Inter-relationships between inflammatory mediators

Transcription

1 Gut 1993; 34: Department of Mediine and the Epithelial Membrane Researh entre, University of Manhester, Hope Hospital, Salford, Manhester T D Wardle L Hall L A Turnberg orrespondene to: T D Wardle, Department of Mediine, linial Sienes Building, Hope Hospital, Eles Old Road, Salford M6 8HD. Aepted for publiation 31 August 1992 Inter-relationships between inflammatory mediators released from oloni muosa in ulerative olitis and their effets on oloni seretion T D Wardle, L Hall, L A Turnberg Abstrat Metabolites of arahidoni aid have been impliated in the pathophysiology of ulerative olitis - they an stimulate intestinal seretion, inrease muosal blood flow, and influene smooth musle ativity. The influene on the muosal transport funtion of ulture medium in whih oloni muosal biopsy speimens had been inubated was investigated using rat stripped distal oloni muosa in vitro as the assay system. oloni tissue from patients with olitis and from ontrol subjets was ultured. Medium from inflamed tissue ontained more prostaglandin E2 (PGE2) and leukotriene D4 (LTD4) and evoked a greater eletrial (seretory) response in rat oloni muosa than ontrol tissue medium. In inflamed tissue, ylo-oxygenase inhibition (indomethain) attenuated PGE2 but inreased LTD4 prodution; onversely lipoxygenase inhibition (II 27968) inhibited LTD4 prodution but enhaned PGE2 output. Eah inhibitor alone enhaned the eletrial response in the rat olon. Inhibition of both enzymes (indomethain plus II 27968) aused a fall in both PGE2 (82%) and LTD4 (89%) prodution and in the eletrial response (57%). Inflamed tissue treated with a phospholipase A2 inhibitor (meparine) produed less PGE2, LTD4, and eletrial responses when ompared with inflamed tissue, either untreated (91%, 92%, and 79% respetively) or treated with ylo-oxygenase and lipoxygenase inhibition. Inubation with bradykinin stimulated eiosanoid release and eletrial response, while a bradykinin antagonist aused a modest inhibition. Analysis of these observations suggests that a ombination of arahidoni aid derivatives aounts for about half the seretory response. Other produts of phospholipase A2 ativity are probably responsible for muh of the remainder, leaving up to 2% the result of types of mediator not determined in this study. (Gut 1993; 34: 53-58) High onentrations of prostaglandins and leukotrienes have been found in stool water and oloni muosal biopsy speimens from patients with ulerative olitis'-7 and these have been impliated in the pathophysiology of inflammatory bowel disease. They may not only be involved in the mediation and amplifiation of the immune response but several have also been shown to stimulate muosal seretion, inrease muosal blood flow, and influene smooth musle ativity, eah of whih may be relevant to the diarrhoea that these patients suffer.8"1 Beause suh a wide variety of inflammatory mediators is liberated in olitis it is diffiult to asertain whih, either alone or in ombination, might be responsible for the assoiated hanges in intestinal funtion. We desribe studies of the influene on intestinal seretion of inflammatory mediators released into the medium in whih biopsy speimens of inflamed oloni muosa were ultured. We used rat oloni muosa in vitro as our 'assay' system for determining seretory responses. Studies of the effet of a variety of inhibitors on these responses, and on the release of a number of mediators, have allowed us to show that a ombination of prostaglandins and leukotrienes are probably responsible for over half of the seretory response, and that other produts of phospholipase A2 ativity are probably responsible for muh of the remainder. Methods 53 PATIENT DETAILS Thirty patients underwent olonosopy after bowel preparation. Preparation onsisted of a three day low residue diet, and one day before the examination a ombination of X prep (purified senna extrat; 1 ml/kg body weight) and 1% mannitol (5 ml) modified aording to the patient's symptoms. Ten of the patients (four men and six women, median age 36 years) had a linial diagnosis of irritable bowel syndrome. They all had endosopially and histologially normal muosa. Twenty patients (13 men and seven women median age 39-4 years) with ative distal protosigmoiditis had biopsy speimens taken from inflamed muosa. Six patients were taking mesalazine (4 mg three times daily), four prednisolone (5 to 15 mg one daily), and seven topial steroids at the time of olonosopy. All biopsy speimens were taken with non-spiked foreps to minimise tissue trauma. Ethial approval for these studies was given by the Salford Health Authority Ethis ommittee. EXPLANT ULTURE A series of ultures was performed for eah patient. Muosal biopsy speimens were immediately plaed in transport medium (L15, with added peniillin G and streptomyin sulphate), transferred to the laboratory, washed gently three times in the Ll5 medium, arefully Gut: first published as /gut on 1 April Downloaded from on 1 Deember 218 by guest. Proteted by opyright.

