Endothelin 1 Induces Leukocyte Adhesion in Submucosal Venules of the Rat Small Intestine

Transcription

1 GASTROENTEROLOGY 1998;114: Endothelin 1 Induces Leukocyte Adhesion in Submucosl Venules of the Rt Smll Intestine MIHÁLY BOROS,* STEFFEN MASSBERG,* LAJOS BARANYI, HIDECHIKA OKADA, nd KONRAD MESSMER* *Ludwig-Mximilins University, Institute for Surgicl Reserch, Klinikum Grosshdern, Munich, Germny; nd Deprtment for Moleculr Biology, Ngoy City University, Ngoy, Jpn Bckground & Aims: The relese of endothelin 1 (ET-1) nd the ctivtion of leukocytes re involved in the pthophysiology of gstrointestinl ischemi/reperfusion injuries. The im of this study ws to define the in vivo reltion between ET-1 nd endothelil cell leukocyte interctions. Methods: Anesthetized rts were studied to chrcterize the microvsculr effects of incresing doses of locl nd systemic infusions of ET-1 in ll lyers of n ilel segment. Leukocyteendothelil interctions were monitored with intrvitl fluorescence videomicroscopy. The ET A receptor selective ntgonist BQ 610, the novel ET A -receptor ntgonist ETR-Pl/fl peptide, nd the ET B -receptor ntgonist IRL 1038 were used to investigte the roles of receptor subtypes. Results: The functionl cpillry density of the mucos ws significntly decresed by 3 nmol/kg intrvenous ET-1. After 30 minutes the rolling frction of leukocytes reched 90% in the postcpillry venules, nd the number of dherent leukocytes ws significntly incresed fter 90 minutes. ETR-Pl/fl peptide inhibited leukocyte rolling by 88%, BQ 610 by 73%, nd IRL 1038 by 30%. Both ET A -receptor ntgonists prevented ET-1 induced firm dhesion. The ET A - receptor ntgonists but not IRL 1038 inhibited the ET-1 induced lymphtic muscle nd mucosl cpillry perfusion filure. Conclusions: ET-1 induces leukocyte rolling nd dherence through predominntly ET A receptor medited mechnism in the submucosl venules of the intestinl microcircultion. The endothelins (ETs) re fmily of 21 mino cid peptides produced by vsculr endothelil cells. 1 Three ctive isoforms (ET-1, ET-2, nd ET-3) nd two specific receptors for ET, the ET A receptor nd the ET B receptor, hve been identified nd cloned. 2 The ET A receptor medites vsoconstriction nd hs high ffinity for ET-1, wheres the ET B receptor medites vsoconstriction (ET B2 ) nd vsodiltion (ET B1 subtype) nd hs equl ffinity for ET-1 nd ET-3. 3 ET-1 is the most powerful vsoconstrictor known to dte, nd it my prticipte in regultion of the smooth muscle tone in mny systems in both physiologicl nd pthophysiologicl settings. 2 The plsm levels of ET-1 re enhnced during experimentl nd clinicl ischemi, hypoxi, sepsis, nd bcteremi, suggesting tht it my medite vsoconstriction of regionl vsculr beds during these conditions. 4 7 However, in ddition to the independent role of ET-1 s dominnt vsoconstrictor, some dt suggest tht ET-1 might contribute to the biologicl ctivities of the polymorphonucler leukocytes (PMNs). ET-1 hs been reported to induce locl leukocyte ccumultion nd PMN ggregtion, to stimulte PMN nd monocyte superoxide production, nd to increse cytosolic-free clcium concentrtion in vitro These properties rise the possibility tht n up-regulted ET relese my be ssocited with the ttchment of ctivted PMNs to the vsculr intim during pthologicl sttes, such s gstrointestinl ischemi-reperfusion injuries. This ssumption might be vlid, but there is no direct experimentl evidence to llow impliction of the in vitro findings to the in vivo vsculr effects of ET. First, to our knowledge, there hve been no studies showing the potency of ET-1 to trigger leukocyte endothelil cell interctions in vivo. Second, severl studies hve questioned the potency of ET-1 to ctivte PMNs or monocytes. In contrst with the positive findings, ET-1 does not induce leukocyte C 2 trnsient or ggregtion, ccumultion, chemotxis, or migrtion of PMNs or monocytes In the present study, the possible role of ET-1 s n inducer of leukocyte dhesion to the vsculr endothelium hs now been investigted by direct observtion of the microvsculr chnges in the smll intestinl microcircultion with fluorescence videomicroscopy system. The circultory effects of ET-1 might depend on the mode of dministrtion, suggesting divergence between the locl vsculr nd systemic hemodynmic responses. Abbrevitions used in this pper: ET, endothelin; FACS, fluorescence-ctivted cell sorter; FITC, fluorescein isothiocynte; HAES, hydroxyethyl strch; MAP, men rteril pressure; PMN, polymorphonucler leukocyte by the Americn Gstroenterologicl Assocition /98/$3.00

2 104 BOROS ET AL. GASTROENTEROLOGY Vol. 114, No. 1 The close-rteril infusion of ET-1 is known to induce extensive mucosl injury to the rt stomch nd intestine, wheres regionl vsodiltion my follow the intrvenous (IV) dministrtion of ET-1. 16,17 Thus, the locl nd systemic vsculr effects of ET-1 were chrcterized seprtely. In line with our gols, we determined whether pretretment with ET-receptor ntgonists would ttenute the responses to exogenous ET-1. ET receptors re found to be present in the gstrointestinl trct, ET-1 is bundnt in the smll intestine, nd the intestinl microcircultion is primry trget orgn of leukocyte-medited circultory disorders in mny types of circultory shock The importnce of endogenous ETs in pthologicl conditions hs creted n intensive serch to find pproprite ntgonists to ET receptors. Recently, Brnyi et l. 21 proposed new pproch to the design of biologiclly ctive peptides, bsed on the ntisense-homology box theory. ETR-Pl/fl is the first ntisense-homology box derived peptide tht hs potent nti ET A -receptor ctivity in vitro. We report the first dt on the in vivo effects of ETR-Pl/fl peptide on exogenous ET-1 induced