Kate Blease, Anne Burke-Ga ney & 1 Paul G. Hellewell

Transcription

1 British Journl of Phrmcology (1998) 124, 229 ± Stockton Press All rights reserved 0007 ± 1188/98 $ Modultion of cell dhesion molecule expression nd function on humn lung microvsculr endothelil cells y inhiition of phosphodiesterses 3 nd 4 Kte Blese, Anne Burke-G ney & 1 Pul G. Hellewell Applied Phrmcology, Imperil College School of Medicine t the Ntionl Hert nd Lung Institute, Dovehouse Street, London, SW3 6LY 1 Expression of cell dhesion molecules (CAM) on the lung microvsculr endothelium is elieved to ply key role in the recruitment of leukocytes in pulmonry in mmtion. Moreover, regultion of CAM expression my e n importnt mechnism through which this in mmtion my e controlled. Experimentl evidence hs suggested tht comined phosphodiesterse (PDE) 3 nd 4 inhiitors increse cyclic AMP levels within cells greter thn inhiition of either isoenzyme lone. In the present study we ssessed the e ect of comintions of roliprm (PDE4 inhiitor), ORG 9935 (PDE3 inhiitor) nd slutmol (-gonist) on CAM expression nd neutrophil or eosinophil dhesion to humn lung microvsculr endothelil cells (HLMVEC). 2 Tumour necrosis fctor- (TNF-)-induced intercellulr dhesion molecule (ICAM)-1, vsculr cell dhesion molecule (VCAM)-1 nd E-selectin expression were mesured on HLMVEC monolyers t 6 h y speci c ELISA technique in the presence of di erent comintions of medium, roliprm, ORG 9935 nd slutmol. 3 Roliprm in comintion with slutmol, ut neither gent lone, inhiited TNF--induced E- selectin expression, whilst ICAM-1 nd VCAM-1 expression were not ected. ORG 9935 hd no signi cnt e ect on CAM expression lone. However, in comintion with roliprm syngergistic inhiition of VCAM-1 nd E-selectin, ut not ICAM-1, expression ws oserved. No further inhiition ws seen in the dditionl presence of slutmol. 4 Neutrophil dhesion to TNF--stimulted (6 h) HLMVEC ws minly E-selectin dependent in this model, s ENA 2 n nti-e-selectin monoclonl ntiody (ma) rogted neutrophil dhesion. Eosinophil dhesion ws E-selectin-, ICAM-1- nd VCAM-1-dependent, s ssessed y the inhiitory ctivity of ENA 2 nd the ility of ma to the ICAM-1 lignd, CD18, nd ma to the VCAM-1 lignd, VLA 4, to ttenute dhesion. 5 Roliprm in the presence of slutmol or ORG 9935 signi cntly inhiited neutrophil dherence to TNF--stimulted HLMVEC. Eosinophil dherence to monolyers ws inhiited only when HLMVEC were ctivted in the presence of roliprm nd ORG Collectively, the ndings presented in this mnuscript suggest tht inhiition of PDE4 with pproprite ctivtion of denylte cyclse is su cient to inhiit induction of E-selectin expression on HLMVEC to level tht hs functionl consequences for neutrophil dhesion. In contrst, comined inhiition of PDE3 nd 4 isoenzymes is necessry to inhiit VCAM-1 nd to hve inhiitory e ects on eosinophil dhesion to ctivted HLMVEC. Upregultion of ICAM-1 expression on HLMVEC does not pper to e modulted y PDE3 nd 4 inhiition. These dt my hve implictions for the use of selective PDE4 inhiitors in lung in mmtion. Keywords: PDE inhiitors; lung microvsculr endothelium; TNF-; E-selectin; vsculr cell dhesion molecule-1; intercellulr dhesion molecule-1; leukocyte dhesion Introduction During in mmtion loclly relesed cytokines such s tumour necrosis fctor (TNF)- induce the expression of cell dhesion molecules (CAM) on endothelil cells (Crlos & Hrln, 1994; Mlik & Lo, 1996). These dhesion molecules include intercellulr dhesion molecule-1 (ICAM-1), E-selectin nd vsculr cell dhesion molecule-1 (VCAM-1) which ind to leukocytes vi their lignds; (i) lymphocyte function-ssocited ntigen-1 (LFA-1; CD11/CD18) nd mcrophge ntigen-1 (Mc-1; CD11/CD18), (ii) L-selectin, silyl-lewis X nd relted crohydrte structures; nd (iii) very lte ntigen-4 (VLA 4 ; 4 1 ), respectively (Crlos & Hrln, 1994; Mlik & Lo, 1996). Evidence from oth in vitro nd in vivo studies 1 Author for correspondence t present ddress: Section of Medicine ± Vsculr Biology, Clinicl Sciences Centre, Northern Generl Hospitl, She eld, S5 7AU suggest tht regultion of CAM expression on endothelil cells plys key role in controlling leukocyte dhesion nd migrtion during in mmtory diseses of the lung including sthm, dult respirtory distress syndrome (ARDS) nd sepsis (Redl et l., 1991; Gosset et l., 1995; Gru et l., 1996; Moss et l., 1996). In ptients with llergic sthm, signi cnt increses in endothelil CAM expression from ronchil mucos iopsies hve een oserved compred to norml sujects (Gosset et l., 1995). This increse in CAM hs een correlted with eosinophil nd totl leukocyte in ltrte mesured in roncholveolr lvge uid (BALF) from these ptients. Pulmonry endothelil cells tken from ptients with ARDS (Gru et l., 1996) lso show signi cnt increses in ICAM-1 nd VCAM-1 expression compred to controls, nd inhiition of E-selectin function with monoclonl ntiody hs protective e ects on lung function in experimentl nd clinicl sepsis (Ridings et l., 1995; Friedmn et l., 1996).

