Leukotriene B4 in inflammatory bowel disease

WF STENSON. Leukotriene B,. in inflammatory bowel disease. Can J Gastro-enterol 1993;7(2):182-186. Leukotriene 14 is an inflammatory mediator produced by metabolism of arachidonic acid through the 5-lipoxygenase pathway in neucrophils, macrophages and mast cells. The major biological effect of leukotriene B4 is the activation of circulating neutrophils to bind to the vascular endothelium and migrate into inflamed tissues. There are markedly elevated levels of leukotriene B4 in inflammatory bowel disease and there is considerable evidence that it is an important neutrophil activator in this disease. Preliminary studies suggest that inhibition of leukotriene 14 synthesis may be therapeutic in inflammatory bowel disease.

T HE PATI IOOENESIS OF INTESTINAL inflammalion in general, anJ in, flammacory bowel disease (IBO) m particular, can be divided into cwo stages.The first stage is the initiating event which trigger:, the mflamrnaton• response, hut m lBD the m1t1acmg event is unknown.The second stage 1s the amplification of the inflammatory response which mvolves a numhcr ol inflammatory cel ls indudmg lymphocytes, mast cells, macrophages and neutrophib.The amplificatton of the inflammatory response is contrnllcJ by solu ble mediators inc.ludmg histamine, serotonin, proJucts of the complement pathway, prostaglandirn, and leukotri• enes.This second stage, the ampltfica• tion of the inflammatory response, ti important 111 the pathogenesis of IBD fo r two reasom.First, it is lhe amplifica• tion of the inflammatory respome, and not the initiating event, which 1s the cause of the tissue destruction and the hisrolog1c and functional changes char, actenmc of IBO.Second, those Jnigi which are effective in the treatment of IBO appear to exert their rhcrapcuuc effects by moJulatmg the rmduction of soluble mediators ( l,2).Moreover, as long as the 111itiating e\'cnt remains unknown, it 1s likely that further advances tn the mcJ1cal thcrary of IBO will result from rharmacnlog1( modulat1on of inflammatory mediators.

PROSTAGLANDINS IN IBO
Few investigations have fuu1sed directly on mediators of the inflammatory process 111 IRD.Prostaglandins, which arc found in mLrease~I concentrations in inflammatory exudates, have received the most attcnrion (3,4).High levels of prostagland1m arc present m rectal muc11sa and 111 scrum tn !BD and high levels of prnstagl.111dinmetabolites arc found 111 the urine.Prostaglandin levels decline when patients with (Bl) arc treated with either corticosteroids or sulrhasalazine.However, prostaglandins a lso declme when IBD pauenrs arL• treated with nonstcroidal anu-inflammatory drugs (eg, mdomethacm), but pancnts do not ihow cl1111cal improvement.There is, 111 fact, some evidence that non-,tcro1dal ,1genrs may mLrease the ,;everity of I BD.These last f1 ndings sug-~cst that prosraglandms m,1y not he important mcJ1acors 111 IBD and that the mechanism of act1un of corticosteroids and sulphasala:mc may nnt relate to the inhibition of rrostaglandin synthesis.
There was good correlation between elevated levds of L TB4 and presence of histolog1c inflammation and diarrhea rroduct1on.AraLhadonate metabolism was alw assayed in a second model of 111test111al 111flammat1on -acetiL acid tulms m the rat.
Although they arc considered to he chronic inflammatory diseases the histologic picture of both Crohn's disease and ulcerative colitis include~ inteme neutrophil infiltration.In vivo studies with radiolabclled peripheral blood neutrophils demonstrate the movement of large numbers of neutrophils out of the circulation into the inflamed mucosa and the intestinal lumen (20).
This process suggests the presence of a potent chemotactic agent in the intestinal mucosa which r,romotcs the passage of infiltrating neutrophils between endothelial cells, through the basement membrane and into the intestinal tissue.
Among the soluble mediators of inflammation that arc important neutrophil chemotactic agents arc: CSa, which is part of the complement cascade; bacterial-derived peptides including formylmethionlleucylphcnylalanine; and the arachidonate metabolites LTB4 (21) and 5-HETE.To determine which, if any, of these compounds was the mediator of neutrophil infiltration of the mucosa in IBD a study was performed using a Boyden chamber with 51Cr-labcllcd neutrophils in the upper chamber and either a chemoattractanr or buffer in the lower chamber (22).Two filters, one on top of the other, separated the two chambers.After a 3 h incubation perio<l, the amount of rndioact1vity present in each filter was determined and the results expressed as the percent of total radioactivity present in the lower of the two filters.When buffer alone was present in the lower chamber almost none of the neutrophils penetrated the upper filter and entered the lower filter.When a chemotactic agent was present in the lower chamber a significant portion of the cells penetrated the upper fi l tcr and entered the lower.A dose-response curve w,1' obtained when various amounts of synthetic L TB4 were placed in the lower chamber.Next, homogenates of human colornc mucosa were added to the lower chamber at concentraunns of 2 mg/ml, 20 mg/ml and 100 mg/ml.The chemotactic response to ulceranvc colitis mucosa was more than 20 times that to normal mucosa and the response to Crohn's colitis mucosa was more than 10 times that to normal mucosa.
To characterize the nature of the chemotactic agent in ulcerative colitis mucosa the chemotactic activity in homogenates of ulcerative colitis mucusa was compared to th,it in the lipid extracts of the homogenates.
Results showed chemotactic activity in the lipid extract to he between 78% and 90% of the chcmotactic activity in the total homogenate, suggesting that much of the chemotactic activity was present as a lipid.Fractions obtained by HPLC separntton of the lipid extracts from ulcerative colitis mucosa were utilized in the chemotaxis assay.Only the fraction that co-eluted with L TB4 contained a significant amount of chemurnctic activity, indicating that L TB4 was the predominant chemotactic agent in ulcerative colitis mucosa!extracts.