microvsculr chnges in the rt smll intestine. As reference we used BQ 610, compound with specific ET A -receptor ntgonist properties. The possible roles of ET B receptors were tested by selective ET B -receptor ntgonist IRL 1038 pretretment. Mterils nd Methods The experiments were performed in ccordnce with the Germn legisltion on the protection of nimls. Seventythree mle Sprgue Dwley rts (verge weight, 180 g), housed in n environmentlly controlled room with 12-hour light-drk cycle, were deprived of food but not wter 12 hours before the experiments. The nimls were nesthetized with chlorl hydrte (360 mg/kg intrperitonelly) fter premediction with tropine (0.1 mg/kg subcutneously), nd plced in supine position on heting pd for mintennce of the body temperture between 36 nd 37 C, mesured vi rectl thermistor. Polyethylene ctheters (PE 50, ID 0.58 mm; F. Portex, Hythe, Englnd) were inserted into the left crotid rtery nd jugulr vein for the recording of men rteril pressure (MAP), hert rte nd centrl venous pressure, blood smpling, nd the injection of fluorescent dyes for intrvitl videomicroscopy. A trcheotomy ws performed, nd the nimls were mechniclly ventilted with n inspired O 2 concentrtion (FiO 2 ) of 0.33 with rodent respirtor (Hrvrd Apprtus, South Ntick, MA). Throughout the experiment, the nimls received n infusion of Ringer s lctte t rte of 40 ml kg 1 h 1 nd 1 mg 100 g 1 h 1 chlorlose (Merck, Drmstdt, Germny), to mintin euvolemi nd nesthesi, respectively. After trnsverse lprotomy, segment of the terminl ileum perfused by single rtery ws selected. The mrginl vessels were divided nd ligted under Leitz dissecting microscope (Leitz, Wetzlr, Germny) to eliminte ny possible connection with the systemic circultion other thn the perfusing ilel rtery. The ileocolic rtery ws cnnulted (PE 50, ID 0.28 mm) just distl to the ilel brnch perfusing the selected segment, to mesure mesenteric blood pressure nd to llow injection of the test compounds. The intestinl segment with intct neurovsculr connections ws plced on specilly designed stge, nd n pproximtely 2-cmlong longitudinl incision ws then mde ntimesentericlly with microcutery to open the distl, borl prt of the segment. The ilel segment ws covered with glss coverslip, nd the microcircultion of the seros, longitudinl nd circulr muscles, submucos, nd mucos could be observed nd nlyzed by vrying the depth of focus of the microscope over the intct orl, or open borl prt of the segment. During the entire in vivo microscopic procedure, the tissue ws constntly bthed in 37 C Ringer s lctte to void drying nd exposure to mbient ir. Intrvitl Fluorescence Microscopy We used modified Leitz-Orthopln microscope with 100-W HBO mercury lmp ttched to Ploemo-Pk illumintor with n I 2/3 blue nd N 2 green filter block (Leitz, Wetzlr, Germny). The intestinl microcircultion ws nlyzed by using n epi-illumintion technique. With 10 (long distnce) nd 25 (wter immersion) objectives (W 25 /0.6; Leitz), the mgnifiction on the video screen (PVM-1442 QM, digonl 33 cm; Sony, Munich, Germny) ws 180 nd 450, respectively. The microscopic imges were recorded by chrge-coupled device video cmer (FK 6990) nd trnsferred to video system for off-line evlution. Contrst enhncement ws chieved by IV injection of 0.2 ml 0.75% fluorescein isothiocynte (FITC)-lbeled hydroxyethylstrch (HAES) (mol wt, 200,000; Levosn, Linz, Austri). Leukocytes were stined in vivo by mens of rhodmine 6G (mol wt, 479, 0.2%, 0.1 ml IV; Sigm Chemicl Co., St. Louis, MO). Video Anlysis Quntittive ssessment of microcircultory prmeters ws performed off-line by frme-to-frme nlysis of the videotped imges. The cpillry density of lymphtic microvessels stined by the FITC-HAES drining into the lymphtic cpillry network ws determined with computer-ssisted imge nlysis system (CAMAS Zeintl, Heidelberg, Germny). Functionl cpillry density, length of perfused nutritive cpillries per observtion re (cm 1 ), nd red blood cell velocity (mm/s) were mesured in five seprte fields of the circulr nd longitudinl muscle lyers of the intestine. Similrly, functionl cpillry density, length of perfused cpillries per villus re (cm 1 ), nd cpillry red blood cell velocity (mm/s) were determined in five fields of the exposed mucos. Leukocyte endothelil cell interction ws nlyzed within five postcpillry nd five intrmurl collecting venules per niml, including the observtion of nondherent, dherent, nd rolling leukocytes. Adherent leukocytes (stickers) were defined in ech vessel segment s cells tht did not move or detch from the endothelil lining within n observtion period of 30 seconds, nd re given s number of cells per

3 Jnury 1998 ENDOTHELIN INDUCED LEUKOCYTE ADHESION 105 squre millimeter of endothelil surfce, clculted from the dimeter nd length of the vessel segment observed, ssuming cylindricl geometry. Rolling leukocytes were defined s cells moving t velocity less thn two fifths of tht of erythrocytes in the centerline of the microvessel, nd their numbers re given s percentge of the number of nondherent leukocytes pssing through the observed vessel segment within 30 seconds. Extrvstion of the fluorescein mrker ws investigted during ech observtion period by clculting the rtio of extrvsculr vs. intrvsculr fluorescence intensity with computerssisted nlysis. Thirty-minute video records were required to complete the mesurements. Experimentl Protocol After stbiliztion period, bsl crdiovsculr prmeters were mesured for 20 minutes nd intrvitl videomicroscopy ws then performed to estblish bseline microvsculr vribles in ll groups. In the first series of experiments, dose responses to ET-1 were obtined t concentrtions of 0 (shm-operted), 0.1, 1, nd 3 nmol/kg