2 54 Wardle, Hall, Turnberg blotted, weighed (range 3-8 mg), and plaed in a 5 m ulture dish ontaining 1 ml of ulture medium (MRL) 166, plus gluose 5 [ig/ml, methionine 1,uM/ml, Tris buffer 2 mm, glutamine 3 [im/ml, 3-retinyl aetate 1 [tg/ml, peniillin G 1 units/ml, streptomyin sulphate 1 ig/ml, gentamiin 5 [ig/ml, and amphoterain 1-25 [ig/ml). Individual muosal biopsy speimens were ultured with either no additives or in the presene of one of the following: indomethain (ylo-oxygenase inhibitor); II (lipoxygenase inhibitor); a ombination of indomethain and II 27968; meparine (phospholipase A2 inhibitor) (all at 1-'M); and bradykinin, or des arg leu bradykinin (bradykinin antagonist) (both at 1-8M). The ulture dishes were plaed in a humidified hamber maintained at 37, supplied with a mixture of 95% oxygen/5% arbon dioxide, and rotated at 1 yles/minute. After 4 hours of ulture the medium was removed and divided into three aliquots for measurement of PGE2, LTD4, and eletrial responses in rat oloni muosa. EIOSANOID MEASUREMENTS PGE2 and LTD4 were measured using ommerially available radioimmunoassay kits (PGE2, du Pont UK, Stevenage, Herts, UK"; LTD4, Amersham, Aylesbury, Buks, UK'2). Eiosanoids were extrated from the ulture medium using solid phase sorbant extration (mini olumns, Amersham). The resultant sample ompetes with a fixed amount of radioatively labelled eiosanoid analogue (iodinated PGE2 or tritiated LTD4) for a limited number of binding sites. The sample PGE2 antibody omplex is separated from the free antigen by polyethylene glyol preipitation and entrifugation, and then ounted in a gamma ounter. Separation of the leukotriene bound antibody omplex was failitated using dextran oated haroal. After entrifugation the quantity of antibody bound radioative ligand was measured on a beta ounter. Assay performane harateristis Assay performane harateristis were as follows. PGE2 intra and interassay variation values were 11 pg/ml and 6 pg/ml respetively; reovery was 96% and sensitivity -8 pg/ml. ross reativity (non-e prostaglandins) was < 4%." LTD4 intra and interassay variation values were 14 pg/ml and 39 pg /ml respetively; reovery was 91% and sensitivity 5 pg/ml. ross reativity (non-sulphidopeptide leukotrienes) was <.1%.12 Eiosanoid onentrations were alulated by interpolation from a standard urve. All results were expressed in pmol/mg wet tissue/ hour. RAT DISTAL OLON PREPARATION An in vitro tehnique modified from that of Ussing and Zerahn was used.'3 Unfasted male Sprague-Dawley rats were killed and the distal olon was removed immediately and bathed in oxygenated buffer. Musle layers were stripped and the two most distal piees of muosa were mounted as sheets, between Perspex flux hambers, with a surfae area of -64 m2 (VT Plastis Ltd, Warrington, UK). The spontaneous, basal transmuosal potential differene (PD) was measured, on a high impedane digital volmeter via fine tipped eletrode bridges (3M K I in 3% agar) onneted to mathed alomel half ells. The short iruit urrent (Is) was delivered by silver/silver hloride eletrodes via