a-4/b-1 and a-l/b-2 integrins mediate cytokine induced lung leukocyte-epithelial adhesion and injury

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1 British Journal of Pharmaology (27) 152, & 27 Nature Publishing Group All rights reserved /7 $3. RESEARCH PAPER a-4/b-1 and a-l/b-2 integrins mediate ytokine indued lung leukoyte-epithelial adhesion and injury LA Parmley 1, ND Elkins 1, MA Fini 1,2, Y-E Liu 3, JE Repine 1,2 and RM Wright 1,2 1 Webb-Waring Institute for Caner, Aging and Antioxidant Researh, University of Colorado Health Sienes Center, Denver, CO, USA; 2 Department of Pulmonary Sienes, The Shool of Mediine, University of Colorado Health Sienes Center, Denver, CO, USA and 3 Department of Biohemistry, Prineton University, Prineton, NJ, USA Bakground and purpose: Injury to the alveolar epithelium is a ritial feature of aute lung injury (ALI). Using a ytokine model of ALI we demonstrated previously that newly reruited mononulear phagoytes (MNP) ontributed to lung inflammation and injury. We hypothesized that ytokines delivered into the alveolar airspae would have multiple effets on the lung that may ontribute to lung injury. Experimental approah: Intratraheal ytokine insufflation and leukoyte adoptive transfer in vivo were ombined with in vitro analyses of lung epithelial ell-mnp adhesion and injury. Lung inflammatory injury was assessed by histology, leukoyte infiltration, and release of LDH and RAGE. Key results: insufflation was assoiated with apparent MNP-epithelial adhesion, up-regulation of alveolar ICAM-1 and VCAM-1, and the release of LDH and RAGE into the bronhoalveolar lavage. Insufflation of small moleule integrin antagonists suppressed adhesion of MNP and modulated release of LDH and RAGE. Adoptive transfer of MNP purified from ytokine insufflated lungs into leukopeni rats demonstrated the requirement of MNP for release of LDH that was not indued by ytokine alone. Corroboration that disrupting the ICAM/LFA1 interation or the VCAM/VLA4 interation bloked MNPepithelial ell interation and injury was obtained in vitro using both bloking monolonal antibodies and the small moleule integrin antagonists, BIO5192 and 143. Conlusions and impliations: MNP reruited following ytokine insufflation ontributed to lung injury. Further, integrin antagonists redued alveolar epithelial ell injury indued during lung inflammation. Intratraheal delivery of small moleule antagonsists of leukoyte-epithelial adhesion that prevent lung injury may have signifiant linial utility. British Journal of Pharmaology (27) 152, ; doi:1.138/sj.bjp.77443; published online 1 September 27 Keywords: aute lung injury; mononulear phagoyte; alveolar epithelium; a-4/b-1; a-l/b-2; integrin antagonist Abbreviations: ALI, aute lung injury; AM, alveolar marophage; A-MNP, alveolar MNP; IFN-g, interferon-g; IL-1, interleukin-1; MNP, mononulear phagoyte; PMN, polymorphonulear phagoyte or neutrophil; RAGE, reeptor for advaned glyation end produts Introdution Inflammation ontributes to many linial lung disorders inluding ventilator-indued lung injury, hroni obstrutive pulmonary disease, aute lung injury (ALI) and aute respiratory distress syndrome (de Boer et al., 2; Belperio et al., 22; Matthay et al., 23; Prine et al., 23; Rose et al., 23). While the immunologial mehanisms mediating lung inflammation may involve ells of both innate Correspondene: Dr RM Wright, Webb-Waring Institute for Caner, Aging and Antioxidant Researh, University of Colorado Health Sienes Center, 42 East 9th Avenue, Campus Box C-322, Denver, CO 8262, USA. rihard.m.wright@uhs.edu Reeived 26 Marh 27; revised 11 June 27; aepted 3 August 27; published online 1 September 27 and adaptive immunity, mononulear phagoytes (MNPs) of innate immunity appear to play deisive roles in many inflammatory states. For example, MNPs and the MNP hemokine, monoyte hemotati protein-1 (MCP-1), ontribute signifiantly to the inflammation assoiated with hroni obstrutive pulmonary disease (de Boer et al., 2), and MNPs are ritially involved in the pathogenesis of ventilator-indued lung injury, ALI and aute respiratory distress syndrome (Belperio et al., 22; Matthay et al., 23; Prine et al., 23; Rose et al., 23; Frank et al., 26). The role played by MNPs in inflammatory disorders is not well understood and is rapidly evolving. In the rat, the resting lung ontains a resident population of mature alveolar marophages (AMs) that are renewed by onstitutive

2 916 Lung leukoyte-epithelial adhesion and injury LA Parmley et al reruitment (Srivastava et al., 25). Following the indution of inflammation, monoytes are reruited to the lung primarily, but not exlusively, in response to the hemokines, MCP-1 and MCP-2, or leukotrienes (Gunn et al., 1997; Maus et al., 21b, 22a). One established in the lung, monoytes rapidly differentiate into marophages by a omplex proess initiated following transendothelial migration out of the blood stream (Valledor et al., 1998; Rosseau et al., 2a; Maus et al., 21a). This effetively defines two subpopulations of MNPs in the inflamed lung: the resident AMs and the newly reruited and differentiating inflammatory alveolar mononulear phagoytes (A-MNPs) (Maus et al., 26). Whether distint biologial roles an be attributed to these different MNP subpopulations is unknown. However, in humans and mie, the AM population and the newly reruited MNPs indued by inflammatory stimuli an be distinguished by distint surfae markers, mehanism of reruitment (Sunderkotter et al., 24; Garn et al., 26; Take and Randolph, 26) and gene expression profiles (Srivastava et al., 25) indiative of distint MNP subpopulations. In the present study, and until the language used in referene to both populations has beome odified, we have adopted the use of the term A-MNPs to refer to the entire population of MNPs, inluding the resident AMs and the inflammatory MNPs, harvested from the lung lavage. AMs refer stritly to the resident MNP population harvested from lungs of untreated and ontrol rats. MNPs in general are potent soures of pro-inflammatory mediators suh as tumor nerosis fator-a, interleukin (IL)-1, IL-8, or reative oxygen speies that promote inflammation (Matthay et al., 23; Prine et al., 23; Rose et al., 23; Desouza et al., 25; Smith et al., 26). MNPs respond to many ativating signals inluding the baterial lipopolysaharide and ytokines suh as IL-1 or interferon-g (IFN-g) (Kovarik et al., 1998; Ehrt et al., 21; Dobrovolskaia and Vogel, 22; Beutler and Rietshel, 23). Upon ativation, MNPs serete ytokines involved in the orhestration of the inflammatory response, ativate the respiratory burst niotinamide adenine dinuleotide phosphate oxidase and release reative oxygen speies. For example, MNPs express and serete proinflammatory hemokines inluding regulated upon ativation, normal T-ell expressed and sereted, marophage inflammatory protein-1a, MCP-1, MCP-2, tumor nerosis fator-a and IL-8 (Luas et al., 1998; Foey et al., 2; Gavrilin et al., 2; Wetzel