body wt ET-1 (Alexis Corp., Läufelfingen, Switzerlnd). Fifty minutes fter the end of the first microscopy, solution of 0.1 ml ET-1 or vehicle ws continuously infused IV into the systemic circultion with syringe pump (WPI. S250i Pump, Srsot, FL) over 1 minute. The nimls were llotted into one or other of the following groups: ET 0 group, shm-operted (n 5), ET nmol/kg IV group (n 5), ET-1 1 nmol/kg IV group (n 5), nd ET-1 3 nmol/kg IV group (n 5). An dditionl group of nimls (n 5) served s positive control nd received 90 nmol/kg IV norepinephrine hydrochloride (Arterenol, Hoechst AG, Frnkfurt m Min, Germny) in 0.1-mL sline infusion. In three seprte groups of nimls, the sme mounts of ET-1 were dministered in close-rteril infusions through the cnnulted ileocolic rtery into the locl circultion of the intestinl segment: ET nmol intr-rteril (IA) group (n 5), ET-1 1 nmol/kg IA group (n 5), nd ET-1 3 nmol/kg IA group (n 3). In ech group, intrvitl videomicroscopy ws performed 30 nd 90 minutes fter the end of infusion of test mterils or vehicle, respectively. In the second series, ET-receptor blockers lone or in combintion with 3 nmol/kg ET-1 infusion were dministered. In these groups, the infusion of ET A -receptor ntgonist BQ 610 (0.3 mol/kg; homopiperidinyl-crbonyl-leu-d-trp- (CHO)-D-Trp-OH; Alexis Corp.), ET B -receptor ntgonist IRL 1038 (0.3 mol/kg; Cys11-Cys15-endothelin-1 [11 21]), or ETR-Pl/fl peptide (0.3 mol/kg; VLNLCALSVDRYRA- VASWRVI; Kurbo Ltd., Osk, Jpn) ws strted fter the end of control microscopy. A bolus of 0.1 mg/kg BQ 610 ws dministered, followed by constnt infusion of 4 g kg 1 min 1 BQ 610 for 30 minutes (n 5). In experiments with ETR-Pl/fl, the peptide ws continuously infused IV for 30 minutes in dose of 25 g kg 1 min 1 (n 5). IRL 1038, specific ET B -receptor blocker, ws infused in dose of 0.3 mol/kg for 30 minutes (n 5). In these groups, 3 nmol/kg ET-1 or vehicle ws infused IV into the systemic circultion 20 minutes fter the end of BQ 610, ETR-Pl/fl peptide, or IRL 1038 pretretment. Blood gs vlues were regulrly mesured throughout the experiments. Leukocyte counts nd hemtocrit vlues were determined from 0.1-mL blood smples withdrwn from the crotid ctheter t the beginning nd t the end of ech experiment using Coulter Counter T 540 (Coulter Electronics, Hileh, FL). At the end of the observtion period, tissue smples were obtined from the intestinl segment, nd the nimls were then killed with n overdose of chlorl hydrte. Endothelin Assy Plsm ET-1 levels were mesured in shm-operted nd in systemic IV ET-1 treted groups. Blood smples of 0.1 ml were drwn from the crotid rtery t the beginning nd t the end of experiments nd were collected in chilled polypropylene tubes contining 1 mg/ml ethylenediminetetrcetic cid (EDTA) nd 500 KIU/mL protinin (Trsylol; Byer, Leverkusen, Germny). The blood smples were centrifuged t 3000g for 10 minutes t 0 C, nd the plsm smples were collected nd stored t 70 C until ssy. ET-1 ws extrcted by using Amprep minicolumns for smple preprtion (Amershm, Buckinghmshire, Englnd). Plsm levels of ET-1 were mesured with rdioimmunossy kit (Endothelin-1,2 125 I high-sensitivity ssy system; Amershm). According to the mnufcturer, the cross-rectivity with ET-3 ws 0.001%. Flow Cytometry The surfce expression of CD11b/c on circulting grnulocytes ws mesured by flow-cytometric nlysis, using phycoerythrin-lbeled mouse nti-rt monoclonl ntibody (OX-42; Phrmingen, Sn Diego, CA). Fifty-microliter blood smples were drwn from the crotid rtery into EDTA contining cells t the beginning nd t the end of ech experiment nd were incubted with sturting mounts (5 L) of conjugted monoclonl ntibody for 30 minutes t 4 C in the drk. For the removl of contminting red blood cells nd for fixing leukocytes, lysing solution (FACS Lysing Solution; Becton Dickinson, Heidelberg, Germny) ws used. The smples were centrifuged, wshed, nd resuspended in phosphte-buffered sline (PBS) before flow-cytometric nlysis ws performed. Negtive controls were obtined by omitting the monoclonl ntibody. The expression of the dhesion molecule on grnulocytes ws determined by using fluorescence-ctivted cell sorter (FACS) Anlyzer Flow Cytometer (Becton Dickinson). PE fluorescence signls were collected fter excittion t nm, using stndrd bnd pss filters. In generl, mximum of 10 4 events ws collected for ech smple. Antibody binding ws determined fter gting for grnulocytes by their chrcteristic volume nd side-sctter properties. Fluorescence histogrms were used to clculte men fluorescence intensities nd to ssess the percentges of positively stined cells. Sttistics Dt nlysis ws performed with sttisticl softwre pckge (SigmStt for Windows, Jndel Scientific, Erkrth, Germny). The Kruskl Wllis test followed by Dunn s method

4 106 BOROS ET AL. GASTROENTEROLOGY Vol. 114, No. 1 ws used for the estimtion of stochstic probbility in intergroup comprisons. Friedmn repeted mesures for nlysis of vrince ws pplied for multiple comprisons with control. Men vlues SEM re given. P vlues of 0.05 were considered significnt. Results Bseline vlues of MAP nd other mcrohemodynmic vribles were not significntly different between the groups (dt not shown). The bseline vlues of lymphtic cpillry density were significntly lower (29 40 cm 1 ) thn the vlues of functionl cpillry density of the muscle nd mucos. The bseline vlues of functionl cpillry density of mucos ( cm 1 ) were significntly higher thn the functionl cpillry density of the muscle; likewise, the functionl cpillry density of the circulr muscle lyer ( cm 1 ) ws significntly higher thn the bseline functionl cpillry density of the longitudinl muscle ( cm 1 ). A mximum of 9.5% nd 24% of