IM NaI in 1% agar bridges. The eletrodes were onneted to a voltage lamp for automati short iruiting. The lamp was orreted for fluid resistane between the PD sensing bridges. Tissue ondutane and resistane were alulated from the PD and Is aording to Ohm's law. Eah muosal sheet was bathed on both sides with 5 ml of isotoni buffer ontaining: Na 146 mm; K 4-2 mm; l mm; HO mm; H2PO4-2 mm; HPO4 1 2 mm; a 1 2 mm; Mg 1-2 mm; and gluose 1 mm, at ph 7-4. The bathing media were stirred and oxygenated via a bubble lift system using 95% 2/5% O2 and were maintained at a onstant temperature of 37. ulture medium (1 1d) was added to the bathing fluid on the serosal aspet of rat oloni muosa after eletrial stability had been reahed, usually after 3 minutes. SERETORY AGONISTS Eiosanoids PGE2 or LTD4, in final onentrations ranging from 1- " to 1-4M, were added to the serosal aspet of stripped rat distal olon. hanges in PD, Is, and resistane were reorded. ulture medium ulture medium (1 il) was added to the serosal aspet of stripped rat olon and eletrial measurements, as desribed above, were reorded. The proess was repeated using either medium inubated with the inhibitors to at as ontrols or medium from biopsy speimens ultured with the inhibitors as detailed above. The resultant rise in Is was ompared with the PGE2 and LTD4 dose response urves. HEMIALS The 5 lipoxygenase inhibitor, II 27968, was kindly supplied by Dr R Dowell, Imperial hemial ompany, Alderley Edge, UK. Prostaglandin E2, bradykinin, des arg leu bradykinin, meparine, and indomethain were obtained from Sigma hemial o, Poole, Dorset, UK. LT D4 was purhased from asade Biohem Ltd, University of Reading, Berks, UK. ALULATIONS All values are expressed as the mean (SEM). Statistial omparisons were performed using paired and unpaired t tests. 4 Gut: first published as /gut on 1 April Downloaded from on 1 Deember 218 by guest. Proteted by opyright.

3 ** + Results 55 w 3 QL 3 N L- 2- D U) E (n L *7 - 'Doo D E *' D' ~~~ *' E D Q= Q- L -.- :E - O _~ Figure 1: Prostaglandins E2 prodution by ultured oloni muosa (pmol and pglmg wet tissue! hour mean (SEM)). n=1 ultureslolumn. =ontrol muosa; I=inflamed muosa; O=ylo-oxygenase inhibition (indomethain); LO=lipoxygenase inhibition (II 27968). *Signifiantly different from untreated ontrol tissue, p<-1; **signifiantly differentfrom untreated inflamed tissue, p< E n4 - ~~II I I I _ 3" 16 ~ - 1 i - 8-3j H ~~~~~~~~~~~~~4 -a L~~~~~~~~~~~~~~~ o~~~~~~~ E aa) E :. -~~~~~~~- -~~~~~~~~~~~2 (D Figure 2: Leukotriene D4 prodution by oloni muosa (pmol and pg/mg wet tissue/hour mean (SEM)) n= 1 ultures/olumn. =ontrol muosa;* I=inflamed muosa;* O=ylooxygenase inhibition (indomethain), LO=lipoxygenase inhibition (II 27968). *Signifiantly different from untreated ontrol tissue, p<-1; **signifiantly different from untreated inflamed tissue, p<-1. ~ i- ~- 1 6 E o i *3 5 2 tō ) M * * io as va * * - ) m me u m~~~~ m- Figure 3: The effet ofinflammatory mediators liberated from ultured oloni tissue on the seretory response ofrat oloni muosa; the effet ofagonists and