2 230 K. Blese et l Recent interest hs focused on intrcellulr signlling mechnisms involved in the regultion of CAM expression s tool for modulting in mmtion. In wide rnge of cells nd tissues, denosine 3' :5'-cyclic monophosphte (cyclic AMP) hs proved to e n importnt trget; incresing levels of this second messenger within cells (to ctivte protein kinse A (PKA) which in turn, phosphoryltes other sustrtes; reviewed y Giemycz & Reurn, 1991) hs een shown to hve nti-in mmtory e ects (reviewed y Teixeir et l., 1997). Incresing cyclic AMP levels within cells y phrmcologicl mnipultion cn e chieved in two wys; y incresing the rte of synthesis of cyclic AMP from ATP vi ctivtion of denylte cyclse, or y decresing the rte of rekdown y hydrolysis to inctive 5'-monophosphtes (Torphy & Undem, 1991). Hydrolysis of cyclic AMP nd cyclic GMP is crried out y fmily of seven distinct phosphodiesterse (PDE) enzymes (Bevo et l., 1994). Selective inhiition of these isoenzymes ppers to e the most e ective mechnism for controlling cyclic AMP levels within cells. The use of speci c PDE inhiitors in in vitro nd in vivo models of in mmtion hs demonstrted vriety of therpeutic nti-in mmtory e ects (Dent et l., 1994; Teixeir et l., 1997). Studies on ronchil smooth muscle hve shown tht PDE3 nd 4 isoenzyme inhiitors re e ective roncho-relxnts nd my e e ective in relieving irwy ostruction in sthmtics in comintion with - gonists such s slutmol (Polson & Strd, 1996). In ddition to their ronchodiltor ction, selective PDE4 inhiitors suppress pro-in mmtory functions of rnge of cells. Amongst their ctions re inhiition of in mmtory cell ctivtion (neutrophils, monocytes, lymphocytes, mst cells nd eosinophils; Torphy & Undem, 1991; Giemycz nd Dent, 1992; Dent et l., 1994), reduction in endothelil cell permeility (Suttorp et l., 1993; 1996), suppression of trnsendothelil leukocyte migrtion (Lidington et l., 1996) nd inhiition of in mmtory cell recruitment into sites of in mmtion (reviewed y Teixeir et l., 1997). In some cells nd tissues the use of PDE3 nd 4 inhiitors in comintion hs een shown to e more e ective t suppressing cell function thn inhiition of PDE 3 or 4 isoenzymes lone. These e ects include inhiition of pulmonry endothelil monolyer permeility, regultion of IL-2 relese from T-lymphocytes nd more e ective relxtion of trchel smooth muscle (Shhid et l., 1991; Giemycz et l., 1996; Suttorp et l., 1996). Endothelil cells hve een shown to express the isoenzymes PDE2, 3 nd 4 (Suttorp et l., 1993; 1996), PDE3 nd 4 eing the mjor cyclic AMP hydrolyzing enzymes. By rising intrcellulr cyclic AMP levels with non-selective PDE inhiitor (IBMX), TNF-induced VCAM- 1 nd E-selectin expression on humn umilicl vein endothelil cells (HUVEC) ws prtly inhiited (Poer et l., 1993; Ghers et l., 1994). Inhiition of PDE4 isoenzyme ctivity in HUVEC (used in comintion with denylte cyclse ctivtors nd protein kinse A ctivtors), hs lso een shown to inhiit E-selectin ut not VCAM-1 expression (Morndini et l., 1996). However, The e ect of PDE3 inhiition lone nd in comintion with PDE4 inhiition on TNF--induced CAM expression on endothelil cells hs not een studied. Therefore, the im of the present study hs een to investigte the e ects of comined PDE3 nd PDE4 inhiition with Org 9935 nd roliprm, respectively, in the sence nd presence of -gonist (slutmol) on CAM expression nd leukocyte dhesion to humn lung microvsculr endothelil PDE3/4 inhiitors nd lung endothelil CAM cells (HLMVEC). With the recent isoltion of humn pulmonry microvsculr endothelil cells (Crley et l., 1992), we hve een le to use this in vitro model to investigte vrious spects of pulmonry microvsculr endothelil cell function. This my e useful s the sis for studies of dhesion molecule trgeted therpies of pulmonry in mmtory disese. Methods Antiodies A nity isolted got nti-(mouse immunogloulins) gmm nd light chin speci c peroxidse conjugte ws otined from TCS Biologicls Ltd. Mouse nti-humn ICAM-1 (ma RR1/1) (Rothlein et l., 1988) ws provided y Dr R. Rothlein (Boehringer Inglheim Phrmceuticls, Ridge eld, CT, U.S.A.). Monoclonl ntiodies BBIG-E1 (nti-e-selectin; Pigott et l., 1991), BBIG-V1 (nti-vcam-1) nd 2B4 (nti- VLA-4; Needhm et l., 1994) were generous gifts from Dr R. Pigott (British Biotech, Oxford). F(') 2 frgments of the nti- CD18 ma 6.5E (Andrew et l., 1993) nd the nti-e-selectin ma ENA 2 (Leeuwenerg et l., 1990) together with the control myelom IgG 1 MOPC21 were generous gifts from Dr M. Roinson (Celltech, Slough). Cell culture HLMVEC used in this study hve een shown to retin numer of properties of EC including the production of humn Fctor-VIII-relted ntigen, uptke of cetylted LDL nd expression of CD31 (Shen et l., 1995). We hve lso con rmed tht these cells were positive for Fctor-VIII-relted ntigen nd CD31 expression (dt not shown). HLMVEC were mintined in EGM-MV medium, modi ction of MCDB 131, supplemented with 10 ng ml 71 humn recominnt epiderml growth fctor, 1 mg ml 71 hydrocortisone, 5% heted-inctivted foetl clf serum (FCS), 50 mg ml 71 gentmicin, 50 ng ml 71 mphotericin-b, ovine rin extrct contining 12 mg ml 71 protein, nd 10 mg ml 71 heprin. Cells used etween pssge 58 ±108did not lter in terms of sl or TNF--induced CAM expression, nd hydrocortisone in the culture medium lso hd no detectle e ect on CAM expression (sl or TNF--stimulted). Con uent cells were wshed with HBSS, trypsinized with 0.025% trypsin +0.01% EDTA nd collected into trypsin neutrlizing solution. Cells were seeded t density of cells per well onto 1% geltin-coted t-ottomed Nunclon 96-well microtitre pltes. Enzyme-linked immunosornt ssy (ELISA) for ICAM-1, VCAM-1 nd E-selectin expression ICAM-1, VCAM-1 nd E-selectin were detected 4 dys fter seeding y n ELISA method (Pigott et l., 1991) y use of mouse nti-humn ICAM-1 (RR1/1), VCAM-1 (BBIG-V1) or E-selectin (BBIG-E1) primry mas, nd peroxide-linked got nti-mouse secondry ntiody. Brie y, con uent HLMVEC in Nunclon 96 well pltes, were incuted for 6 h with medium or TNF- (0.1 or 1 ng ml 71 ) in the sence or presence of comintions of roliprm (1 ± 10 mm), slutmol (10 mm) nd ORG 9935 (1 ± 10 mm). The vehicle, DMSO (0.1%) hd no e ect on sl ICAM-1 or TNF--induced CAM expression (dt not shown). CAM expression ws mesured t 6 h s previous kinetic nlysis demonstrted tht

3 K. Blese et l signi cnt levels of ICAM-1, VCAM-1 nd E-selectin were expressed t this time (Blese et l., 1996). Following removl of stimuli, cells were wshed three times with PBS contining C 2+,Mg 2+ nd 0.1% BSA nd incuted for 45 min with 1 mg ml 71 RR1/1, BBIG-V1 or BBIG-E1. Primry ntiody ws removed y wshing, nd HLMVEC monolyers were incuted (45 min) with 1 : 1000 dilution (in PBS+10% got serum) of got nti-mouse peroxidse conjugte followed y incution with ABTS, peroxidse-sensitive sustrte (1 mg ml 71, in 0.2 M citrte/phosphte u er, ph 5, contining 0.1% H 2 O 2 ) for 30 min. The rection ws terminted y ddition of 0.2 M citrte. All incutions were crried out t room temperture. Chromophore development ws determined y mesuring opticl density (O.D.) t 405 nm (OD 405 ) y use of Titretec MCC/340 Multiscn microplte reder. Bckground sornce ws determined from monolyers incuted without primry ntiody nd this vlue ws then sutrcted from the sornce redings. The dt presented PDE3/4 inhiitors nd lung endothelil CAM 231 re derived from O.D. redings which fll long the liner portion of the development curve. Seprtion of humn peripherl lood neutrophils or eosinophils Grnulocytes were isolted from peripherl lood of norml or mildly topic dult donors for neutrophils or eosinophils respectively, y the method of Hslett et l. (1985). Brie y, lood ws collected to totl volume of 40 ml into 3.8% citrte nd spun for 20 min t 300 g. The pltelet-rich plsm ws removed, underlyed with 90% Percoll nd spun t 2000 g for Figure 1 E-selectin expression on HLMVEC following 6 h exposure to () 0.1 ng ml 71 or () 1 ng ml 71 TNF- in the sence nd presence of roliprm (10 mm) nd/or slutmol (10 mm). Results re shown s mens+s.e.men of 5 experiments. Sttisticl nlysis ws performed y use of one-wy ANOVA followed y Dunnett's test which compres test vlues to control (cell monolyers incuted with TNF-). + P50.01 or ++ P show signi cnt