nondherent leukocytes rolled long the endothelil lining of the submucosl collecting nd postcpillry venules, respectively, during bseline conditions. In the shm-operted control group, no significnt chnge ws observed in lymphtic cpillry density, muscle, nd villus functionl cpillry density or red blood cell velocity. Similrly, there were no significnt chnges in the number of rolling nd dherent leukocytes in the control group throughout the experiments. Study 1: Effects of Systemic IV Administrtion of ET-1 An pproximtely 60% decrese in serosl lymphtic cpillry density ws the result of 0.1 nmol/kg ET-1 nd induced significnt reduction in the functionl cpillry density of the longitudinl muscle lyer 90 minutes fter IV ET-1 infusion. A 10-fold elevtion of the dose resulted in significnt decreses in subserosl lymphtic cpillry density, functionl cpillry density, nd red blood cell velocity of both longitudinl nd circulr muscle lyers 30 nd 90 minutes fter infusion. In this group, the functionl cpillry density nd red blood cell velocity in the mucosl villi were likewise significntly ltered. IV infusion of 3 nmol/kg ET-1 induced 62% nd 63% decreses in the cpillry red blood cell velocity in the longitudinl nd circulr muscles, respectively. Similrly, the functionl cpillry density of the mucosl villi ws decresed by 34% nd 41% 30 nd 90 minutes fter ET infusion, respectively, with concomitnt 35% decrese in the villus cpillry red blood cell velocity by the end of the observtion period (Tble 1). Dt on primry nd secondry leukocyte-endothelil cell interctions in the submucosl postcpillry venules re presented in Figures 1 nd 2. Dt on leukocyte interctions in the submucosl collecting venules re shown in Tble 2. The number of firmly dherent nd rolling leukocytes pssing collecting nd postcpillry venule vessel segments ws slightly enhnced 90 minutes fter the dministrtion of 0.1 nmol/kg IV ET-1. However, the percentge of rolling leukocytes in the submucosl collecting venules reched 40% 90 minutes fter the dministrtion of 1 nmol/kg ET-1. Simultneously, significnt increse in the number of firmly dherent leukocytes ws observed 90 minutes fter the infusion of 1 nmol/kg ET-1. Elevtion of the dose to 3 nmol/kg induced ner-mximl effect on the leukocyte rolling phenomenon. In this group, ggregtes of 2 4 leukocytes were occsionlly observed in the collecting venules, nd the rolling frction of circulting leukocytes ws 90% nd 85% in the postcpillry nd collecting venules, respectively, 30 minutes fter ET-1 infusion (Figure 1). Tble 1. Microcircultory Prmeters 90 Minutes After Administrtion of ET-1 or Vehicle Shm-operted 0.1 nmol/kg IV ET-1 1 nmol/kg IV ET-1 3 nmol/kg IV ET nmol/kg IA ET-1 1 nmol/kg IA ET-1 Mucos FCD (cm 1 ) b,c b,c b,c RBCV (mm/s) c c c Circulr muscle FCD (cm 1 ) c b,c b,c b b,c RBCV (mm/s) Longitudinl muscle FCD (cm 1 ) c RBCV (mm/s) b,c b,c c c Subseros LCD (cm 1 ) c c c b,c FCD, functionl cpillry density; LCD, lymphtic cpillry density; RBCV, red blood cell velocity. Complete cesstion of microvsculr flow. b P 0.05 vs. shm-operted control. c P 0.05 vs. bseline vlues. Kruskl Wllis nd Friedmn test. 3 nmol/kg IA ET-1

5 Jnury 1998 ENDOTHELIN INDUCED LEUKOCYTE ADHESION 107 Figure 1. Primry leukocyte endothelil cell interction in submucosl postcpillry venules of shm-operted (n 5) controls nd nimls tht received n IV ET-1 (gry brs) or locl, intr-rteril (white brs) ET-1 infusion. Leukocytes were stined in vivo with rhodmine 6G nd clssified s rolling, dherent, or nondherent cells. Numbers of rolling leukocytes re given s percentge of the number of ll leukocytes pssing through the observed vessel segment within 30 seconds. Observtions were mde during bseline conditions, then 30 nd 90 minutes fter dministrtion of 0.1 nmol/kg IV ET-1 (n 5), 1 nmol/kg IV ET-1 (n 5), 3 nmol/kg IV ET-1 (n 5), 0.1 nmol/kg IA ET-1 (n 5), 1 nmol/kg IA ET-1 (n 5), or 3 nmol/kg IA ET-1 (n 3). *Complete cesstion of microvsculr flow. Vlues re mens SEM. # P 0.05 vs. shm-operted group. P 0.05 vs. bseline vlues. Kruskl Wllis one-wy nlysis of vrince on rnks nd Friedmn test., ET-1, intrvenous; b, ET-1, close-rteril. These vlues correspond to 15- nd 22-fold elevtions in the rolling frction of the leukocytes compred with the bseline vlues. The percentge of rolling leukocytes ws slightly decresed 90 minutes fter ET-1 infusion, but concomitntly significnt increse in the number of sticking leukocytes ws observed in both vessel types studied (Figure 2). Although there ws tendency for the bckground fluorescence to increse with time in groups to which ET-1 ws dministered, the chnge ws sttisticlly not significnt throughout the experiments. Muscle nd villus functionl cpillry density nd red blood cell velocity decresed 30 nd 90 minutes fter the dministrtion of 90 nmol/kg norepinephrine; these chnges were comprble to those observed fter 3 nmol/kg ET-1 (dt not shown). There were no significnt chnges in the number of rolling nd dherent leukocytes in this group throughout the experiments. The rolling frction of leukocytes ws nd in the postcpillry nd collecting venules, respectively, 90 minutes fter the infusion. Figure 2. Secondry leukocyte endothelil cell interction in submucosl postcpillry venules. Adherent leukocytes (stickers) were determined in ech vessel segment s cells tht did not move or detch from the endothelil lining within n observtion period of 30 seconds nd re given s numbers of cells per squre millimeter of endothelil surfce. *Complete cesstion of microvsculr flow. Vlues re mens SEM. # P 0.05 vs. shm-operted group, P 0.05 vs. bseline vlues. Kruskl Wllis one-wy nlysis of vrince on rnks nd Friedmn test., ET-1, intrvenous; g, ET-1, close-rteril.