et al., 2; Ragno et al., 21; Carvalho-Pinto et al., 22). Reently, A-MNPs were found to ontribute to neutrophil (polymorphonulear phagoyte or neutrophil (PMN)) immigration, reruitment and infiltration following intratraheal instillation of baterial lipopolysaharide or tumor nerosis fator-a in mie (Maus et al., 22b, 23; Woo et al., 25), or ytokine in rats (Wright et al., 24). As soures of both peptide (IL-8, Cin) and lipid (LTB 4 ) hemotati fators, A-MNPs may ontribute to expansion of the inflammatory ell population. The extent to whih A-MNPs may also ontribute to lung injury per se is unknown. However, the resident AMs speifially appear to exert little injury to the lung in the absene of an inflammatory induer (Wright et al., 24). Furthermore, lung injury during inflammation may result from ontributions by AMs (Frank et al., 26), the inflammatory A-MNP population, PMNs (Patton et al., 1995) or reflet the ombined effets of ytotoxi agents released into the inflammatory environment. Instillation of aute-phase ytokines into the airway of rodents has been used to study the speifi effets of ytokines found in the lavage fluid of linial ALI/aute respiratory distress syndrome patients (Tsan et al., 1992; Patton et al., 1995; Guidot et al., 1996; Kang et al., 1996; Maus et al., 21a; Hybertson et al., 23; Matthay et al., 23; Wright et al., 24). We previously demonstrated the vigorous inflammatory response indued by intratraheal insufflation of IL-1 (Leff et al., 1992, 1994a, 1995) or IL-1 in ombination with IFN-g (Wright et al., 24). Both PMNs and MNPs were inreased rapidly in the alveoli following ytokine insufflation; and lung leak, oxidative stress and alveolar epithelial ell apoptosis were onomitantly inreased following ytokine-indued lung inflammation. Diret evidene that A-MNPs partiipated in leukoyte reruitment and inflammation was obtained in adoptive ell transfer experiments using A-MNPs obtained from the bronhoalveolar lavage (BAL) of rats previously insufflated with ytokine. We observed high-level expression of the reative oxygen speies generator, xanthine oxidoredutase, in the newly reruited A-MNPs, but none in the resident AMs. Furthermore, inhibition of xanthine oxidoredutase in the newly reruited A-MNPs prevented reruitment of PMNs during adoptive transfer demonstrating a ritial role for xanthine oxidoredutase in A-MNP-mediated neutrophil reruitment (Wright et al., 24). These data, therefore, identify another potential distintion between the resident AMs and the newly reruited A-MNPs. While the role of PMN epithelial interation in hroni obstrutive pulmonary disease airway inflammation and injury has been learly identified (Pettersen and Adler, 22), muh less is known about the A-MNP epithelial interation in lung inflammation and injury. The potentially omplex role played by newly reruited MNPs in lung inflammation was suggested by the inrease in alveolar epithelial adhesion by both PMNs and MNPs observed following hypoxiaindued airway inflammation (Bek-Shimmer et al., 21). In the present experiments, we hypothesized that ytokines in the alveolar airspae would have omplex effets on the lung inluding the modulation of surfae moleules that promote MNP adhesion where the ontribution to lung inflammation and injury may be enhaned. Data shown here indiate that A-MNPs and A-MNP epithelial adhesion ontributes to ytokine-indued inflammatory lung injury and that insufflation of small moleule integrin antagonists into the alveolar air spae an redue ytokine-indued inflammatory lung injury. Methods Animal proedures The use of rats in this study was approved by the University of Colorado Institutional Review Board under the protool number (4)1E. Healthy male Sprague Dawley rats (3 4 g body weight; Saso, Omaha, NE, USA) were used in all studies. British Journal of Pharmaology (27)

3 Lung leukoyte-epithelial adhesion and injury LA Parmley et al 917 Previous experiments had optimized lung inflammation indued by IL-1 at 5 ng (Leff et al., 1994a). Insufflation of IFN-g alone at doses from to 1 ng promoted apparent dose-dependent leukoyte adhesion that was maximal at 1ng (not shown). Unless otherwise speified, 5 ng of reombinant rat IL-1a and/or 1 ng of IFN-g (hereafter referred to as ytokine) in.5 ml of normal saline were delivered intratraheally into the airway of rats previously anesthetized with ketamine/xylazine (Wright et al., 24) and lung tissues were harvested 24 h later. Control rats were insufflated with normal saline alone. Differential and total inflammatory ell ounts were determined on bronhoalveolar lavage fluid (BALF) obtained 24 h following ytokine insufflation as desribed (Wright et al., 24). Histology and immunofluoresene were performed on lungs harvested 24 h following ytokine insufflation. Lungs were perfused until blood free, removed surgially and divided. One fration was immediately fixed in paraformaldehyde for histology or immunofluoresene staining. Another fration was frozen immediately in liquid N 2 for subsequent biohemial analyses. BALF ells were olleted from separate rats by pumping 7. ml of normal saline into the trahea. Lavage fluid was pumped in and out of the lung three times before being olleted. Leukoyte depletion To onfirm the role of adoptively transferred MNPs in lung injury it was neessary to remove the effet of irulating leukoytes that would be drawn to the lung by ytokine or MNP insufflation. Aordingly, endogenous irulating leukoytes were depleted with intraperitoneal injetion of 165 mg kg 1 of ylophosphamide in sterile normal saline as desribed (Ikezumi et al., 23a, b). A suessful depletion of 498% irulating leukoytes was observed after 48 h from the point of ylophosphamide administration. Leukopeni rats to be used for adoptive transfer of MNPs were treated with ylophosphamide and 48 h later insufflated with ytokine or saline. Adoptive MNPs transfer was then onduted 24 h later as desribed in Figure 5. The persistene of ylophosphamide-indued leukopenia was onfirmed throughout the 96 h of the experiment and was onsistent with published reports (Ikezumi et al., 23a, b). Tissue fixation, immunohistohemistry and mirosopy Lung tissue was fixed in 1% neutral-buffered formalin overnight and embedded in paraffin. Two and four miron setions were prepared and hydrated by exposure to xylene for 3- to 5-min periods followed by two sequential 1 min exposures to 1%, 95% ethanol, dh 2 O, and finally for 5 min in phosphate-buffered saline (PBS). Hydrated setions were either stained with hematoxylin and eosin or prepared for immunohistohemistry using a modified version of the Cell Signaling protool for paraffin setions (Cell Signaling, Beverly, MA, USA). Setions prepared for immunohistohemistry were digested in a pepsin digestion solution for 3 min in a 37 1C humidity hamber (1 ml.1 N HCl þ 9 ml distilled water (dh 2 O) þ.1 ml of 1 g ml 1 pepsin). Setions were washed in dh 2 O three times for 5 min eah, inubated in 1% H 2 O 2 for 1 min