DRUG THERAPY OF IBO AND THE UPOXYGENASE PATHWAY
The two most widely used medical therapies for IBO arc corticosteroids and sulphasalazinc.Each Llf them block the synthesis of lipoxygcnase products in vitro, but each also has other r,harmacologic properties unrelated to arnchidonatc metabolism which may contribute to their therapeutic efficacy.Corticosteroids induce the synthesis of lipomodulin which blocks phospholipase AZ and thus limits the availability of arachidonatc, the substrate for the cyclooxygenasc and lipoxygenase pathways (1).
Sulphasalazine is metabolized to 5-ASA and sulphapyridine.While it is thought that sulphapyridinc is responsible for the unde:,irable side effect:, of sulphasalazinc and that 5-ASA is the therapeutic agent, there is substantial evidence that tht: parent compound, ,ulphasalazine, possesses pharmacologic properties dbtinct from those of 5-ASA.Both sulphasalazme and 5-ASA inhibit cyclonxygenase and 5ltpoxygenase.In addimm, sulphasalazinc mhibits thromboxane ~ynthcrnse.One of the difficulties in determining therapeutically rcle\'ant pharmacologic effects is determ 111mg the appropri:He concentrations of the:,c compounds for study.In treated patients the concentration:, of these cnmpLlunds in stool are enormous, ie, l mm for sulr,hasalazinc and 1 OmM for 5-ASA (2 3 ).However, they are poorly ahsnrbed and the serum concentrations me quite low.Thus, high concentral inns nf thc~e agents arc observed on the lumenal side of the inflamed mucosa while, ar the same time, concentrations in the capillaries are minimal.The concentration of drugs to which relevam cells in the mucosa arc exposed is unclear.
When rested in in vitro assay systems at concentrations found in the colonic lumen these compound:, exert many pharmacologic effects, including inhihirion of arachidonic metabolism; however, when tested at concentrations found in the serum, their pharmacol,lgic effecb arc relatively minimal.For example, sulphasalazine inhibits a numhcr of steps in both the cyclooxygenllsc pathway aml the lipoxygenasc pathway at a concentration of approximately l mM, which is the cnncenmuion found in the colonic lumen hut ntit m the bloodstream.However, hoth corticosteroids and sulphasalazinc have other pharmacologic properties unrelated to arachidonate metabolism which may contribute to their therapeutic efficacy in IBD.Corticosteroids affect lymphocyte proliferation whereas sulphasalazine blocb lymphocyte cytoroxicity (24 ).
Either of these effects could contribute to their therapeutic efficacy in IBD.
ACKNOWLEDGEMENT: This work wa, supported by Nil-I gram AM33 l 65 ,ind ,1 grant from the National Foundau on fur llc1t1s and Coliw,.