6 108 BOROS ET AL. GASTROENTEROLOGY Vol. 114, No. 1 Tble 2. Leukocyte Endothelil Cell Interctions in Submucosl Collecting Venules of Shm-Operted Controls nd After ET-1 Infusions Shm-operted 0.1 nmol/kg IV ET-1 1 nmol/kg IV ET-1 3 nmol/kg IV ET nmol/kg IA ET-1 1 nmol/kg IA ET-1 3 nmol/kg IA ET-1 Rolling leukocytes Bseline min b b,c c min b b,c c Adherent leukocytes Bseline min b,c min b,c b,c NOTE. Observtions were mde during bseline conditions, then 30 nd 90 minutes fter dministrtion of 0.1 nmol/kg IV ET-1 (n 5), 1 mmol/kg IV ET-1 (n 5), 3 nmol/kg IV ET-1 (n 5), or 0.1 nmol/kg IA ET-1, 1 nmol/kg IA ET-1 (n 5), 3 nmol/kg IA ET-1 (n 3), respectively. Vlues re mens SEM. Complete cesstion of microvsculr flow. b P 0.05 vs. shm-operted control. c P 0.05 vs. bseline vlues, Kruskl Wllis nd Friedmn test. Effects of Close-rteril ET-1 Doses of 0.1 nd 1 nmol/kg close-rteril ET-1 induced 70% nd 80% decreses in lymphtic cpillry density, respectively, nd no lymphtics could be observed fter the dministrtion of doses higher thn 1 nmol/kg (Tble 1). Locl infusions of 0.1 nmol/kg ET-1 induced pproximtely 20% reductions in muscle nd mucosl red blood cell velocity nd functionl cpillry density, similr to those observed fter 1 or 3 nmol/kg of IV pplied ET-1. Incresing the ET-1 dose to 1 nmol/kg resulted in pproximtely 50% decreses in mucosl functionl cpillry density nd red blood cell velocity by the end of the observtion period. When the dose of the peptide ws incresed to 3 nmol/kg, complete microcircultory rrest ws observed in ll lyers of the intestinl segment throughout the observtion period. Cpillry perfusion could not be observed in the longitudinl, or circulr muscle lyers, or in the mucosl villi even 90 minutes fter ET-1 dministrtion. The hemodynmic chnges induced by 0.1 nmol/kg close-rteril ET-1 were not ccompnied by significnt ltertions in the numbers of endothelil cell leukocyte interctions in the postcpillry nd collecting venules; only slight increse in dherent leukocytes ws observed. Elevtion of the dose of IA ET-1 to 1 nmol/kg resulted in significnt increse in the number of rolling leukocytes in the postcpillry venules 30 minutes fter the end of closerteril infusion (Figure 1). However, the increses in the numbers of rolling nd firmly dherent leukocytes were significntly lower thn those observed fter 1- or 3-nmol/kg doses of ET-1 dministered IV. In this group, the number of rolling leukocytes in the collecting nd postcpillry venules decresed towrd the bseline levels t the end of the experiments (Figure 1 nd Tble 2). Study 2: Effects of ET-Receptor Antgonist Pretretment Administrtion of ET-receptor ntgonists lone did not influence the microhemodynmic prmeters in either cse (dt not shown). In the subseros, ET-1 infusion resulted in prompt decrese in lymphtic cpillry density. The specific ET A -receptor ntgonist BQ 610 inhibited this chnge only t the first observtion time point, but the ET-1 induced impirment in lymphtic cpillry dringe ws completely prevented by pretretment with ETR-Pl/fl peptide. The ET B -receptor ntgonist IRL 1038 did not influence the decrese of this vrible; the lymphtic cpillry density vlues were even lower compred with the ET-treted group (Tble 3). In the BQ 610 pretreted group, no significnt chnge in mucosl functionl cpillry density could be shown 30 nd 90 minutes fter ET-1 dministrtion. Similrly, ETR-Pl/fl peptide pretretment completely prevented the ET-induced cpillry perfusion filure in the smll intestinl mucos. The cpillry red blood cell velocity ws significntly decresed fter dministrtion of ET-1, nd this effect ws significntly ttenuted by BQ 610 t the 30-minute observtion point. However, ETR-Pl/fl peptide pretretment completely prevented the ET-1 induced impirment of the cpillry red blood cell velocity. Pretretment of the nimls with IRL 1038 did not significntly influence the ET-1 induced decreses in mucosl functionl cpillry density nd red blood cell velocity (Tble 3). The functionl cpillry density vlues for the longitudinl nd circulr muscle lyers were not significntly different from the bseline vlues in the two groups involving ET A -receptor ntgonist pretretment. The ET B -receptor ntgonist IRL 1038 did not significntly

7 Jnury 1998 ENDOTHELIN INDUCED LEUKOCYTE ADHESION 109 Tble 3. Microcircultory Prmeters 90 Minutes After IV Administrtion of 3 nmol/kg ET-1 in Groups Pretreted With ET-Receptor Antgonists Shm-operted ETR-PI/II peptide pretretment BQ 610 pretretment IRL 1038 pretretment Mucos FCD (cm 1 ) ,b RBCV (mm/s) ,b b Circulr muscle FCD (cm 1 ) b RBCV (mm/s) b Longitudinl muscle FCD (cm 1 ) b RBCV (mm/s) b Subseros LCD (cm 1 ) b FCD, functionl cpillry density; LCD, lymphtic cpillry density; RBCV, red blood cell velocity. P 0.05 vs. shm-operted control. b P 0.05 vs. bseline vlues, Kruskl Wllis nd Friedmn test. modify this effect of ET-1 tretment. The red blood cell velocity in the muscle lyers ws mintined t the bseline level fter ETR-Pl/f1 pretretment, wheres BQ 610 nd IRL 1038 did not influence the ET-1 induced chnge in this microvsculr prmeter. None of the ET receptor ntgonists lone cused significnt chnges in the endothelil cell leukocyte interctions (dt not shown). Thirty minutes fter 3 nmol/kg ET-1 infusion, BQ 610 nd in prticulr ETR-Pl/fl peptide inhibited the leukocyte rolling evoked by exogenous ET-1 (Tble 4). Thirty minutes fter ET-1 dministrtion, ETR-Pl/fl ttenuted the leukocyte rolling in the postcpillry nd collecting venules by 88% nd 75%, respectively, vs. the 3 nmol/kg ET-1 only group. Seventy-three percent nd 68% inhibition, respectively, ws observed in nimls pretreted with BQ 610. At 90 minutes, the percentge of inhibition of ET-1 induced rolling ws 83% nd 73% in the postcpillry nd collecting venules fter ETR-Pl/fl pretretment, nd 57% nd 8% following BQ 610 pretretment, respectively. The selective ET B -receptor ntgonist IRL 1038 lso trnsiently prevented the ET-1 induced leukocyte rolling. Pretretment of the nimls with IRL 1038 led to 30% nd 43% inhibition of the increse in the rolling frction of the leukocytes 30 minutes fter ET-1 dministrtion in the postcpillry nd collecting venules, respectively, compred with the 30% nd 6% inhibition observed fter 90 minutes. Both ET A blockers were effective in preventing ET-1 induced firm leukocyte dherence throughout the 90-minute observtion period. The ET B -receptor ntgonist IRL 1038 did not influence the number of sticking leukocytes in the submucosl postcpillry venules but trnsiently reduced the number of stickers in the collecting venules 30 minutes fter ET-1 dministrtion (Tble 4). Tble 4. Leukocyte Endothelil Cell Interctions in Submucosl Collecting nd Postcpillry Venules Before nd After IV Infusion of 3 nmol/kg ET-1 in Groups Pretreted With ET-Receptor Antgonists ETR-PI/fI peptide pretretment BQ 610 pretretment IRL 1038 pretretment Collecting venules Postcpillry venules Collecting venules Postcpillry venules Collecting venules Postcpillry venules Rolling leukocytes Bseline min b 21 5 b 90 min b b 48 6 b Adherent leukocytes Bseline min b min ,b ,b NOTE. Observtions were mde during bseline conditions, then 30 nd 90 minutes fter dministrtion of 3 nmol/kg IV ET-1. The nimls were pretreted with ETR-PI/fI peptide (n 5), BQ 610 (n 5), or IRL-1038 (n 5). Vlues re mens SEM. P 0.05 vs. shm-operted control. b P 0.05 vs. bseline vlues, Kruskl Wllis nd Friedmn test.