antagonists. Is=short iruit urrent (ia.m 2- mean (SEM)). n=4 piees of stripped rat distal oloni muosalolumn. M=medium alone, =ontrol muosa; I=inflamed muosa; O=ylo-oxygenase inhibition (indomethain); LO=lipoxygenase inhibition (II 27968). *Signifiantly different from untreated ontrol tissue, p<-1; **signifiantly differentfrom untreated inflamed tissue, p<-o. EIOSANOID MEASUREMENTS Inflamed tissue produed signifiantly more PGE2 and LTD4 than ontrol tissue (2-79 (- 11) v 54 (-4); :1[73 (-11) v -44 ( 4) respetively, p<l) (all values are given in pmol/mg wet tissue weight/hour). The prodution rate of eiosanoids is also expressed graphially in both pmol and pg/mg wet tissue weight/hour; for example in inflamed tissue PGE2 prodution is 2-79 pmol or 112 pg/mg wet weight/hour and LTD4 is 1-73 pmol or 837 pg/mg wet weight/ hour. (Figs 1 and 2). ylo-oxygenase inhibition (indomethain, 1-5M) signifiantly redued PGE2 prodution when values were ompared with those in the untreated groups (inflamed -511 (-6) v 2-79 ( 11); ontrol -17 ( 2) v -54 (-4); p<l), whereas the yield of LTD4 was inreased (inflamed 2 4 ( 17) v 1-73 (- 11): ontrol 74 (-7) v 44 ( 4), p<oo1). A signifiant inrease in PGE2 prodution, by all groups, followed lipoxygenase inhibition (II 27968, 1-'M) (inflamed 3-9 (-22) v 2-79 (1 1); ontrol-91 (- 13)v -54 ( 4), p<-1), while LTD4 generation was redued (inflamed 16 ( 7) v 1-73 (-11); ontrol - 1 ( 1) v 44 (4), p<l). ombined inhibition of ylo-oxygenase and lipoxygenase produed almost idential PGE2 results to those found after indomethain alone (Fig 1), and LTD4 results to those found after II alone (Fig 2). In inflamed tissue, phospholipase A2 inhibition (meparine) appreiably redued the prodution of both PGE2 and LTD4 when ompared with values in untreated muosa (PGE2 21 ( 3) v 2-79 (411); LTD4 414 (-4) v 1-73 (-11) respetively; p< 1). The redution after meparine was also greater than that after ombined ylo-oxygenase and lipoxygenase inhibition (PGE2 21 (-3) v 5 (-5); LTD4-14 (-4) v -19 (-3) respetively, p<4). Meparine also signifiantly attenuated eiosanoid output by ontrol tissue (PGE2-11 (-14) v -54 (-4); LTD4-1 (-8) v -44 (-4); p<o-1). In omparison with the untreated group, bradykinin stimulated a signifiant inrease in both PGE2 prodution (ontrol 1-1 (- 18) v -54 (-4); inflamed 4-1 (-24) v 2-79 (-11); p<oool) and LTD4 prodution (ontrol 1-1 (-12) v -44 (-4); inflamed 4-1 (-17) v 1-7 (- 1 1); p<o-oo1). Inhibition of bradykinin resulted in a fall in PGE2 and LTD4 generation by all groups but only the reduiion found in the inflamed group reahed statistial signifiane (inflamed PGE2 2-3 (-14) v 2-79 (-11); LTD4 1-1 ( 1) v 1-73 (-11) respetively; p<ool, ontrol PGE2-34 (-5) v -54 (-4); LTD4-29 (-5) v -44 (-4); NS). EFFET OF ULTURE MEDIUM ON STRIPPED RAT DISTAL OLON Fresh ulture medium applied to the serosal half hamber did not produe any hange in baseline eletrial ativity (Fig 3). ulture medium inubated with the inhibitors did not influene Gut: first published as /gut on 1 April Downloaded from on 1 Deember 218 by guest. Proteted by opyright.