and washed three times for 5 min eah in dh 2 O. Setions were finally washed in PBS for 5 min and bloked for 1 h at room temperature in 5% bloking solution (either donkey or goat serum in PBS). The setions were then inubated overnight at 4 1C with the appropriate antibody diluted in bloking solution. Subsequently, the setions were washed in PBS three times for 5 min eah. The appropriate seondary antibody diluted in bloking solution was added to eah setion and inubated for 3 min at room temperature. One again, the setions were washed in PBS three times for 5 min and the setions were inubated with the 3,3 -diaminobenzidine peroxidase substrate reagent aording to the manufaturer s instrutions. The setions were rinsed in dh 2 O and subsequently stained with hematoxylin. Setions were then dehydrated by inubating twie in 95 and 1% ethanol for 1 s eah. Finally, two inubations in xylene for 1 s eah were performed. Slides were then mounted with O-phenylene diamine (OPDA) and sealed. Positive ells were visualized with a Nikon Diaphot inverted mirosope using visible light. Purifiation of mononulear phagoytes Resident AMs were purified from the lungs of ontrol rats by lavage as desribed (Wright et al., 24). A-MNPs omprising both the resident AM population and the newly reruited MNPs were purified by lavage from lungs of rats insufflated with ytokine 24 h before. Cells were washed in RPMI medium and were then plated into flasks with RPMI medium and allowed to adhere for 1 h. Non-adherent ells were removed and the remaining adherent ells were washed four times with PBS and harvested from the plates by sraping into PBS/ethylenediaminetetraaeti aid (Davies and Gordon, 25). Post-lavage mononulear phagoytes (PL-MNPs) present in the lung tissue were subsequently purified from the lungs of rats following removal of the A-MNPs. Lungs were lavaged to obtain the A-MNP population, perfused blood free and ells not released by lavage were prepared as desribed (Steinmuller et al., 2). Briefly, lungs were inubated under moderate agitation for 6 min in RPMI media ontaining 1 U ml 1 or ollagenase type 1 and 5 U ml 1 DNAse at 37 1C. After inubation, the digested tissue was passed through a sterile sieve to remove tissue fragments, and the ells obtained in the flow through were adhered to flasks as desribed above. Adherent ells were suspended in saline to a final onentration of ells ml 1. MNPs omprised 495% of the ells reovered from adhesion as determined by the mirosopi examination of Wright s-stained ells (Wright et al., 24) from both the A-MNPs and the PL-MNPs preparations. Fluoresene-ativated ell sorter analysis with the MNPspeifi markers CD68 and CD163 onfirmed that these ells were MNPs. Fluoresene-ativated ell sorter analysis was performed on purified MNP populations exatly as desribed previously (Wright et al., 24), using fluoresein isothioyanate-onjugated goat anti-mouse immunoglobulin (Ig)G 1 as a seondary antibody and mouse-derived primary antibodies to rat CD68 (ED1) and CD163 (ED2). Cultivation of both populations in the presene of M-CSF for 7 days resulted in differentiation of 98 1% of the ells into British Journal of Pharmaology (27)

4 918 Lung leukoyte-epithelial adhesion and injury LA Parmley et al marophages, again onfirming the absene of PMNs in these preparations. Cell ulture, adherene and injury in vitro L2 rat lung alveolar type II ells (ATCC, Manassas, VA, USA, number CCL-149) were grown in Ham s F12K medium with 1% heat inativated fetal bovine serum at ph 7.4 and 37 1C as indiated by the supplier. Cells were seeded in 48-well plates at ells per well. After 24 h, monolayers were washed and exposed to ytokines as indiated. After 24 h of exposure to ytokine, ells were washed and resupplied with ytokine-free medium at whih point purified MNPs were added either in the presene or absene of bloking monolonal antibodies, the integrin antagonists, BIO5192 or 143, at 1 mm. Non-adherent ells were removed 3 min later by washing twie in PBS. Adherent ells were quantitated exatly as desribed (Bek-Shimmer et al., 21). Latate dehydrogenase (LDH) ativity was quantitated in the ell-free medium after 24 h of oultivation. Latate dehydrogenase determination LDH was quantitated in the ell-free BALF using the CytoTox96 kit, a non-radioative ytotoxiity assay, exatly as indiated by the supplier (Promega Corporation, Madison, WI, USA). To preserve ativity of LDH, samples were assayed from fresh, unfrozen BALF within 1 h of lavage. Cell-free BALF was maintained on ie until the assays were performed. LDH was quantitated from the ell-free supernatants of L2 monolayers and from L2/MNP oultures. Total L2 ell ontent was determined by lysis of six independent onfluent L2 monolayers. Total L2 LDH per well was IU. LDH isozyme ontent was determined in fresh BALF and in oultures using the TITAN GEL-LD agarose eletrophoresis as desribed by the supplier (Helena Laboratories, Beaumont, TX, USA). Freshly isolated BALF was onentrated by sequential preipitation in 4 and 7% (NH 4 ) 2 SO 4, resuspended in sodium phosphate buffer at ph 7.5, dialysed overnight in sodium phosphate buffer, ph 7.5 and isotype eletrophoresis performed. MNPs and neutrophils were isolated from ytokine-insufflated rats on Peroll gradients as desribed (Eisenhauer et al., 1989; Ross et al., 1998), lysed and LDH isotype determined. Fresh erythroytes and serum were prepared from rats at the time of death and LDH isozyme distribution was determined for eah. Sodium dodeyl sulphate-polyarylamide gel eletrophoresis and western immunoblot analysis Cell lysates were prepared as desribed previously (Wright et al., 24), and the protein onentration determined by using the biinhonini aid assay. Aliquots ontaining 5 mg of protein or a onstant volume of ell-free BAL (2 ml) were inubated with equal amounts of loading buffer (5% b-meraptoethanol, 95% Laemmli loading dye) for 1 min at 371C, and then boiled for 5 min. Samples were then separated by eletrophoresis on 7.5% sodium dodeyl sulfate-polyarylamide gel eletrophoresis or 4 2% gradient sodium dodeyl sulfate-polyarylamide gel eletrophoresis gels for 4 min at 1 V, transferred to polyvinylidene difluoride membranes (Whatman In, Florham Park, NJ, USA). Membranes were bloked overnight at 41C in 5% nonfat dried milk in Tris-buffered saline (ph 7.6) ontaining.1% Tween. Membranes were then inubated with antibodies as indiated. Antigen-antibody omplexes were deteted by reation with an enhaned hemiluminesene western blotting detetion kit aording to the manufaturer s instrution (Amersham Life Sienes, Pisataway, NJ, USA). Eah experiment was run in tripliate, and representative immunoblots are shown. Statistial analyses Data are expressed as the mean and standard error of the mean and were assessed for signifiane using the Student s t-test or analysis of variane. Po.5 was onsidered signifiant. Reagents Most reagents, buffers, substrates and inhibitors were purhased from Sigma Chemial Company (St. Louis, MO, USA). Reombinant rat IL-1a (5-RL-5) and IFN-g (285-IF-1) were purhased from R&D Systems (Minneapolis, MN, USA). Sterile normal saline (.9% NaCl, ph 6.) was purhased from Baxter Health Care (Deerfield, IL, USA). Mouse antibodies to the rat-speifi MNP surfae markers CD68 (MCA341R) and CD163 (MCA342R) were obtained from Serote (Raleigh, NC, USA). Fluoresein isothioyanateonjugated goat anti-mouse IgG 1 (STAR7) was also obtained from Serote. Goat polylonal IgG intraellular adhesion moleule-1 (ICAM-1) and vasular ell adhesion moleule-1 (VCAM-1), donkey anti-goat IgG-HRP and goat anti-mouse IgG-HRP were purhased from Santa Cruz Biotehnology (Santa Cruz, CA, USA). Anti-reeptor for advaned glyation end produts (RAGE) antibody smab1179 was purhased from R&D Systems. Normal goat serum was obtained from ICN Biomedials (Aurora, OH, USA). Donkey serum was purhased from Researh Diagnostis (Flanders, NJ, USA). The 3,3 -diaminobenzidine peroxidase substrate kit was obtained from Vetor Laboratories (Burlingame, CA, USA). The ICAM/lymphoyte funtion-assoiated protein 1 (LFA-1) integrin antagonist, 143, was obtained from Hoffmann- La Rohe (Nutley, NJ, USA). The VCAM/very late antigen-4 (VLA-4) integrin antagonist, BIO5192, was obtained from Biogen In. (Cambridge, MA, USA). Cylophosphamide was from Sigma. Results insufflation promoted apparent leukoyte epithelial adhesion and injury Our previous experiments demonstrated that ytokine insufflation promoted the rapid influx of MNPs and PMNs into the alveolar airspae that was assoiated with histologial evidene of tissue injury, alveolar wall terminal deoxynuleotide UTP nik-end labeling staining and lung aspase-3 ativation, suggesting the development of British Journal of Pharmaology (27)

5 Lung leukoyte-epithelial adhesion and injury LA Parmley et al 919 signifiant tissue injury and apoptosis following ytokine insufflation (Wright et al., 24). We observed that apparent adhesion of leukoytes was also promoted by ytokine insufflation, and ytokine-insufflated lungs revealed ollapse of the inflamed alveolar walls and inreased fragmentation or fragility of the alveoli themselves (Figure 1a). b LDH Ativity (I.U.) Relative Band Intensity 12 1 Saline Saline Insufflation *** To further haraterize injury assoiated with ytokine insufflation, we quantified the levels of the intraellular enzyme, LDH, reovered in the ell-free BALF. Consistent with previous observations, lung LDH release was signifiantly inreased 24 h following ytokine insufflation (Figure 1b). The LDH isozyme profile was determined from the ell-free BALF, Peroll gradient purified MNPs and PMNs, erythroytes and serum from rats following ytokine insufflation. Whole-ell lysates from ultured rat lung epithelial ells, L2 ells, were used for omparison. The LDH isozyme profile from the ell-free BALF was very distint from that obtained from purified MNPs, PMNs, erythroytes or from serum (Table 1). Although minor ontributions from LDH1, LDH2 and LDH3 were evident in the BALF, the ratio of LDH4 to LDH5 ontent was losest to that obtained from wholeell lysates of L2 rat lung alveolar epithelial ells and markedly different from that obtained from lavage leukoytes, erythroytes or serum (Table 1). The protein alled RAGE is expressed in both human and rat lung alveolar type-i ells (Dahlin et al., 24; Shirasawa et al., 24) and was reently identified as a marker for lung alveolar ell injury and inflammation, inluding lung injury that arises in human ALI (Morbini et al., 26; Uhida et al., 26). We observed an average 11.2-fold inrease in RAGE reovered in the ell-free BALF following ytokine insufflation in rats ompared to saline insufflation alone (Figure 1). Immunohistohemial staining onfirms the presene of MNPs in the apparently adherent leukoytes Considerable evidene supports the role of PMNs in injury to the lung in both hroni obstrutive pulmonary disease airway inflammation and following ytokine insufflation (Leff et al., 1994a, b, 1995; Pettersen and Adler, 22). Sine ytokine insufflation ould promote adhesion of either PMNs or MNPs to the respiratory epithelium in vivo, paraffin-embedded lung tissue speimens were stained for immunoreativity to the MNP-speifi proteins, CD68 and CD163. CD68 and CD163 are surfae markers that reflet RAGE Insufflation: d CD68 CD163 Saline Saline b a a b a Figure 1 insufflation indued apparent leukoyte adhesion and lung injury. (a) Rats were insufflated with the saline vehile or ytokine, and 24 h later lungs were perfused free of blood harvested, and prepared for histologial examination. Tissue setions were stained by hematoxylin and eosin and representative setions photographed under light mirosopy. (b) LDH levels were quantitated in the ell-free BALF reovered from rats insufflated as above with saline or ytokine. Six rats were used in eah group for quantitation of LDH (***Po.1 by Student s t-test). () Western immunoblot analysis of RAGE from the ell-free BALF of individual rats insufflated with either saline or ytokine. Three rats were randomly seleted from eah group of six used in panel a. Twenty miroliters of ell-free BALF was loaded in eah lane, and band intensity obtained from the western blot analysis was quantitated using Kodak imaging software. (d) Lung tissue speimens from rats insufflated with either saline or ytokine were stained for the MNP-speifi markers CD68 and CD163 and ounterstained with hematoxylin. The presene of MNPs in adherent (a), alveolar (b) and interstitial tissue ompartments is indiated (). BALF, bronhoalveolar lavage fluid; LDH, latate dehydrogenase; MNPs, mononulear phagoytes; RAGE, reeptor for advaned glyation end produts. British Journal of Pharmaology (27)

6 92 Lung leukoyte-epithelial adhesion and injury LA Parmley et al Table 1 LDH isozyme profile LDH1 LDH2 LDH3 LDH4 LDH5 LDH4/LDH5 BALF MNP PMN 172. ND RBC 172. ND L Serum Abbreviations: BALF, bronhoalveolar lavage fluid; LDH, latate dehydrogenase; MNP, mononulear phagoyte; RBC, red blood ell. LDH isozymes were separated and identified as desribed in Materials and methods using ell-free BALF or whole-ell lysates of MNP, PMN, erythroytes (RBC) and serum from ytokine insufflated rats 24 h following ytokine treatment. Lysates of L2 ells were prepared from onfluent monolayers. Isozyme bands were quantitated by sanning dosimetry following eletrophoresis (see Materials and methods). Individual isozyme bands are expressed as perent ontribution to the total isozyme ontent that was set at 1%. Data show the mean7s.e.m. of six independent determinations. VCAM-1 ICAM-1 Saline Figure 2 Immunohistohemial staining of alveolar ICAM-1 and VCAM-1. Lungs were insufflated with either saline or ytokine and 24 h later lungs were perfused free of blood, the BALF removed and the tissues stained for immunoreative VCAM-1 or ICAM-1. Staining of alveolar ICAM-1 took on an apparently foal harater (arrows) following ytokine insufflation (shown in two randomly seleted fields). BALF, bronhoalveolar