increses in E-selectin expression from control nd *P50.01 or **P show signi cnt decreses in E-selectin expression from levels induced y TNF-. Figure 2 E ect of roliprm lone nd in comintion with slutmol on TNF-induced () ICAM-1 nd () VCAM-1 expression on HLMVEC. Endothelil cells were stimulted for 6 h with TNF- (1 ng ml 71 ), with TNF- nd roliprm (0.1 ± 10 mm) or with TNF-, roliprm (0.1 ± 10 mm) nd slutmol (10 mm). Results re expressed s % of TNF--induced CAM expression nd re mens of 5 experiments; verticl lines show s.e.men. Bsl nd TNF- induced ICAM-1 were (OD 405 ) nd , respectively while TNF--induced VCAM-1 expression ws Sttisticl nlysis ws performed y use of one-wy ANOVA followed y Dunnett's test which compres test vlues to control (cell monolyers incuted with TNF-).

4 232 K. Blese et l 20 min t room temperture (temperture used unless otherwise stted) to produce pltelet-poor plsm (PPP). To the lower u y cot produced y the rst spin, 5 ml of 6% dextrn ws dded nd the volume mde up to 50 ml with 0.9% sline. This ws llowed to stnd for 30 min for erythrocyte sedimenttion to occur. The leukocyte-rich superntnt ws removed nd centrifuged t 300 g for 8 min. The pellet ws resuspended in PPP nd lyered onto freshly prepred discontinuous Percoll-plsm grdients (42 nd 51% Percoll in PPP) nd centrifuged for 10 min t 260 g. The grnulocyte nd ws collected, wshed in PPP nd resuspended in KRPD u er (4.8 mm KCl, 3.1 mm NH 2 PO 4, 12.5 mm N 2 HPO 4, 5% glucose nd 2% FCS). Grnulocytes otined from norml donors were 498% neutrophils. Grnulocytes otined from mildly topic donors were used to purify eosinophils nd were incuted with nti-cd16 microeds to remove neutrophils (1/10 dilution when 40 l of eds were dded per 10 8 grnulocytes for 45 min). A Type-CS MACS seprtion column ws set up in mgnetic seprtor nd microedlelled grnulocytes pplied to the top of the column. Eosinophils (497% pure) were eluted in 35 ml KRPD u er. Eosinophils or neutrophils ( cells ml 71 ) were lelled (30 min, 378C) with uorescent dye, clcein-am (10 mm dissolved in 1% DMSO in KRPD without FCS). Cells were wshed twice in KRPD without FCS nd resuspended t cells ml 71 in KRPD contining 2.5% FCS, 0.93 mm CCl 2 nd 1.2 mm MgSO 4 for the dhesion ssy. Mesurement of neutrophil or eosinophil dhesion to lung microvsculr endothelil cells HLMVEC monolyers grown on 96-well pltes were pretreted with TNF- (1 ng ml 71 ) nd/or comintions of PDE inhiitors with or without slutmol for 6 h. Monolyers were wshed 36 with PBS (contining C 2+ /Mg 2+ ) to remove stimuli efore crrying out the dhesion ssy. One hundred microlitres of MOPC21, 6.5E, ENA 2 or KRPD with C 2+ /Mg 2+ were dded per well, followed y 100 ml of clcein-am lelled neutrophils or eosinophils nd the plte ws incuted t 378C for 30 min. Fluorescence ws mesured with Biolite F1 plte reder (excittion wvelength of nm nd emission wvelength of nm) efore nd fter wshing the plte twice with PBS contining 1% horse serum to remove non-dherent cells. % dhesion ws expressed s uorescence fter wshing the plte minus the ckground uorescence, divided y uorescence efore wshing plte minus ckground Cell culture regents HLMVEC, prepred y Clonetics, (Cliforni, U.S.A.), were otined s cryopreserved third pssge (3) cultures from TCS PDE3/4 inhiitors nd lung endothelil CAM Biologicls Ltd (Buckinghm). Microvsculr endothelil growth medium (EGM-MV), 0.025% trypsin+0.01% EDTA, HBSS, trypsin neutrlizing solution were lso otined from TCS Biologicls Ltd. Dulecco's PBS (+C 2+ /Mg 2+ ) ws purchsed from Gico Lortories (Pisley, Scotlnd). Cytokines nd other regents Humn recominnt (hr) TNF- ws otined from Boehringer Mnnheim (Lewes, Est Sussex; speci c ctivity umg 71 ). The following products were purchsed from Sigm Chemicl Compny Ltd. (Poole, Dorset): 2,2'- zinois-(3-ethylenthizoline-6-sulfonic cid; ABTS), geltin, got serum, horse serum, foetl clf serum (FCS), hydrogen peroxide (H 2 O 2 ) nd dimethylsulphoxide (DMSO). Percoll nd dextrn were otined from Phrmci Biotech Ltd. (St. Alns), sterile norml sline (0.9%) from FL (Mnufcturing) Ltd., Fresenius Helth Cre Group (Bsingstoke) nd