4 56 Figure 4: Prostaglandin E2 dose response urve in stripped rat distal olon. Values are mean (SEM). Number of rat muosal preparations under eah point. the basal Is. ulture fluid from inflamed tissue evoked a signifiantly larger Is inrease than fluid from ontrol muosa (31 (2 6) v 6-3 (1-3) [A.m-2; p<-1). ylo-oxygenase inhibition Medium from ontrol biopsy speimens ultured with or without indomethain produed similar rises in eletrial measurements. However, a signifiantly greater inrease in Is ourred with fluid derived from inflamed muosal biopsy speimens treated with indomethain, ompared with the untreated group (4 (2-5) v 31 (2-6) [ta.m-2; p<-1). Lipoxygenase inhibition Lipoxygenase inhibition with II did not influene the modest rise in eletrial ativity seen withthe ontrol biopsy medium. However, after lipoxygenase inhibition medium from inflamed tissue produed a signifiantly greater Is response than that from untreated ontrol tissues (43 5 (5) v 31 (2 6) [ta.m-2; p<'1). ombined lipoxygenase and ylo-oxygenase inhibition There was no signifiant differene between the short iruit response evoked by ulture medium from untreated ontrol tissue and ontrol biopsy speimens exposed to ombined ylo-oxygenase and lipoxygenase inhibition. ulture medium from inflamed tissue treated in the same way, however, produed a signifiantly lower Is response than medium from untreated tissues (13-4 (2 1) v 31 (2 6),uA.m-';p<25). Phospholipase A2 inhibition ontrol biopsy speimens inubated with meparine produed media whih provoked a signifiantly smaller Is response than untreated muosal media (2-5 (1) v 6-3 (1-3) ia m-2; p<o1). Medium from inflamed tissue exposed to meparine evoked an Is response that was signifiantly lower than that from either medium from untreated tissue or from tissues exposed to E a) 2 -.r5 (n -1 -j 12 1 E Wardle, Hall, Turnberg the ombined effets of ylo-oxygenase and lipoxygenase inhibition. (6A4 (1 1) v 31 (2 6) and 13 4 (2d1) [ia.m-2; respetively, p< 5). Bradykinin agonist The addition ofbradykinin to the ulture medium bathing both types of tissue produed a highly signifiant inrease in Is ompared with values in the untreated groups (ontrol 17 5 (2 8) v 6 3 (1-3): inflamed 66-1 (4 9) v 31 (2 6) [ta.m-2; p<l). Bradykinin antagonism Bradykinin reeptor blokade produed a fall in the Is for both tissue types, but only in the inflamed group did the hange reah statistial signifiane (31 (2 6) v 19-3 (3<1) [ta.m-2; p<l). DOSE RESPONSE URVES PGE2 PGE2 added to the serosal, but not the muosal, side of stripped rat distal olon aused a rapid inrease in Is, whih peaked after 2½/2 to 3 minutes. The dose response urve for PGE2 gave an E5 of 5 x 1-7M (Fig 4). PGE2 generated a parallel but smaller inrease in PD and a modest rise in tissue ondutane. LTD4 LTD4 added to the serosal aspet of stripped rat distal olon, evoked a rapid rise in Is, whih peaked after 2/2 minutes (Fig 5). The E5 for this response was 8 x 1-7M. The transmuosal PD also inreased while ondutane rose to a modest extent. ombined PGE2 and L TD4 At the peak Is response to PGE2 at 1-'M and 1-8M, LTD4 1-5M and 1-8M respetively were added to the serosal hamber. The ombined Is for 1-5M was 43.4 pa.m2 and for 1-8M it was 2-8 [ta.m2. These values were not signifiantly different from those expeted from the dose response urves (41*5 ta.m2: o 1-3- J -2- L) ' t - 6 (I) -1-4 f lo Leukotriene D4 (M) Figure 5: Leukotriene D4 dose response urve in stripped rat distal olon. Values mean (SEM). Number of rat muosal Prostaglandin E2 (M) preparations under eah point. 1 3 Gut: first published as /gut on 1 April Downloaded from on 1 Deember 218 by guest. Proteted by opyright.