lavage fluid; ICAM-1, intraellular adhesion moleule 1; VCAM-1, vasular ell adhesion moleule-1. differentiation and loalization of MNPs in rats. While CD163 exhibits preferred binding to MNPs in the lung tissue and CD68 is expressed largely but not exlusively on A- MNPs, neither marker is expressed on PMNs (Covin et al., 1998). Fluoresene-ativated ell sorter analysis had previously identified high-level CD68 expression on A-MNPs reovered from the lung lavage of rats insufflated with ytokines 24 h before (Wright et al., 24). CD68 and CD163 immunoreativity were deteted in the A-MNPs and in MNPs found in the perivasular tissue spae, the interstitial alveolar ompartment and in the MNPs loalized at the alveolar epithelium 24 h following ytokine insufflation (Figure 1). These data onfirm that many of the apparently adherent ells were MNPs. Intratraheal ytokine insufflation stimulated alveolar ICAM-1/ VCAM-1 expression Adhesion and migration of MNPs into the alveolar ompartment is thought to depend in part upon epithelial ICAM-1/ MNP LFA-1 interation and epithelial VCAM-1/MNP VLA-4 interation, and alveolar epithelial ells are known to express ICAM-1 and VCAM-1 (Bek-Shimmer et al., 21; Williams, 23), while MNPs express LFA-1 and VLA-4 (Rabb et al., 1994; Li et al., 1998). Immunohistohemial analysis of ytokine-insufflated lungs showed enhaned staining of alveolar ICAM-1 and markedly inreased staining of alveolar VCAM-1 24 h following ytokine treatment ompared to saline-insufflated sham ontrols (Figure 2). Staining of alveolar epithelial ICAM-1 aquired an intense foal harater 24 h following ytokine insufflation in ontrast to VCAM staining that appeared more uniform (Figure 2). Small moleule integrin antagonists suppressed ytokine-indued lung injury The small moleule antagonist of a-l/b-2 integrin interation, 143, bloks ICAM-LFA-1 interation and leukoyte adhesion by tight binding to the a-hain I-domain, restriting ICAM to an inative onformation that an inhibit inflammation in vivo (Lin et al., 1999; Welzenbah et al., 22; Shimaoka et al., 23; Li et al., 24). We insufflated rats onurrently with ytokines and 143 and 24 h later lungs were harvested, leukoytes ounted, and the ell-free BALF assayed for LDH and RAGE ontent. We observed that 143 oinsufflation did not prevent leukoyte infiltration (Figure 3a). In ontrast, LDH release was signifiantly redued by 143 oinsufflation at both high- and British Journal of Pharmaology (27)

7 Lung leukoyte-epithelial adhesion and injury LA Parmley et al 921 A BALF ells ml -1 x B LDH Ativity (I.U.) Saline 1 mm with 1mM /143 Insufflation 1 mm with 1 mm Sham 1 mm with 1 mm 1 mm /143 Insufflation with 1 mm C Relative Band Intensity a b RAGE Insufflation: Saline E Differential Count (% of Total Cells) AM MNP PMN Saline 1 mm with 1 mm 1 mm with 1 mm /143 Insufflation Figure 3 A small moleule antagonist of the a-l/b-2 integrin bloks ytokine-indued lung injury. Rats were insufflated with either saline, ytokine, 143, or oinsufflated with ytokine and 143. After 24 h, BALF was harvested, inflammatory leukoytes ounted, and LDH and RAGE levels quantitated. (A) The effet of 1 and 1 mm 143 on the reovery of inflammatory ells from the BALF was determined by hemoytometer ounting after Wright s staining. (B) The effet of high- and low-dose 143 on LDH ativity found in the ell-free BALF was determined as desribed above. Statistial differenes were alulated for omparison between rats reeiving ytokine and no 143 or ytokine and 1 or 1 mm 143 (**Po.2 by Student s t-test). (C) Western immunoblot analysis of RAGE obtained in the ell-free BALF. Twenty miroliters of BALF was run per lane as desribed in Figure 1. Two rats from eah group were randomly seleted for illustration and similar results were obtained for all six rats in eah group. Band intensity was normalized to the saline ontrol. (D) The effet of 143 on the histologial arhiteture. Both low-dose (box b) and high-dose (box ) 143 suppressed evidene of inflammation and leukoyte adherene ompared to ytokine insufflation without 143 (box a). (E) Differential ell ounts obtained from the BALF were determined following Wright s staining. Data shown are the mean and standard error for six rats in eah group and represent the perentile ontribution of eah group to the total ell population, whih was set at 1%. Note: an essentially pure AM population is routinely obtained from sham insufflation with saline alone. BALF, bronhoalveolar lavage fluid; LDH, latate dehydrogenase; RAGE, reeptor for advaned glyation end produts. British Journal of Pharmaology (27)

8 922 Lung leukoyte-epithelial adhesion and injury LA Parmley et al low-dose treatment (Figure 3b), and RAGE levels reovered in the ell-free BALF were also dramatially redued by 143 oinsufflation (Figure 3). Histologial examination of lung tissues from ytokine-insufflated rats with either high- or low-dose 143 oinsufflation revealed preservation of lung tissue arhiteture and diminished alveolar inflammation (Figure 3d). Thus, the a-l/b-2 antagonist, 143, dereased evidene of lung injury in ytokine-insufflated rats. We observed that 143 given alone promoted a small but onsistent PMNs infiltration (Figures 3a and e) without signifiantly inreasing LDH release or reduing the AM levels. 143 treatment alone did not inrease the level of newly reruited A-MNPs (Figure 3e). Small moleule antagonists of the a-4/b-1 integrin interation, BIO1211 and BIO5192, have been used in rats and sheep to blok experimentally indued airway inflammation and autoimmune enephalomyelitis (Lin et al., 1999; Abraham et al., 2; Leone et al., 23; Theien et al., 23). Both intraperitoneal and intratraheal administration of BIO1211 redued airway inflammation and LDH release in animal models of inflammation (Lin et al., 1999; Abraham et al., 2). BIO5192 is a highly seletive, and potent inhibitor of a- 4/b-1 (VLA-4) interation with VCAM-1 and possesses a K D of o1 pm in vitro (Leone et al., 23; Theien et al., 23). We examined the apaity of BIO5192 to blok ytokineindued injury in rats when insufflated diretly into the airway. BIO5192 had no signifiant affet on ytokineindued reruitment of leukoytes when oinsufflated with ytokine (Figure 4a). In ontrast, BIO5192 dose-dependently suppressed LDH release when oadministered with ytokines (Figure 4b). At doses of 1 mg and below, BIO5192 began to lose effetiveness 24 h following ytokine insufflation. However, LDH release was redued to sham-treated levels at a dose of 1mg. Higher doses of BIO5192 promoted LDH release. Reovery of RAGE in the ell-free BALF was redued over threefold by oinsufflation of BIO5192 (Figure 4). While 143 did not produe the finely graded dose response observed here for BIO5192, both high- and lowdose 143 generated statistially signifiant redution in LDH release and reovery of RAGE in the ell-free BALF. Thus, antagonists of both a-l/b-2 and a-4/b-1 bloked LDH and RAGE release when oadministered with ytokine. LDH release