very low endotoxin BSA from Byer Ltd (Bsingstoke). MACS CS seprtion columns nd CD16 microeds were purchsed from Miltenyi Biotech (Cmerley, Surrey). Slts for Kres Ringer phosphte dextrose (KRPD) u er were otined from Sigm nd BDH. Clcein-AM ws otined from Cmridge Bioscience (Cmridge). Roliprm ws gift from Dr N. Cooper, Chiroscience (Cmridge) nd Org 9935 (4,5 - dihydro - 6-(5,6 -dimethoxy-enzo[]thien-2-yl)-5-methyl- 1(2H)-pyridzinone) ws gift from Dr Shhid, Orgnon Lortories (Scotlnd). Sttistics Results re expressed s men+s.e.men of n experiments. Sttisticl nlysis ws crried out using one-wy nlysis of vrince (ANOVA) followed y the Dunnett's test or Bonferroni selected comprison test s indicted in gure legends. Results were considered signi cnt if P Results E ect of roliprm nd slutmol on TNF- induced CAM expression Stimultion of HLMVEC with TNF- (0.1 nd 1 ng ml 71 ) for 6 h cused signi cnt increse in E-selectin expression (Figure 1 nd, respectively). In the presence of roliprm (10 mm) or slutmol (10 mm), responses to oth concentrtions of TNF- were not signi cntly ltered. However, comintion of roliprm with slutmol either olished or cused 64% inhiition of E-selectin expression induced y Tle 1 E ect of roliprm in the presence nd sence of slutmol on TNF-±induced ICAM-1 nd VCAM-1 expression on HLMVEC ICAM-1-expression VCAM-1-expression Roliprm (mm) 7Slutmol +Slutmol 7Slutmol +Slutmol HLMVEC were incuted for 6 h with TNF- (0.1 ng ml 71 ) with or without the ddition of roliprm nd slutmol (10 mm). ICAM- 1 nd VCAM-1 expression re expressed s percentge of responses to TNF--lone; sl ICAM-1 ws (OD 405 ) , TNF-induced ICAM-1 ws nd TNF--induced VCAM-1 ws Vlues re men+s.e.men of 4 experiments.

5 K. Blese et l PDE3/4 inhiitors nd lung endothelil CAM nd 1 ng ml 71 TNF-, respectively (Figure 1, ). Concentrtion-response curves estlished tht 10 mm roliprm provided the mximl inhiitory e ect (dt not shown). Bsl ICAM-1 expression (OD ) ws not ltered fter 6 h incution with roliprm (0.1 ± 10 mm) lone or in comintion with slutmol (10 mm) (dt not shown). In ddition, TNF- (1 ng ml 71 ) induced ICAM-1 ( ) or VCAM-1 ( ) expression ws not ected y roliprm lone or roliprm plus slutmol (Figure 2, ). Responses to the lower concentrtions of TNF- (0.1 ng ml 71 ) were lso un ected y these compounds lone or in comintion (Tle 1). E ect of Org 9935 lone nd in comintion with roliprm on TNF--induced CAM expression The PDE3 inhiitor, Org 9935 (0.1 ± 10 mm), lone or in comintion with roliprm (10 mm), hd no e ect on sl ( ) or TNF--(1 ng ml 71 ; ) induced ICAM-1 expression (Figure 3). However, comintion of Org 9935 (3 nd 10 mm) nd roliprm (10 mm) cused synergistic inhiition of TNF--(1 ng ml 71 ) induced VCAM-1 nd E-selectin expression (Figure 3 nd c). Addition of slutmol (10 mm) in the presence of Org 9935 (10 mm) did not lter the e ect of Org 9935 lone, nd slutmol did not further reduce the inhiitory e ect of Org 9935 plus roliprm on TNF--induced E-selectin or VCAM-1 expression (dt not shown). Contriution of CAM expression to neutrophil nd eosinophil dhesion to HLMVEC c Before the functionl consequences of PDE inhiition on TNF-induced CAM expression were determined, the CAM requirement for dhesion of unstimulted neutrophils or eosinophils to HLMVEC monolyers ws ssessed y use of function-locking mas. Bsl dhesion of neutrophils to untreted HLMVEC ws signi cntly (P50.01) incresed following stimultion of HLMVEC for 6 h with TNF- (1 ng ml 71 ; Figure 4). Compred to the control ntiody (MOPC21, 10 mg ml 71 ), the nti-e-selectin ma (ENA 2, 10 mgml 71 ) reduced the dhesion of neutrophils to the level seen on untreted HLMVEC (Figure 4). In contrst, the nti-cd18 ma (6.5E, 10 mg ml 71 ; concentrtion tht hs een shown to e su cient to lock dhesion, see elow) hd no e ect on neutrophil dhesion. These dt suggest tht under the conditions of the ssy, the dhesion of unstimulted neutrophils which is incresed s result of TNF--ctivtion of HLMVEC is solely due to E-selectin nd tht CD18/ICAM- 1 interctions were not involved. Eosinophil dhesion to TNF- stimulted HLMVEC ws signi cntly inhiited y the