5 Inter-relationships between inflammatory mediators releasedfrom oloni muosa in ulerative olitis and their effets on oloni seretion [A.m2 respetively - that is, there was no evidene of potentiation at either maximal or half maximal onentration. Disussion In these studies we have shown that inflamed tissue releases more eiosanoids into the ulture medium than ontrol muosa, and that medium from any of the tissues will evoke an eletrial response in stripped rat distal olon. A rise in eletrial potential differene and Is in this muosal preparation is assoiated with seretion of hloride ions and we have used a rise in eletrial measurements as a proxy for anion seretion. Inflamed tissue produed signifiantly more PGE2 and LTD4 than ontrol muosa, but the differene was not as great as that shown by retal dialysis.'5 16 Although the degree of indomethain indued ylo-oxygenase inhibition is variable,7 all groups showed signifiant attenuation of PGE2 prodution and a reiproal, signifiant inrease in LTD4 generation. onversely, inhibition of lipoxygenase ativity resulted in a suppression of LTD4 generation and stimulation of PGE2 synthesis. Presumably, the inreased availability of arahidonate for one enzyme system when the alternative route was bloked is responsible for these reiproal effets on PGE2 and LTD4 prodution. Beause PGE2 and LTD4 have eah been shown to stimulate intestinal seretion'8-21 it is not surprising to find that inhibiting the prodution of one of these did not redue the seretory (eletrial) response to medium, in whih a reiproal rise in the other had ourred. More diffiult to explain is the greater Is response to medium in whih one stimulant was appreiably depressed while the other was only moderately inreased. It is possible that the inhibition of the lipoxygenase or ylo-oxygenase pathways aused the synthesis ofmore potent seretagogues than simply the two measured in this study. Other potential ontenders for this role inlude prostaylin, a more potent seretagogue than PGE222 in the ase of lipoxygenase inhibition, and other leukotrienes (B4, 4) in the ase of ylo-oxygenase inhibition.2' This type of analysis leads to the onlusion that there is a finely balaned prodution of the major metabolites of arahidoni aid and that subtle alterations in the relative ativities of the enzymes onerned will ause variable responses. Blokade of a major pathway, as in this study, an thus produe unpreditable hanges in funtional responses. The failure of early attempts to treat ulerative olitis with ylooxygenase inhibitors may be expliable on this basis Simultaneous blokade of ylo-oxygenase and lipoxygenase pathways redued both PGE2 and LTD4 prodution, as might be expeted, and also attenuated the eletrial response of rat oloni muosa to this ulture medium. However, the 82% redution in PGE2 prodution and the 89% redution in LTD4 prodution was assoiated with only a 57% redution in transmuosal Is suggesting that other fators are probably involved in provoking the eletrial response. One possibility is that potentiation between the effets of low onentrations of these metabolites might our. Suh a potentiation would have to be greater at low than at high onentrations to aount for this observation and we did not find evidene of potentiation between PGE2 and LTD4 in our study. Thus, this seems a less likely explanation than the simultaneous generation of other types of seretory agonists. In favour of this are the results of phospholipase A2 inhibition with meparine. Here blokade of the enzyme responsible for arahidoni aid liberation aused a muh greater fall in the eletrial response of rat oloni muosa (from 57% after ombined lipoxygenase and ylo-oxygenase inhibition to 79% after phospholipase A2 inhibition). PGE2 and LTD4 prodution were also further redued (82% to 92% and 89% to 91% respetively), but these falls were muh less impressive than the fall in Is. It is interesting to ompare the eletrial responses to ulture media (with their measured eiosanoid onentrations) with the dose response urves for PGE2 and LTD4. It is lear that the onentrations of both eiosanoids measured in the ulture media are muh lower than those that might be expeted to provoke the eletrial responses whih were observed if their effets were simply additive. The mean eiosanoid onentrations derived from ulture medium from inflamed tissue were in the order of 4-45x 1-"M for PGE2, and 2 77x 1-"M