requires both ytokine priming and the presene of MNPs As reported for the insufflation of endotoxin in mie (Maus et al., 26), ytokine insufflation in rats produes rapid influx into the lung of newly reruited MNPs and PMNs that effetively dilute the resident AM population. We observed a BALF ells ml -1 x BIO5192 Dose (ug) b LDH Ativity (I.U.) BIO5192 Dose (ug) Relative Band Intensity RAGE Insufflation: Saline + BIO5192 Figure 4 A small moleule antagonist of the a-4/b-1 integrin bloks ytokine-indued lung injury. Rats were insufflated with ytokine in the presene or absene of BIO5192 over a broad onentration range; and after 24 h, BALF was harvested, inflammatory leukoytes ounted and levels of LDH and RAGE quantitated in the ell-free BALF. (a) The effet of a range of doses of BIO5192 on ytokine-indued BALF ell ounts was determined as desribed in Figure 3. (b) The effet of BIO5192 dose on ell-free BALF LDH levels. Six rats were used in eah group of both (a) and (b), exept for the highest dose of BIO5192 where two rats were used. Statistial differenes were alulated for omparison between rats reeiving ytokine and no BIO5192 or ytokine and BIO5192 (***Po.1 determined by Student s t-test). () Representative western immunoblot analysis of two rats from eah group. BIO5192 was used at 1 mg for rats reeiving both ytokine and BIO5192. BALF, bronhoalveolar lavage fluid; LDH, latate dehydrogenase; RAGE, reeptor for advaned glyation end produts. British Journal of Pharmaology (27)

9 Lung leukoyte-epithelial adhesion and injury LA Parmley et al 923 no detetable LDH or RAGE release in the BALF of salineinsufflated rats, expressing an essentially pure AM population. On the other hand, LDH and RAGE were released during ytokine insufflation, whih may reflet injury indued by PMNs, AMs, or newly reruited A-MNPs. Sine ytokine insufflation neessarily mixes resident AMs with newly reruited MNPs, MNPs were purified from ytokine-insufflated rats, omprising A-MNPs, and from the post-lavage lung tissue, omprising PL-MNPs. The A-MNP population ontains both the newly reruited MNPs and the resident AMs present before ytokine insufflation, whereas MNPs purified from the post-lavage lung tissue (PL-MNPs) ontain few or no AMs. Fluoresene-ativated ell sorter analyses of A-MNPs and post-lavage PL-MNPs using antibodies to CD68 and CD163 onfirmed the absene of PMNs or AMs in the purified PL-MNPs (not shown). A-MNP and PL-MNP populations were purified from the lungs of rats insufflated with ytokine 24 h before, and ells were then insufflated into naïve rats without ytokine priming. Treatment: CyPh i.p. 165mg/Kg b Time Line for Adoptive Transfer of inflammatory MNP MNP Final Insufflation Transfer Harvest Hours: LDA Ativity (I.U.) Hr Blood WBC x 1 6 ml Hr Blood & BALF 72Hr Blood, BALF, Lung Tissue Cirulating leukoytes 96Hr Blood, BALF, Lung Tissue Crtl Hours post treatment CyPh 72hr 96hr A-MNP + PL-MNP + Cell-free BALF was prepared from these rats with adoptively transferred MNPs 24 h later and LDH levels were quantitated in the ell-free BALF. Although a minor trend in injury produed was apparent, no statistially signifiant differene between PL-MNPs, A-MNPs or ontrol treatments was observed (data not shown). Thus, in rats not previously primed with ytokine, transfer of neither population of insufflated MNPs was able to indue signifiant LDH release. Sine the A-MNPs omprise both AMs and newly reruited MNPs, we infer that neither population indued lung injury in the absene of ytokine insufflation. Cylophosphamide pretreatment was used to deplete irulating leukoytes using the regimen indiated (Figure 5a). As desribed previously (Ikezumi et al., 23a, b), ylophosphamide treatment produed a persistent and profound leukopenia throughout the subsequent 96 h following treatment. At no point in this period did the irulating leukoyte population exeed 5% of the native level (Figure 5b). We treated 15 rats with ylophosphamide or with no ylophosphamide, and 48 h later 12 rats were insufflated with ytokine. Twenty-four hours following ytokine insufflation, three rats were insufflated with A- MNPs and three rats were insufflated with PL-MNPs. LDH was measured from rats reeiving either ylophosphamide or ylophosphamide and ytokine at 72 h (i.e. 24 h exposure to ytokine). LDH was measured from rats reeiving either ylophosphamide or ylophosphamide at 48 h, followed by ytokine and 24 h later insufflated with A-MNPs or PL-MNPs. Thus, these rats were exposed to ylophosphamide for 96 h, ytokine and no ells for 24 h, and subsequently MNP ells for an additional 24 h (total of 96 h). Rats reeiving ytokine but no ylophosphamide were harvested Figure 5 Adoptive transfer of MNPs into rats rendered leukopeni by pretreatment with CyPh demonstrated that MNPs but not ytokine alone promote LDH release in vivo. (a) The time-line used for CyPh treatment and adoptive transfer of MNPs is illustrated. (b) Total irulating leukoytes were quantitated at various points following CyPh treatment, and these data onfirm the persistene and degree of leukopenia obtained by the CyPh treatment used in panel. () Fifteen rats were treated with 165 mg kg 1 CyPh or three rats with no CyPh (CyPh ). Forty-eight hours later, 12 rats were insufflated with ytokine (ytokine þ ). Twenty-four hours following ytokine insufflation, three rats were insufflated with purified A-MNPs and three rats were insufflated with purified PL-MNPs. LDH was measured in the ell-free BALF from rats reeiving either CyPh or CyPh and ytokine at 72 h (i.e. 24 h exposure to ytokine). LDH was measured from rats reeiving either CyPh or CyPh at 48 h, followed by ytokine and 24 h later insufflated with A-MNPs or PL- MNPs. Thus, these rats were exposed to CyPh for 96 h, ytokine and no ells for 24 h, and subsequently MNP ells for an additional 24 h (total of 96 h). Rats reeiving ytokine but no CyPh were harvested 96 h following ytokine insufflation. LDH data were analysed by analysis of variane and only salient points are indiated (***Po.2 relative to 96 h CyPh with or without ytokine). No statistially signifiant differene was observed between 72 or 96 h CyPh with or without ytokine insufflation, and no statistially signifiant differene was observed between rats reeiving adoptively transferred A-MNPs, PL-MNPs in the presene of ytokine and CyPh and those insufflated with ytokine in the absene of CyPh treatment. A-MNPs, alveolar mononulear phagoytes; CyPh, ylophosphamide; LDH, latate dehydrogenase; MNPs, mononulear phagoytes; PL-MNPs, post-lavage mononulear phagoytes. British Journal of Pharmaology (27)

