillard JW, protein necessary for leukotriene production. Nature 343:278, 1990 45. Musch MW, Miller RJ, Field M, et al: Stimulation of colonic secretion by lipoxygenase metabolites of arachidonic acid. Science 2171255, 1982 46. Musser JH, Kreft A F 5-Lipoxygenase: Properties, pharmacology, and the quinolinyl (bridged)aryl class of inhibitors. J Med Chem 35:2501, 1992 47. Nielsen OH, Ahnfeit-Ronne I, Thomsen MK, et al: Effect of the leukotriene LTD,/ LTE, antagonist, SR 2640, in ulcerative colitis: An open clinical study. Prostaglandins Leukotrienes Essent Fatty Acids 42181, 1991 48. Nielsen OH, Bukhave K, Elmgreen J, et a1 Inhibition of 5-lipoxygenase pathway of arachidonic acid metabolism in human neutrophils by sulfasalazine and 5-aminosalicylic acid. Dig Dis Sci 6:577, 1987 49. Peskar BM, Dreyling KW, Peskar BA, et al: Enhanced formation of sulfidopeptideleukotrienes in ulcerative colitis and Crohn’s disease: Inhibition by sulfasalazine and 5-aminosalicylic acid. Agents Actions 18381, 1986 50. Podolsky DK Inflammatory bowel disease (first of two parts). N Engl J Med 325:928, 1991 51. Podolsky DK Inflammatory bowel disease (second of two parts). N Engl J Med 325:1008, 1991 52. Prasit P, Belley M, Blouin M, et al: A new class of leukotriene biosynthesis inhibitor: The development of MK-0591. J Lipid Mediat 6:239, 1993 53 Rachmilewitz D, Simon PL, Schwartz LW, et al: Inflammatory mediators of experimental colitis in rats. Gastroenterology 97326, 1989 54. Racusen LC, Binder HI: Effects of prostaglandin on ion transport across isolated colonic mucosa. Dig Dis Sci 25:900, 1680 55. Rask-Madsen J: Eicosanoids and their role in the pathogenesis of diarrhoea1 diseases. Clin Gastroenterol 15:545, 1986 56. Rask-Madsen J: The role of eicosanoids in the gastrointestinal tract. Scand J Gastroenterol 127(suppl):7, 1987 57. Rouzer CA, Ford-Hutchinson AW, Morton HE, et al: MK-886, a potent and specific leukotriene biosynthesis inhibitor blocks and reverses the membrane association of 5lipoxygenase in ionophore-challenged leukocytes. J Biol Chem 265:1436, 1990 58. Rouzer CA, Kargman S Translocation of 5-lipoxygenase to the membrane in human leukocytes challenged with ionophore A23187. J Biol Chem 263:10980, 1988 59. Rouzer CA, Samuelsson B: On the nature of the 5-lipoxygenase reaction in human leukocytes: Enzyme purification and requirement for multiple stimulatory factors. Proc Natl Acad Sci USA 82:6040, 1985 60. Samuelsson B: Leukotrienes: Mediators of immediate hypersensitivity reactions and inflammation. Science 220568, 1983 61. Samuelsson B, Dahlen S, Lindgren JA, et al: Leukotrienes and lipoxins: Structures, biosynthesis, and biological effects. Science 2371171, 1987 62. Serhan CN, Fridovich J, Goetzl EJ, et al: Leukotriene B, and phosphatidic acid are calcium ionophores: Studies employing arsenazo 111 in liposomes. J Biol Chem 2574746, 1982 63. Sharon P, Ligumsky M, Rachmilewitz D, et al: Role of prostaglandins in ulcerative colitis: Enhanced production during active disease and inhibition by sulfasalazine. Gastroenterology 75638, 1978
-
332
YANG
64. Sharon P, Stenson WF: Metabolism of arachidonic acid in acetic acid colitis in rats: Similarity to human inflammatory bowel disease. Gastroenterology 8855, 1985 65. Sharon P, Stenson W F Enhanced synthesis of leukotriene B4 by colonic mucosa in inflammatory bowel disease. Gastroenterology 86453, 1984 66. Sigal E The molecular biology of mammalian arachidonic acid metabolism. Am J Physiol 260:L13, 1991 67. Siolander A, Schippert A, Hammarstrom S A human epithelial cell line, intestine 407, can produce ^<-hydroxyeicosatetraenoicacid and leikotriene 8,. Prostaglandins 4585, 1993 68. Smith WL, Meade EA, DeWitt D L Pharmacology of prostaglandin endoperoxide synthase isozymes-1 and -2. Ann NY Acad Sci 714:136, 1994 69. Stenson WF: Role of eicosanoids as mediators of inflammation in inflammatory bowel disease. Scand J Gastroenterol 172(suppl):13, 1990 70. Stenson WF, Lobos EA: Sulfasalazine inhibits the synthesis of chemotactic lipids by neutrophils. J Clin Invest 69:494, 1982 71. Travis SPL, Jewel1 DP: Salicylates for ulcerative colitis-their mode of action. Pharmacol Ther 63:135, 1994 72. Wallace JL, Keenan CM: An orally active inhibitor of leukotriene synthesis accelerates healing in a rat model of colitis. Am J Physiol258:G527, 1990 73. Wallace JL, MacNaughton WK, Morris GP, et a 1 Inhibition of leukotriene synthesis markedly accelerates healing in a rat model of inflammatory bowel disease. Gastroenterology 9699, 1989 74. Williams C N Overview of 5-ASA in therapy of inflammatory bowel disease. Can J Gastroenterol8:379, 1994 75. Woods JW, Evans JF, Ethier D, et a1 5-Lipoxygenase and 5-lipoxygenase-activating protein are localized in the nuclear envelope of activated human leukocytes. J Exp Med 1781935,1993 76. Yamamoto S Mammalian lipoxygenases: Molecular structures and functions. Biochim Biophys Acta 1128117, 1992 77. Zingarelli B, Squadrito F, Graziani P, et al: Effects of zileuton, a new 5-lipoxygenase inhibitor, in experimentally induced colitis in rats. Agents Actions 39:150, 1993 78. Zipser RD, Nast CC, Lee M, et al: In vivo production of leukotriene B, and leukotriene C, in rabbit colitis: Relationship to inflammation. Gastroenterology 9233, 1987 Address reprint requests to Vincent W. Yang, MD, PhD Ross 918 The Johns Hopkins University School of Medicine 720 Rutland Avenue Baltimore, MD 21205