nti-cd18 ma, 6.5E (10 mg ml 71 ; P50.05) nd the nti-vla 4 ma, 2B4 (10 mg ml 71 ; P50.05). A comintion of these two mas further reduced the dhesion of eosinophils to the level pproching tht seen on unstimulted HLMVEC (P50.01; Figure 4). The nti-e-selectin ma (ENA 2,10mgml 71 ) lso Figure 3 E ect of Org 9935 lone nd in comintion with roliprm on TNF--induced () ICAM-1, () VCAM-1 nd (c) E-selectin expression on HLMVEC. Endothelil cells were stimulted for 6 h with TNF- (1 ng ml 71 ), with TNF- nd Org 9935 (0.1 ± 10 mm) or with TNF-, Org 9935 (0.1 ± 10 mm) nd roliprm (10 mm). Results re expressed s % of TNF--induced CAM expression nd re mens of 5 experiments; verticl lines show s.e.men. Bsl nd TNF- induced ICAM-1 were (OD 405 ) nd , respectively, while TNF--induced VCAM-1 nd E-selectin expression were nd , respectively. Sttisticl nlysis ws performed y use of one-wy ANOVA followed y Dunnett's test which compres test vlues to control (cell monolyers incuted with TNF-). *P50.01 or **P denotes signi cnt decreses in TNF- induced CAM expression compred to control levels.

6 234 K. Blese et l PDE3/4 inhiitors nd lung endothelil CAM Figure 4 Adhesion of () neutrophils nd () eosinophils to TNF- stimulted HLMVEC nd the e ect of dhesion molecule mas. HLMVEC were stimulted with medium or TNF- (1 ng ml 71 ) for 6 h followed y ddition of neutrophils or eosinophils nd control ma MOPC21, nti-cd18 ma 6.5E, nti-e-selectin ma ENA 2, nti-vla-4 ma 2B4 or comintion of 6.5E nd 2B4. All mas were used t nl concentrtion of 10 mg ml 71. After 30 min, nondherent grnulocytes were removed y wshing nd the percentge dhesion clculted. Vlues re men+s.e.men of 4 determintions in 2 experiments. Sttisticl nlysis ws performed y use of onewy ANOVA followed y Dunnett's test which compres test vlues to control. ++ P50.01 denotes signi cnt increses in dhesion compred to sl levels nd *P50.05 or **P50.01, denotes signi cnt decreses in dhesion compred to TNF- induced levels. Figure 5 E ect of comintion of roliprm, Org 9935 nd slutmol on () neutrophil nd () eosinophil dherence to HLMVEC monolyers. HLMVEC were stimulted for 6 h with medium or TNF- (1 ng ml 71 ) for 6 h in the sence or presence of roliprm (10 mm) or roliprm plus Org 9935 (10 mm). Incutions were lso crried out in the presence of slutmol (10 mm). After 30 min, non-dherent grnulocytes were removed y wshing nd the percentge dhesion clculted. Vlues re men+s.e.men of 3 ± 6 determintions in 2 ± 3 experiments. Sttisticl nlysis ws performed y use of one-wy ANOVA followed y the Bonferroni selected comprison. ++ P50.01 denotes signi cnt increses in dhesion compred to sl levels nd *P50.05 or **P50.01, denotes signi cnt decreses in dhesion compred to TNF- induced levels. hd signi cnt e ect when tested lone (Figure 4) lthough it did not further reduce the inhiitory ctions of 6.5E nd 2B4. These dt indicte tht eosinophil dhesion ws dependent on E-selectin, ICAM-1 nd VCAM-1. E ect of comined PDE3 nd 4 inhiition on eosinophil nd neutrophil dhesion to HLMVEC Incution of unstimulted HLMVEC with roliprm, slutmol or Org 9935 lone or in comintion, hd no e ect on sl dherence of neutrophils or eosinophils (dt not shown). Adhesion of neutrophils to HLMVEC stimulted with TNF- (1 ng ml 71 ) in the presence of comintion of roliprm (10 mm) nd slutmol (10 mm) ws signi cntly reduced compred with TNF- lone (P50.05; Figure 5). A similr reduction ws seen on HLMVEC stimulted in the presence of roliprm plus Org 9935 (10 mm) (P50.01) nd this ws not further inhiited in signi cnt mnner y the ddition of slutmol (Figure 5). Inhiition of neutrophil dhesion correltes with chnges in E-selectin expression in the presence of comintions of roliprm, Org 9935 or slutmol s s shown in Figures 1 nd 3c. Eosinophil dhesion to TNF--treted HLMVEC in the presence of roliprm ws not signi cntly reduced nd ddition of slutmol hd no further e ect (Figure 5). In contrst, ctivtion of HLMVEC in the presence of rolpirm plus Org 9935 resulted in signi cnt suppression of eosinophil dhesion nd this ws not further inhiited, in signi cnt mnner, y the ddition of slutmol (Figure 5).