10 924 Lung leukoyte-epithelial adhesion and injury LA Parmley et al 24 h following ytokine insufflation, typial of the leukonormi model routinely used. We observed that LDH release was not indued by ytokine insufflation in rats pretreated with ylophosphamide (Figure 5). However, signifiant inrease in LDH release was produed when rats were pretreated with ylophosphamide, insufflated with ytokine 48 h later, and subsequently insufflated with PL-MNPs or A-MNPs (Figure 5). These data demonstrate that while ytokine alone was unable to indue LDH release, in ytokine-primed lungs adoptively transferred MNPs from Relative Band Intensity VCAM-1 -Atin Relative Band Intensity ICAM-1 -Atin Adherent ells per field Control IL-1 IFN-γ IL-1 + IFN-γ Control treated d Adherent ells per field Control IL-1 IFN-γ IL-1 + IFN-γ Control treated MNP ICAM-Ab VCAM-Ab e 7 MNP BIO LDH Ativity (I.U.) L2 A-MNP L2+ L2+ L2+ L2+ L2+ L2+ L2+ Control Control A-MNP BIO A-MNP +A-MNP +A-MNP +BIO British Journal of Pharmaology (27)

11 Lung leukoyte-epithelial adhesion and injury LA Parmley et al 925 either A-MNP or PL-MNP populations promoted statistially signifiant release of LDH ompared to rats treated in the same way but without adoptive transfer of MNPs. Furthermore, the level of LDH measured in the ell-free BALF of ylophosphamide-treated rats insufflated with MNPs was not signifiantly different from that obtained by ytokine insufflation in ylophosphamide-untreated rats. Epithelial ell LDH release in vitro was indued by MNPs and bloked by 143 and BIO5192 To onfirm that MNPs reovered from the lungs of ytokineinsufflated rats ould promote epithelial injury, A-MNPs were oultivated on monolayers of L2 ells, a rat type II alveolar epithelial ell line. Both ICAM-1 and VCAM-1 expression were inreased in L2 ells by 24 h of ytokine treatment (Figures 6a and b). Adhesion of purified A-MNPs to L2 monolayers was stimulated by ytokine treatment and inhibited by treatment with bloking monolonal antibodies to ICAM-1 or VCAM-1 (Figure 6). Similarly, the small moleule integrin antagonists, 143 and BIO5192, also bloked A-MNP adhesion to L2 monolayers in vitro (Figure 6d). We observed that both antibody to ICAM-1 and VCAM-1 as well as 143 and BIO5192 redued MNP adhesion to ontrol L2 ells, and this most likely reflets the pre-existing level of ICAM-1 and VCAM-1 expressed by ultured L2 ells. Spontaneous release of LDH from L2 ells (12 IU of ativity) over the ourse of 24 h represented approximately 1% of the total LDH ontent of L2 ells ( IU), and this was not signifiantly elevated by 24 h exposure to ytokine (Figure 6e). Similarly, spontaneous release of LDH from A-MNPs ultivated alone for 24 h (9.8 IU of ativity) represented approximately 15% of the total A-MNP LDH ( IU of ativity). Coultivation of ytokinetreated L2 ells with A-MNPs reovered from the lungs of ytokine-insufflated rats stimulated LDH release to a level of approximately 51% of the total L2 ell LDH after 24 h of oultivation (Figure 6e). Signifiant redution in LDH release was observed in L2/A-MNP oultures by treatment with BIO5192 or 143. Coultivation of L2 ells and A- MNPs in the presene of both integrin antagonists aused substantial dissoiation of the L2 monolayer after 24 h and onsequently LDH was not measured in these experiments. We observed that BIO5192 alone, but not 143, promoted modest release of LDH from L2 ells. Nonetheless, A-MNPs reovered from the lungs of ytokine-insufflated rats stimulated alveolar epithelial ell LDH release in vitro that was inhibited by BIO5192 and 143. Disussion and onlusions Injury to the alveolar epithelial barrier is a ritial omponent of pathogenesis in many lung inflammatory disorders that may be targeted for therapeuti intervention. However, the relatively poor understanding of injury has limited potential therapeuti opportunities. Our present experiments demonstrate that ytokine-indued lung inflammation promoted alveolar MNP adhesion and lung injury, and that insufflation of a-4/b-1 and a-l/b-2 integrin antagonists disrupted MNP adhesion and lung injury. Data shown here demonstrate that ytokine-indued inflammation is assoiated with inreased lung injury. Histologi evidene of alveolar inflammation and tissue damage as well as elevation of RAGE and LDH found in the ell-free BALF were indued 24 h following ytokine insufflation. While MNPs and PMNs omprise 99% of the leukoytes present in the alveolar spae following ytokine insufflation, the LDH isozyme profile obtained in the ellfree BALF was distint from that found in either MNPs or PMNs purified from the same rats. Furthermore, while haemorrhage may be expeted in ytokine-insufflated lungs, it is apparently aompanied by negligible haemolysis sine the LDH isozyme profile was also different from that obtained from erythroytes. While the ratio of LDH4 to LDH5 found in the ell-free BALF was similar, obtained from L2 rat lung epithelial ells, the lear presene of low levels of Figure 6 Effets of L2/A-MNP oulture on L2 ICAM/VCAM expression, leukoyte adhesion and ell injury. (a) Western immunoblot analysis of VCAM-1 expression in ytokine-treated L2 ells. L2 ells were grown to 8 9% onflueny and treated with either 1 ng ml 1 rril-1a, 25 ng ml 1 rrifn-g, both ytokines ombined or were untreated. After 24 h, whole-ell lysates were prepared and used in western blot analysis for VCAM-1 expression. Band intensity for representative immunoblots was quantitated and normalized to the b-atin ontrol. (b) Western immunoblot analysis of ICAM-1 expression in ytokine-treated L2 ells. Whole-ell lysates from (a) were used for immunoblot analysis of ICAM- 1 expression. () Adherene assay with purified A-MNPs and bloking monolonal antibodies to ICAM-1 or VCAM-1. L2 ells were grown in 48- well plates and were treated with IL-1 and IFN-g or were untreated. After 24 h, ells were washed in PBS and the growth medium replaed. Five mirograms of bloking antibody was then added per well, and purified A-MNPs were added 3 min later. Following 3 min of adherene, media was removed, the non-adherent ells were removed by washing twie with.5 ml PBS and the remaining adherent MNPs were ounted from two high-power fields per well. Data show the mean and standard error for six wells in eah group. (d) Adherene assay with purified A-MNPs and 143 or BIO5192. L2 ells and A-MNPs were oultivated exatly as desribed in panel exept that 1 mm 143 or BIO5192 was used instead of the bloking monolonal antibodies. (e) LDH was quantitated in the ell-free medium from ells grown as follows. L2 ells were grown for 72 h alone (L2 ontrol) or grown for 24 h, washed, treated with IL-1 and IFN-g for 24 h, washed and grown for an additional 24 h (L2 þ ytokine). L2 ells oultivated with MNPs were grown for 24 h, washed, treated with ytokine or vehile for 24 h, washed, and MNPs or MNPs with 1 mm BIO5192 or 1 mm 143 were added as indiated. LDH was quantitated in the ell-free medium 24 h later. Purified A-MNP ontrol ells were ultivated alone in RPMI with 1% heat inativated FBS for 24 h (A-MNP ontrol). L2 ells were grown alone for 48 h and then exposed to BIO5192 or 143 for 24 h after whih LDH was quantitated in the ell-free medium. Eah point represents the average of six independent experiments and data show the mean LDH ativity7s.e.m. (***Po.2 for omparison between L2 þ ytokine þ A-MNPs in the presene of BIO5192 or 143). A-MNPs, alveolar mononulear phagoytes; FBS, fetal bovine serum; ICAM, intraellular adhesion moleule; IFN-g, interferon-g; IL-1, interleukin-1; LDH, latate dehydrogenase; MNPs, mononulear phagoytes; PBS, phosphate-buffered saline; VCAM, vasular ell adhesion moleule. British Journal of Pharmaology (27)

