eosinophils on the responsiveness of the bovine isovolumic

Transcription

1 Br. J. Phrmcol. (1993), 19, '." Mcmilln Press Ltd, 1993 Br. J. Phrmcot. (1993), 19, Mcmilln Press Ltd, 1993 A comprison of the effects of polyrginine nd stimulted eosinophils on the responsiveness of the bovine isovolumic bronchil segment preprtion Ther Omri, Mlcolm P. Sprrow, *Mrtin K. Church, *Stephen T. Holgte & 'tclive Robinson 'Deprtment of Physiology, University of Western Austrli, Nedlnds 69, Western Austrli; *Immunophrmcology Group, Southmpton Generl Hospitl, Southmpton S9 4XY nd tdeprtment of Phrmcology & Clinicl Phrmcology, St George's Hospitl Medicl School, Crnmer Terrce, London SW17 ORE 1 The bovine isovolumic bronchil segment preprtion hs been used to study the sensitivity nd responsiveness of bronchil smooth muscle fter vrious mnipultions. 2 Addition of cetylcholine (ACh) to the lumen of the segments elicited n increse in intrluminl pressure s result of contrction of the irwy smooth muscle. However, the increses in intrluminl pressure were greter when the ACh ws dded to the dventitil surfce of the preprtion. 3 Addition of polyrginine to the bronchil lumen for 6-12 min resulted in n incresed mgnitude of response nd greter thn 1 fold increse in sensitivity to ACh dministered into the lumen. Depolriztions induced by KCI were similrly enhnced when the solution ws dded into the lumen. In contrst, the sensitivity nd responsiveness to ACh or K'-induced depolriztion dministered dventitilly ws unchnged. 4 The mechnicl disruption of the epithelium produced 32 fold increse in sensitivity to ACh introduced vi the lumen, wheres the sensitivity to ACh dded dventitilly remined unltered. 5 Addition of polyrginine to the dventitil bthing medium resulted in no chnge in the responsiveness or sensitivity to ACh, irrespective of whether the ACh ws given intrluminlly or dventitilly. 6 Histologicl exmintion reveled tht polyrginine cused extensive disordering of the norml rchitecture of the bronchil epithelium. Tken together with the unltered responsiveness to dventitil ACh (i.e. lck of chnge in intrinsic muscle sensitivity) these observtions suggest tht the effect of polyrginine ws most likely due to disruption of diffusion brrier. 7 In contrst to the effects of polyrginine, the only effect of stimulted eosinophils ws to produce smll diminution in the responsiveness to ACh tht hd been dded dventitilly. Keywords: Polyrginine; bronchil responsiveness; eosinophil; bronchil segment preprtion Introduction In diseses such s bronchil sthm or following certin virl infections, the consequences of the mucosl immune response result in the cquisition of enhnced bronchil responsiveness (Empey et l.,1977; Jennings et l., 1987; Cockroft, 1989). The mechnistic reltionships between the functionl mnifesttion of bronchil hyperresponsiveness nd histologicl chnges occurring in the irwy mucos re currently unknown. However, the pprent ssocition between the presence of ctivted inflmmtory cells, injury of the irwy epithelium nd the ppernce of bronchil hyperresponsiveness hve fostered the belief tht irwy inflmmtion is of primry nd cusl significnce in its pthophysiologicl development. Superficilly, this viewpoint is ttrctive becuse inflmmtory cells certinly do hve the potentil to liberte chemicl meditors, such s free rdicls, cytotoxic proteins nd proteolytic enzymes tht re known to elicit cell injury (Gleich et l., 1988; Gleich, 199). This site-directed injury might result in the exposure of intrepithelil nerve endings, cuse reduction in the production or relese of epithelium-derived inhibitory fctors nd fcilitte the destruction of cellulr diffusion brrier; events which together might ccount for ugmented bronchil responsiveness. In studies of ptients with mild sthm Besley nd collegues (1989) reported n inverse correltion between the ' Author for correspondence. number of exfolited epithelil cells in broncholveolr lvge fluid nd the provoction concentrtion to methcholine. Other workers hve reported the existence of n inverse correltion between the proportion of eosinophils present in broncholveolr lvge fluid (Lm et l., 1987; Wrdlw et l., 1988) or the irwy mucos (Ohshi et l., 1992) nd the degree of bronchil responsiveness to inhled methcholine. Likewise, in sthmtic ptients treted with topiclly ctive corticosteroid, dt hve been presented which pprently link reduction in irwy eosinophil numbers of diminution (but not bltion) of bronchil hyperresponsiveness (Djuknovic et l., 1992). Similr evidence ssociting inflmmtory cell infiltrtion with heightened bronchil responsiveness exists in experimentl nimls (Mrsh et l., 1985; Murphy et l., 1986). On the bsis of these studies it hs been rgued tht there is likely to be cusl link between eosinophils, epithelil injury nd the cquisition of heightened bronchil responsiveness. However, severl investigtions in mn nd experimentl niml models hve provided contrry evidence which dissocites the presence of incresed numbers of eosinophils in the irwy from the development of bronchil hyperresponsiveness, or t lest csts doubt on the obligtory nd dominnt role of the eosinophil in this process (Snjr et l., 199; Chpmn et l., 1991; Djuknovic et l., 199; 1992; Elwood et l., 1992). The impliction of these experiments is tht irwy inflmmtion is not cuslly relted to bronchil hyperresponsiveness, or tht the link between them is more complex thn originlly believed. Thus, the reltionships