8 110 BOROS ET AL. GASTROENTEROLOGY Vol. 114, No. 1 ET-1 Plsm Levels The bsl ET-1 concentrtion in the plsm ws pmol/l. In the control group, the plsm ET-1 concentrtion remined unchnged up to the end of the experiments. Doses of 0.1 nd 1 nmol/kg IV ET-1 did not significntly influence the ET-1 plsm levels 90 minutes fter the end of ET-1 infusion. A 3-nmol/kg IV dose of ET-1 incresed the plsm ET-1 concentrtion from to pmol/l by the end of the experiments. PMN Vlues In the control group no significnt chnge in the number of PMNs ws observed in blood smples drwn from the crotid rtery ( bseline nd t the end of the experiments). Similr vlues were obtined in groups prticipting in IV nd close-rteril ET-1 infusion nd ET-receptor ntgonist pretretment, nd no significnt differences were found between groups. Flow Cytometry The surfce expression of CD11b/c on grnulocytes ws detected. No significnt chnge in CD11b/c binding ws observed in blood smples obtined from nontreted nimls (from bseline to ). A slight, sttisticlly not significnt increse from to nd from to ws noted in response to 1 nd 3 nmol/kg IV ET-1 tretment, respectively. Qulittively nd quntittively similr effects on CD11b/c presenttion were observed fter pretretment with ET-receptor ntgonists. No significnt difference ws found between the ET-1 ntgonist pretreted nd nontreted groups with regrd to the expression of CD11b/c integrin. Discussion Impirment of nutritive perfusion nd ttchment of circulting leukocytes to the endothelil wll re key events in the development of cute inflmmtory response or ischemi-reperfusion injuries. Although it is cler tht cellulr responses to hypoxi or ischemi reperfusion involve multiplicity of fctors, our present study estblished the possibility of n instrumentl role of ET-1 in these microcircultory rections. The results show tht ET-1 my hve significnt effects on neutrophil ttchment nd intestinl microcircultory derngement t concentrtions higher thn those mesured in the serum, but coincide with those used to produce vsoconstrictor effect in experimentl systems. However, the hlf-life of ET-1 in the circultion is extremely short nd exogenous ET-1 is rpidly clered by the lung nd kidneys nd by binding to receptors disseminted in the circultion In pthologicl conditions, lrge mounts of ET re relesed into the regionl circultion; thus, the concentrtion of ET-1 could be very high in the locl environment. ET-1 is secreted predominntly in n bluminl direction, 2 nd thus it my be tht even smll chnges in endogenous ET-1 plsm levels elicit significnt involvement of the peptide in locl vsculr rections. In our experiments, plsm levels of ET-1 were unchnged up to the end of the 90-minute observtion period following the IV dministrtion of 0.1 or 1 nmol/kg ET-1, nd IV infusion of 3 nmol/kg exogenous ET-1 resulted in twofold increse in circulting ET-1. Becuse prolonged, pproximtely 5 10-fold increse in endogenous ET levels hs been reported fter experimentl ischemi or hemorrhge, 4 the enhnced leukocyte rolling induced by 1 3 nmol/kg of exogenous ET-1 might be considered comprble to the microcircultory consequences of endogenous ET relese during pthophysiologicl conditions. Rolling is necessry first step in leukocyte dhesion, nd we hve shown the bility of ET-1 to induce both primry (leukocyte rolling) nd secondry (leukocyte sticking) endothelil cell PMN interctions. Severl possibilities should be considered to explin the mechnisms involved in ET-1 induced leukocyte rolling in the smll intestinl microcircultion. First, endothelil cell derived ET-1 relese is prticulrly sensitive to hemodynmic stimuli, such s chnges in flow-induced sher stress. 25 Correspondingly, endothelil cell leukocyte interctions might be significntly enhnced during period of low flow or fter decrese in sher rte. 26 It my pper tht ET-1 induced vsoconstriction nd microcircultory perfusion filure would led to leukocyte rolling in the submucosl postcpillry nd collecting venules secondry to decrese in sher stress. This effect of ET-1 could minly be medited by ET A receptors present in the intestine, becuse BQ 610 nd ETR-Pl/Fl peptide significntly inhibited both ET-1 induced endothelil-leukocyte interctions nd decreses in cpillry perfusion. Becuse ET B -receptor ntgonist IRL 1038 pretretment ws less effective, it my be postulted tht n ctivtion of vsoconstriction-mediting ET receptors on vsculr smooth muscle cells (e.g., ET A nd ET B2 receptors) leds to immedite hemodynmic chnges, nd the proficiency of ET-receptor ntgonist compounds in inhibiting leukocyte interctions is relted to their cpcity to ntgonize these hemodynmic effects. However, the leukocyte endothelil cell interction ws not incresed fter norepinephrine infusion, when microvsculr perfusion filure ws evident. Similrly,

9 Jnury 1998 ENDOTHELIN INDUCED LEUKOCYTE ADHESION 111 the percentge of dherent leukocytes ws pproximtely twice s high fter 1 nmol/kg IV dministered ET-1 compred with the effects of locl, close-rteril infusion. Close-rteril 0.1 nmol/kg ET-1 induces significnt decrese in microvsculr perfusion prmeters in ll lyers of the smll intestine. On sequentil elevtion of the mount of intr-rteril ET-1, ner-mximl vsoconstrictor effect ws reched t dose of 1 nmol/kg, nd higher locl doses of the peptide induced complete microcircultory rrest. This suggests tht the intestinl microcircultion is extremely sensitive to ET effects, nd ET-induced endothelil cell leukocyte interctions my proceed only if the ET-medited locl perfusion filure does not exceed certin degree. This lso suggests tht the prodhesive effects of ET-1 could not be ttributed solely to significnt impirment of microvsculr perfusion. The second possibility is n interction of ET-1 with endothelil cell membrnes in the systemic circultion nd the relese of secondry chemottrctnt meditors linked to ET A /ET B -receptor ctivtion. Once in the systemic circultion, ET-1 might induce phospholipse A 2 ctivtion, or indirectly ctivte PMNs through pltelet-ctivting fctor nd leukotriene B 4 relese from mcrophges or endothelil cells. 