12 926 Lung leukoyte-epithelial adhesion and injury LA Parmley et al LDH1, LDH2 and LDH3 may indiate either a ontribution from serum leakage into the lung or from other unidentified ells. However, the LDH isozyme profile obtained in the ellfree BALF was different from that obtained from serum. These observations are onsistent with the lung tissue being the primary soure of LDH found in the ell-free BALF of ytokine-insufflated rats as previously reported in both human linial speimens (Cobben et al., 1999a) and for rat lung inflammation indued by either hemial means or by pathogen (Shultze et al., 1994; Cobben et al., 1999b). Elevation of RAGE in the ell-free BALF is also indiative of injury to alveolar epithelial ells, and these data reinfore the observation that lung alveolar apoptosis, oxidative stress and tissue damage were also inreased by ytokine-indued inflammation (Wright et al., 24). The alveolar epithelial adhesion moleules, ICAM-1 and VCAM-1, were stimulated by ytokine insufflation and this was assoiated with apparent inreased alveolar MNP adhesion. Both type I and type II alveolar epithelial ells express ICAM-1 onstitutively (Williams, 23) and alveolar epithelial ICAM-1 is induible in vitro with both IL-1 (Krakauer, 2) and IFN-g (Look et al., 1992). Importantly, type II ell alveolar epithelial ICAM-1 was indued in vivo by IFN-g in both mie (Kang et al., 1996) and rats (present data), and the ombined ation of IL-1 and IFN-g further stimulated ICAM-1 staining in rat lungs. stimulation of epithelial ICAM-1 is signifiant in the ontext of lung injury beause ICAM-1 failitates both adhesion and migration of MNPs over the epithelial surfae (Chang et al., 22; Paine et al., 22) and lung inflammation indued by baterial endotoxin (Bek-Shimmer et al., 22). The role of alveolar epithelial VCAM-1 is less learly delineated in ytokine-indued lung inflammation than that of ICAM-1. However, both ICAM-1 and VCAM-1 were required for effiient migration of MNPs through an alveolar epithelial monolayer (Rosseau et al., 2b), and both ICAM and VCAM an be indued on rat alveolar epithelial ells to promote MNP adhesion (Rabb et al., 1994; Bek-Shimmer et al., 21). In vitro, IL-1 and IFN-g markedly upregulated VCAM expression in astroytoma monolayers and also promoted MNP adhesion (Rosenman et al., 1995). The present data are onsistent with these observations in asmuh as VCAM was indued in rat lung epithelial ells both in vitro and in vivo by ytokine insufflation. These observations suggest that VCAM may play a signifiant role in mediating MNP epithelial interation in ytokine-indued lung inflammation as it does in mediating endotoxinor antigen-indued airway inflammation (Rabb et al., 1994; Li et al., 1998). Roles for ICAM/LFA-1 and VCAM/VLA-4 interations in inflammatory disorders are frequently established using monolonal antibodies direted against ICAM or VLA-4 (Rabb et al., 1994; Li et al., 1998). However, the observation that statins an blok inflammatory proesses at the level of integrin funtion has led to the development of small moleule, statin-like antagonists with high speifiity and effiient binding harateristis that avoid some of the disadvantages attributed to the in vivo use of monolonal antibodies. We employed the LFA antagonist, 143 (Welzenbah et al., 22; Shimaoka et al., 23), to inhibit ICAM/LFA-1 interation in the lung, and our data revealed that disruption of ICAM/LFA-1 interation in vivo redued histologi evidene of inflammation and injury as well as the release of LDH and RAGE in the ell-free BALF. Immunohistohemial analysis also demonstrated ativation of alveolar epithelial VCAM following ytokine insufflation, suggesting that it may also be an important omponent of MNP/alveolar epithelial interation. Several small moleule antagonists of a 4 b 1 -integrin (VLA-4) interation with VCAM have been synthesized and used to blok airway inflammation in vivo (Lin et al., 1999; Abraham et al., 2). Importantly, these ompounds (espeially BIO-1211) proteted the airway from injury when delivered either by an intravenous or intratraheal route. We used a derivative of BIO-1211 to inhibit VLA-4/VCAM interation in our model of ytokine-indued lung inflammation. This ompound, BIO5192, is a very seletive and potent inhibitor of a 4 b 1 -interation and has been used in vivo in rats to blok experimentally indued autoimmune enephalomyelitis (Leone et al., 23; Theien et al., 23). Rats insufflated onomitantly with ytokine and BIO5192 showed a dosedependent derease in LDH release and redution of RAGE in the ell-free BALF, onsistent with a role for VLA-4/VCAM interation in ytokine-indued lung injury. While a statistially signifiant and dose-dependent redution in LDH release was produed by 143 or BIO5192, it is important to note that, at all doses, both ompounds produed a mild neutrophil alveolitis that was independent of ytokine insufflation (data not shown). While the present impression of these ompounds is one of monovalent and antagonisti binding (Lin et al., 1999; Abraham et al., 2; Leone et al., 23; Theien et al., 23), it is possible that the inhibitors are also apable of produing a hemotati signal for PMNs. It is unknown how suh unexpeted signaling would arise. We imagine that in the absene of a speifially ativating signal, PMNs onomitantly reruited to the lung during ytokine insufflation do not promote LDH release and this is supported by the absene of LDH in BALF from rats insufflated with 143 or BIO5192 alone. Nonetheless, despite the presene of PMNs in the ytokine-insufflated lung, LDH and RAGE release were redued by eah ompound in ytokineinsufflated rats. Clearly, further study is required to establish the signifiane of the low-level neutrophil alveolitis indued by intratraheal use of these ompounds. Apparent adhesion of MNPs to the respiratory epithelium was assoiated with enhaned lung injury, and disruption of this interation redued evidene of lung injury inluding histologi evidene of inflammation and tissue damage and release of RAGE and LDH into the ell-free BALF. Adoptive transfer of MNPs into the lungs of rats not previously exposed to ytokine failed to promote LDH release, suggesting that injury required both ytokine stimulation and the presene of MNPs. Depletion of irulating leukoytes with ylophosphamide (Ikezumi et al., 23a, b) showed that LDH release depended on both ytokine priming and the presene of inflammatory MNPs, whereas ytokine alone did not promote LDH release. These observations indiated that the alveolar epithelial ell/mnp interation mediated lung injury and that inhibitors of ICAM-1/LFA-1 and VCAM-1/ British Journal of Pharmaology (27)