for LTD4. As indiated by the dose response urves, no hange in Is would be expeted with these amounts, even if these onentrations were summated. The mean rise in Is evoked by medium from inflamed ultures was 3 [ta.m-2 whih would be expeted at onentrations of some 1-5M PGE2, or 1-6M LTD4, if these were the only mediators present. learly other mediators must also be involved in the eletrial response, but the degree of inhibition by meparine (79%) suggests that most are likely to be produts of phospholipase A2 ativation. It may be onluded from these observations that ylo-oxygenase and lipoxygenase produts aount for most (57%) of the eletrial response indued by medium from ultured oloni muosa. Only PGE2 and LTD4 were measured in these studies and sine they ould only be held responsible for part of the response, other ylooxygenase and lipoxygenase produts are likely to have ontributed. Moreover, other phospholipase A2 metabolites are probably responsible for a further 22% of the eletrial response. It seems most likely that this is due to a non-arahidoni aid derivative, platelet ativating fator being a reasonable ontender for this role. The remaining 21% of the eletrial response produed by medium from ultured inflamed biopsy speimens ould be aused by a variety of other mediators suh as histamine, 5-hydroxytryptamine and, possibly, transmitters liberated from neural tissues. Beause muh of the eletrial, seretory response to ulture medium ould be asribed to the release of produts of phospholipase A2 ativity it was of interest to investigate the Gut: first published as /gut on 1 April Downloaded from on 1 Deember 218 by guest. Proteted by opyright.

6 58 Wardle, Hall, Turnberg influene of one potentially important stimulus to phospholipase A2 ativity. Inflammatory ells, in partiular marophages have reeptors for bradykinin, a potent seretagogue ating almost entirely by stimulating arahidoni aid release via phospholipase A2 ativation. Its effet on intestinal muosa, at least in the rat ileum, is indiret, its site of ation being on subepithelial ells.2829 In our study bradykinin aused a notieable rise in eiosanoid release from ontrol and inflamed biopsy speimens and ulture medium from these aused a greater rise in Is in the rat oloni muosa model. Bradykinin reeptor blokade with des arg leu bradykinin aused a fall in eiosanoid output and the assoiated Is response, suggesting that bradykinin may be a stimulus to endogenous phospholipase A2 ativity in biopsy speimens of normal and inflamed oloni muosa. The fall in PGE2 and LTD4 prodution after bradykinin reeptor blokade was, however, less than that found after diret phospholipase A2 inhibition with meparine or after ombined lipoxygenase and ylo-oxygenase inhibition. Reasons for this smaller effet inlude the possibility that reeptor blokade with des arg leu bradykin was inomplete in these speimens. It is also likely that the other stimuli to phospholipase A2 ativity suh as interleukin 1,3 interleukin 8, and other monoyte derived growth fators,3' are liberated in these tissues. Although we have foussed on the seretory effets of these inflammatory mediators, it is lear that they are likely to have a number of other effets that will ontribute to the pathophysiology of the disease. The effets, on seretion, desribed here, however, provide one indiator of the tissue response in inflammatory disease. In onlusion, we have provided evidene in favour of the view that eiosanoids are the major inflammatory mediators ausing seretory responses in oloni muosa and that their prodution an be signifiantly redued by ombined ylo-oxygenase and lipoxygenase inhibition or by meparine or by a bradykinin antagonist. The maximal redution in eiosanoid release and seretory response was ahieved with meparine and this may warrant further linial evaluation of its therapeuti role in inflammatory bowel disease. are should be taken in the assessment of drugs whih influene inflammatory mediator metaboli pathways sine disturbane of these omplex interations and balanes may produe unpreditable results. We are grateful to Dr R Dowell of the Imperial hemial Industries for donating the 5-lipoxygenase inhibitor. We are indebted to the Ileostomy Assoiation for their finanial support and interest in our work. We would also like to thank arol MDonna for typing the manusript. 1 Gould SR. 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