25,27,28 Furthermore, the effects of ETs my interfere with the ntidhesive ctions of endothelil cell derived ntiggregtory meditors, i.e., nitric oxide nd/or prostcyclin. NO regultes the interction of ET-1 with its receptor both by directly displcing bound ET-1 from the ET A receptor nd by interfering with postreceptor pthwys relted to clcium mobiliztion. 29 During ischemi-reperfusion injuries, the endothelil production of NO is compromised nd the vsculr responsiveness is ltered. 30 The number of ET A binding sites is incresed in response to reperfusion, nd the responsiveness to the vsoconstrictor effects of ET-1 is enhnced. 31,32 It could be hypothesized tht n incresed locl ET level nd reltive lck of NO ply concerted role in the process of neutrophil dherence nd ccumultion in the intestinl microcircultion. Another possibility is direct ctivtion of endothelil cells nd/or PMNs through up-regultion of dhesion molecules on the surfce of the cell membrnes. The in vivo effects of ET-1 on PMN functions re fr from clrified, becuse most of the dt on ET-induced chnges re from studies using isolted cell lines or high ET-1 doses, nd the evidence is lrgely circumstntil. Lopez Frre et l. 10 found tht 10 7 mol/l ET-1 promotes the expression of the CD11/CD18 integrin complex on humn neutrophils in vitro. However, incresed neutrophil dhesiveness or CD11/CD18 expression ws not observed on the use of mol/l ET-1. Nevertheless, in vitro dt from FACS nlysis my not fully reflect the in vivo conditions. Moreover, ctivted cells will probbly dhere nd will no longer be within the systemic circultion. In our study, no significnt increse in CD11b/c expression on circulting grnulocytes ws observed fter IV infusion of 1 or 3 nmol/kg ET-1, respectively. Although the properties of ET-1 seem to fvor role for the peptide in the regultion of microenvironment rther thn cting s circulting hormone, further studies re necessry to investigte the role of potentil PMN surfce receptors in this respect. The short lg period when the leukocyte dhesion reched plteu does not support role for the de novo synthesis of PMN dhesion molecules, but receptor trnsloction from intrcellulr stores nd conformtionl chnges of CD11b/CD18 could lso occur in response to PMN ctivtion. 33 The possibilities of n up-regultion of endothelil receptors must lso be ddressed. Some recent dt suggest tht ET-1 could led to n increse in P-selectin expression on ET-1 treted pltelets nd induces the relese of von Willebrnd fctor from cultured endothelil cells. 34 Exocytosis from the Weibel Plde bodies, the secretory grnules of the vsculr endothelil cells, my cuse rpid relese of von Willebrnd fctor nd the cell surfce expression of P-selectin, membrne protein involved in leukocyte rolling. Our results show tht ETR-Pl/Fl redily nd most competently suppresses the regionl vsculr responses elicited in the smll intestine by nnomolr doses of ET-1, nd the effects might persist for t lest 90 minutes fter ET-1 dministrtion. Antgonism of ET A receptors, i.e., tretment with BQ 610 or ETR-Pl/fl peptide, ws more effective in ttenuting the leukocyte rolling induced by 3 nmol/kg ET-1 thn the ET B -receptor ntgonism of IRL ETR-Pl/fl peptide is frgment of the humn ET A receptor, nd it binds to specific regions of the ET A receptor responsible for the mintennce of protein shpe. 21 In this study, the dministrtion of ETR-Pl/fl peptide ttenuted the PMN responses to ET-1 more powerfully thn did pretretment with the specific ET A -receptor ntgonist BQ 610. The liner tripeptidic compound BQ 610 is 30 times more potent thn BQ 123, generlly used ET A -receptor ntgonist. However, selective blockde of ET B receptors with IRL 1038 ws prtilly effective in reducing ET-1 induced chnges. This suggests tht the residul responses to ET-1 in the presence of BQ 610 my hve been medited by unblocked ET B receptors nd, consequently, lso tht ETR-Pl/fl peptide is nonselective inhibitor of ET receptors: i.e., it hs dul ET A -ET B receptor ntgonist properties. This ssumption is highly conceivble, be-

10 112 BOROS ET AL. GASTROENTEROLOGY Vol. 114, No. 1 cuse the peptide exhibits close mino cid sequence similrity to n ntisense homology box present in the ET B receptor (Brnyi L, unpublished dt, 1996). The ET B receptor ntgonist ctivity could explin the superior effectiveness of the ETR-Pl/fl peptide in inhibiting ET-1 induced PMN dhesions. Becuse ET-1 hs higher ffinity for ET A - thn ET B -receptor subtypes, these results show tht, lthough n ET A -ET B receptor subtype-dependent mechnism is involved in PMN endothelil cell interctions, the ET A receptor is more importnt for producing these chnges. The intestine is the primry trget of nonocclusive tissue ischemi fter circultory shock, nd ETs could medite the erly phse of vsoconstriction nd hypoperfusion of the intestine during such conditions. 6,35 ET A - receptor blockers or nonspecific nti-et serum could reduce the tissue dmge in these settings. 4,6 Similrly, severl fctors ssocited with occlusive gstrointestinl ischemi-reperfusion disese sttes cn stimulte ET-1 production in the endothelium. 36,37 However, only few rticles report direct observtions on the effects of ETs on the microcircultion, nd, to our knowledge, no previous studies hve investigted the microvsculr ctions of ET-1 sequentilly in ll ntomicl lyers of the smll intestine. Recently, we showed significnt decrese in lymphtic cpillry density fter cold storge nd reperfusion fter smll intestinl trnsplnttion. 38 An interesting finding of the present study is the observtion of similr decrese in lymphtic cpillry density fter ET-1 dministrtion. ET-1 my directly constrict the mesenteric lymphtic vessels, 39 nd it seems tht the serosl lymphtic cpillry density correltes well with both the functionl cpillry density of the underlying tissue lyers nd the number of dherent leukocytes in the submucosl venules. The intestinl lymph flow is extremely sensitive to ischemi-reperfusion, nd there is strong evidence for n involvement of ctivted PMNs in lymphtic dysfunction during such conditions. 