7 Discussion K. Blese et l Chrcteriztion nd cellulr distriution of cyclic phosphodiesterse isoenzymes together with the development of speci c inhiitors hs llowed the mechnisms nd functionl consequences of regultion of cyclic nucleotides within cells to e investigted. Within the lung (ovine trchel smooth muscle, humn ronchi nd porcine pulmonry rtery EC; Shhid et l., 1991; de Boer et l., 1992; Suttorp et l., 1993), PDE isoenzymes 1-5 hve een identi ed. Of these isoenzymes, PDE3 nd 4 re the mjor cyclic AMP hydrolyzing enzymes (Bevo et l., 1994). Evidence from in vitro nd in vivo models of pulmonry in mmtion hve demonstrted tht ctivtion of endothelil cells to express CAM is key step in the process of in mmtion within the lung (Gosset et l., 1995; Ridings et l., 1995; Gru et l., 1996). Hence, identi ction of intrcellulr signlling mechnisms involved in CAM regultion on EC my llow the development of phrmcologicl gents to inhiit lung in mmtion. Modultion of cyclic AMP levels within endothelil cells hs een shown to hve ene cil nti-in mmtory e ects, such s inhiition of EC permeility (Suttorp et l., 1996) nd cell migrtion (Frnzini et l., 1995). The mjority of cyclic AMP hydrolyzing ctivity within endothelil cells hs een ttriuted to PDE3 nd 4 isoenzymes (Suttorp et l., 1993; 1996) nd these enzymes re therefore key trgets in order to increse endothelil cyclic AMP levels. The e ects of comined PDE3 nd 4 isoenzyme inhiition hs een studied on endothelil permeility nd cell migrtion, with ene cil nti-in mmtory e ects eing greter thn individul isoenzyme inhiition (Suttorp et l., 1996). However, the e ect of PDE3 nd comined PDE3 nd 4 inhiition on CAM expression on EC hs not een studied, especilly those derived from the humn pulmonry microvsculture. The present study demonstrted tht inhiition of PDE4 isoenzymes with the selective inhiitor roliprm resulted in n ttenution of TNF--induced E-selectin expression on HLMVEC monolyers, ut only when co-incuted with slutmol (n denylte cyclse ctivtor). This suggests tht cyclse ctivity is normlly low such tht inhiition of PDE4 y itself is insu cient to elevte intrcellulr cyclic AMP to levels tht hve functionl consequences on CAM expression. No e ects of roliprm in comintion with slutmol on TNF--induced ICAM-1 or VCAM-1 expression were oserved. These results concur with previous work y Morndini et l. (1996) on HUVEC, where TNF--induced E-selectin (ut not VCAM-1 or ICAM-1) expression ws inhiited y 5 fold higher concentrtion of roliprm, ut only in comintion with forskolin. Our dt nd tht of Morndini et l. (1996) contrst with the oservtions of Poer et l. (1993) who found tht elevtion of intrcellulr cyclic AMP inhiited oth E- selectin nd VCAM-1 expression in response to TNF-. However, in the ltter study the non-speci c PDE inhiitor isoutylmethylxnthine (IBMX) ws used in comintion with forskolin. Thus, the inhiitory e ect on VCAM-1 expression could hve een consequence of multiple PDE inhiition, including PDE3 nd 4. At vrince with ll this work is the study of Deisher et l. (1993), who were unle to lter TNF-induced E-selectin or VCAM-1 expression on HUVEC y use of the non-selective PDE inhiitor, pentoxy lline or diutyryl cyclic AMP. However, one explntion for the di erence ws tht comintion of these gents ws not tested nd therefore the intrcellulr cyclic AMP concentrtions my not hve een elevted su ciently. In the present study comined PDE3 nd 4 inhiition resulted in synergistic inhiition of TNF--induced E-selectin PDE3/4 inhiitors nd lung endothelil CAM 235 nd VCAM-1 expression, whilst no e ect ws seen on ICAM-1 expression. Thus, lthough roliprm nd slutmol filed to modify VCAM-1 signi cntly, roliprm plus Org 9935 ws e ective nd ddition of slutmol did not produce further inhiition. In comprison, E-selectin expression ws ttenuted to similr extent y either roliprm plus slutmol or roliprm plus Org These oservtions suggest tht E- selectin is more sensitive to chnges in cyclic AMP levels thn VCAM-1. Levels of cyclic AMP induced y comined PDE3 nd 4 inhiition my e greter thn those induced y PDE4 inhiition lone, reching su cient levels to inhiit VCAM-1 expression. Tht the comined e ect of PDE3 nd 4 inhiition results in higher levels of cyclic AMP is supported y work in the guine-pig trche nd T lymphocytes, where this tretment resulted in more e ective inhiition of totl cyclic AMP hydrolytic ctivity thn either isoenzyme lone (Hrris et l., 1989; Giemycz et l., 1996). The di erence in sensitivity etween E-selectin nd VCAM-1 my e due to the presence of cyclic AMP response elements identi ed in mrna E-selectin promoter regions (De Luc et l., 1994), the e ect of cyclic AMP eing t the level of trnscription, s no e ect of cyclic AMP on mrna stility hs een oserved (Morndini et l., 1996). These results suggest tht the vrile e ects on VCAM-1 expression oserved y Morndini et l. (1996) y PDE4 inhiition lone my not e due to di erences in culture conditions ut rther to insu cient levels of intrcellulr cyclic AMP. The e ects of PDE inhiition on CAM expression were re ected in the functionl ssys. Activtion of HLMVEC in the presence of roliprm plus slutmol resulted in signi cnt decrese in neutrophil ut not eosinophil dhesion. This e ect correlted with reduction in E-selectin expression nd function, s the E-selectin ma ENA 2 cused signi cnt inhiition of neutrophil ut not eosinophil dhesion. Comined PDE3 nd 4 inhiition lso resulted in reduced neutrophil dhesion nd this ws not further reduced in the presence of slutmol, gin re ecting the E-selectin expression dt. Eosinophil dhesion ws inhiited when the HLMVEC were ctivted in the presence of roliprm nd Org Eosinophils, unlike neutrophils, express the lignd for VCAM-1 (VLA 4 ; Wlsh et l., 1991), nd re therefore sensitive to chnges in VCAM-1 expression in ddition to ltertions in E-selectin nd ICAM-1. Eosinophils lso utilized ICAM-1 to dhere, since dhesion ws prtly reduced y the nti-cd18 ma. However, since ICAM-1 expression ws not ltered y PDE inhiition, the chnges in eosinophil dhesion to HLMVEC in the presence of roliprm nd Org 9935 re therefore considered to e medited vi suppression of oth E- selectin nd VCAM-1. In conclusion, we hve demonstrted tht comined PDE3 nd 4 isoenzyme inhiition results in synergistic inhiition of TNF--induced E-selectin nd VCAM-1 expression, which we speculte is medited vi incresed levels of cyclic AMP. Inhiition of E-selectin expression resulted in reduction of TNF--induced neutrophil dhesion, whilst eosinophil dhesion ws reduced y inhiition of E-selectin nd VCAM-1 expression. The results of this study suggest tht comined PDE3 nd 4 inhiition my result in more e ective inhiition of totl cyclic AMP hydrolytic ctivity of lung microvsculr endothelium thn inhiition of either isoenzyme lone. This my hve implictions for the use of PDE4 inhiitors in lung in mmtion. We re grteful to the British Hert Foundtion (K.B.; grnt # FS/ 95072) nd the Ntionl Asthm Cmpign (A.B.G. nd P.G.H.) for supporting this work.

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9 K. Blese et l TEIXEIRA, M.M., GRISTWOOD, R.W., COOPER, N. & HELLEWELL, P.G. (1997). Phosphodiesterse (PDE4) inhiitors: nti-in mmtory drugs of the future? Trends Phrmcol. Sci., 18, 164 ± 171. TORPHY, T.J. & UNDEM, B.J. (1991). Phosphodiesterse inhiitors: new opportunities for the tretment of sthm. Thorx., 46, 512 ± 523. PDE3/4 inhiitors nd lung endothelil CAM 237 WALSH, G.M., MERMOD, J-J., HARTNELL, A., KAY, A.B. & WARD- LAW, A.J. (1991). Humn eosinophil, ut not neutrophil, dherence to IL-1-stimulted humn umilicl vsculr endothelil cells is 4 1 (very lte ntigen-4) dependent. J.Immunol., 146, 3419 ± (Received Septemer 15, 1997 Revised Jnury 29, 1998 Accepted Ferury 17, 1998)