40,41 The decrese in lymphtic cpillry density could be prevented by ET A -receptor ntgonists, wheres it ws deteriorted by IRL 1038 pretretment. In line with these results, Allcock et l. 42 hve shown tht blockde of the ET B receptors ctully potentites the regionl nd systemic vsoconstrictor responses cused by ET-1. This suggests tht the lymphtic effects of ET-1 re primrily medited by the ET A -receptor subtype. Our model llows the in vivo visuliztion of the intestinl microcircultion, so tht leukocyte-endothelil interctions nd microvsculr chnges cn be observed simultneously. Reductions in subserosl lymphtic cpillry density nd mucosl functionl cpillry density were mong the erliest microcircultory consequences of systemic ET-1 dministrtion. Elevtion of the dose of the peptide produced mrked decreses in perfusion prmeters in ll lyers of the smll intestine. This ction of ET-1 in the intestinl microcircultion ws lrgely medited by ET A receptors, but ET B -receptor subtypes (probbly ET B2 receptors) might lso be involved. Although the results presented in this study estblish role for ET-1 in the development of perfusion filure in the smll intestine, the mechnism tht cuses the functionl cpillry density decrese remins uncler. ET-1 is more potent vsoconstrictor thn ngiotensin II, norepinephrine, or vsopressin, nd ET-1 is likely to ct by stimulting smooth muscle cell ctivity. However, Filep et l. hve shown tht ET-1 infusion induces dosedependent increses in lbumin lekge through ctivtion of the ET A receptor, reflecting disruption of the endothelil brrier. 20,43 Thus, in concert with n incresed precpillry resistnce, n enhnced microvsculr permebility my led to microvsculr hemoconcentrtion, nd the subsequent increse in cpillry blood viscosity might promote perfusion filure. However, Khimenko et l. 44 hve recently shown tht, in the lung, ET-1 did not produce n incresed microvsculr permebility per se. Similrly, in our study, ET-1 did not significntly increse the microvsculr permebility, s shown by the unchnged bckground fluorescence intensity. However, we used only qulittive method to ssess ET-1 induced fluid shifts nd the contribution of ET-1 to microvsculr permebility chnges in the intestine needs n in-depth investigtion. In conclusion, our results clerly show tht ET-1 might ct s promoter of leukocyte rolling in the intestinl microcircultion. In the present study, no single cuse of ET-1 induced leukocyte endothelil cell interctions could be defined. We propose tht microvsculr flow relted ltertions nd secondry meditors relesed during erly stges of microvsculr impirment cused by endothelinemi my together stimulte PMN ttchment to crete n optiml milieu for further leukocyte endothelil cell interctions. The moleculr determinnts of this rection remin obscure, nd further studies re needed to evlute the sequence of events leding to enhnced leukocyte rolling fter ET-1 dministrtion. However, these dt, together with the observtions of elevted plsm levels during certin pthologies, suggest therpeutic potentil for ET-receptor ntgonists for the inhibition not only of microvsculr dysfunction but lso of interctions of PMNs with endothelil cells. References 1. Yngisw M, Kurihr H, Kimur S, Tonobe Y, Kobyshi M, Mitsui Y, Yzki Y, Goto K, Mski T. A novel potent vsoconstric-

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12 114 BOROS ET AL. GASTROENTEROLOGY Vol. 114, No Fujimoto K, Price VH, Grnger DN, Specin R, Bergstedt S, Tso P. Effect of ischemi-reperfusion on lipid digestion nd bsorption in rt intestine. Am J Physiol 1991;260:G595 G Kubes P, Hunter J, Grnger DN. Ischemi/reperfusion-induced feline intestinl dysfunction: importnce of grnulocyte recruitment. Gstroenterology 1992;103: Allcock GH, Wrner TD, Vne JR. Roles of endothelin receptors in the regionl nd systemic vsculr responses to ET-1 in the nesthetized gnglion blocked rt: use of selective ntgonists. Br J Phrmcol 1995;116: Filep JG, Földes-Filep E, Rousseu A, Fournier A, Sirois P, YnoM. Endothelin-1 enhnces vsculr permebility in the rt hert through the ET A receptor. Eur J Phrmcol 1992;219: Khimenko PL, Moore TM, Tylor AE. Blocked ET A receptors prevent ischemi nd reperfusion injury in rt lungs. J Appl Physiol 1996;80: Received Mrch 18, Accepted August 21, Address requests for reprints to: Konrd Messmer, M.D., Ph.D., Institute for Surgicl Reserch, Ludwig Mximilins University, Klinikum Grosshdern, Mrchioninistrsse 15, D Munich, Germny. Fx: (49) Supported by reserch grnt Biomed 2 Contrct No. BMH4-CT (DG12-SSMA). Dr. Boros is Fellow of the Alexnder von Humboldt-Foundtion from Szent-Györgyi Albert Medicl University, Szeged, Hungry. Mnson of Schistosom mnsoni Sir Ptrick Mnson ( ) ws born ner Aberdeen, Scotlnd, the son of n ffluent lird nd bnker. Excessively vigorous exertion in youth left him with prtil prlysis of his right rm. Thereupon he turned to less physiclly demnding pursuits nd chose the study of medicine, first t Aberdeen nd lter t Edinburgh. Attrcted to the Fr Est, he secured ppointment s hed of Bptist Missionry Hospitl. Lter he settled in Hong Kong where the income from thriving privte prctice llowed him to indulge his voction of investigting exotic diseses. In 1877 he showed tht filrisis ws trnsmitted to humns by the mosquito Culex ftigns. In 1890 he moved to London where he ws instrumentl in founding wht becme worldrenowned school of tropicl medicine. There he identified the fluke tht cused common form of schistosomisis. Mnson encourged nd dvised Ronld Ross in his investigtion of the mosquito s vector of mlri; however, Ross refused to shre ny credit with Mnson when Ross ws wrded Nobel Prize in A mn of gret chrm nd compssion, Sir Ptrick retired in 1914 to enjoy his hobbies of fishing nd grdening, s well s to wrm both hnds before the fire of life.