2 554 T. OMARI et l. between eosinophils, epithelil dmge nd smooth muscle responsiveness require further pprisl. In the present pper we report the results of experiments in which we hve continued our mechnistic investigtions into mucosl injury nd its reltionship to functionl chnges in the irwy. Specificlly we hve compred the effects on smooth muscle responsiveness of tretment with the polybsic polymer polyrginine ( surrogte for the rginine-rich eosinophil grnule proteins), tretment with eosinophils or mechnicl denudtion of the epithelium. A preliminry ccount of this work ws presented to the British Phrmcologicl Society (Omri et l., 1991). Methods Isovolumic bronchil segment preprtion Fresh bovine lungs were obtined from locl bttoir nd used not more thn 1 h post mortem. On rrivl t the lbortory, bronchil segments, pproximtely 2 cm in length nd with distl internl dimeter of.3 cm, were dissected from the upper lobes nd cnnulted t both ends. The segments were plced in modified orgn bth (Omri & Sprrow, 1992) nd bthed t 37 C in Krebs solution (composition, mm: NCl 121, KCI 5.4, MgSO4 1.2, NH2PO4 1.2, NHCO3 25, glucose 11.5 nd CCl2 2.5) nd gssed with 5% CO2 in 2. Krebs solution ws lso perfused through the lumen of the segment from n externl reservoir. The Krebs solution bthing both the luminl nd dventitil surfces ws exchnged for fresh solution t regulr intervls throughout the experiment. Chnges in muscle tone were recorded by closing off proximl nd distl tps leving the segment in series with differentil pressure trnsducer (type MPXIODP, Motorol Semiconductors, Phoenix, USA). Responses were recorded on Rikdenki R-5 chrt recorder. Agonists were introduced either on to the dventitil surfce of the segments or into the lumen, by use of syringe nd 3-wy tp situted t the distl end of the preprtion. The segments were lso prepred to permit electricl field stimultion (EFS) with pltinum ring electrodes nd Grss 15 E Oi\ _1 e Resting pressure (cmh2o) Figure 1 Responses of the bovine isovolumic bronchil segment preprtion to electricl field stimultion (6V,.5ms, 2Hz) vi pltinum ring electrodes fter being mintined t rnge of resting intrluminl pressures. Dt re men ± s.e.men of 4 seprte experiments. S44 stimultor. The tissues were mintined t resting intrluminl pressure of 6 cmh2, this being determined s optiml by studying the reltionship between EFS stimultion nd response t different resting pressures (Figure 1). Cumultive responses were recorded to cetylcholine (ACh) dded to the dventitil surfce, while responses to ACh introduced into the lumen were recorded non-cumultively. In some cses the tissues were lso chllenged with KCI depolrizing solution tht contined 121 mm KCI in plce of NCl. In ll experiments control responses to gonists nd EFS were first determined before undertking vriety of mnipultions when the tissues hd resumed their resting tone nd showed consistent responses to EFS. Mechnicl injury of bronchil epithelium As control for other mnipultions, we exmined the responsiveness of bronchil segments tht hd been subjected to mechnicl injury of their epithelium. The epithelium ws dmged by inserting into the lumen cotton pplictor tip soked in Krebs solution nd rubbing it long the length of the segment. Tretment with polyrginine Polyrginine ws dissolved in Krebs solution ech dy nd diluted to pproprite concentrtions for use. To determine the effect of polyrginine when dded to the lumen, exposures of 1 mgml-' for 1 h s well s 1 nd 1 ig ml-' for 2 h were tested. In experiments compring the dventitil nd luminl dministrtion of polyrginine, n exposure of 1 igml-l ws used. In this set of experiments, fter obtining control responses to EFS nd ACh, the segments were exposed to polyrginine vi the dventitil surfce nd the effects on the responses of the tissue observed. After complete recovery, the lumen ws then exposed to polyrginine nd the responses to ACh studied once gin. Thus, in contrst to tissues in which the epithelium hd been injured mechniclly, these studies incorported within segment rther thn between segment control responses. Experiments with inflmmtory cells For ech experiment, pired segments from the sme lung were used. After control responses hd been obtined, ech segment ws exposed to either stimulted or unstimulted cells tht hd been obtined s described below. Stimultion of eosinophils nd neutrophils ws chieved with 5,UM ionophore A23187, concentrtion tht we hve previously shown to ctivte these cell types (Herbert et l., 1991). The ionophore (or its dimethylsulphoxide (DMSO) vehicle t finl concentrtion of 1.6%) ws dded to the cells 1 min before they were introduced into the lumen of the segments. After n exposure period of 1 h the segments were then flushed with fresh Krebs solution nd the perfuste collected for the mesurement of leukotriene B4 by rdioimmunossy. Preprtion of inflmmtory cells A peritonel eosinophili ws estblished in guine-pigs by injecting mle Dunkin-Hrtley strin nimls i.p. with 1 mg ml-' polymyxin B in sline twice weekly for 4 weeks. At 24 h fter the lst injection, the nimls were nesthetized with metofne nd 16G cnnul (Critikon Ltd, Ascot, Berks.) inserted into the peritonel cvity. The cvity ws lvged with 5 ml sterile sline nd the lvge return collected into polypropylene tube. The lvge fluid ws centrifuged t 15 g for 1 min t room temperture nd resuspended in 2 ml of Dulbecco's phosphte buffered sline (DPBS, C2+- nd Mg2+-free). Erythrocytes were lysed by use of hypotonic sline nd n initil differentil cell count then performed by

3 POLYARGININE AND BRONCHIAL RESPONSIVENESS 555 use of eosin/pontmine sky blue. In some experiments the cells were resuspended in n pproprite volume of Krebs solution nd used without further purifiction. In those experiments which used purified eosinophils, further enrichment ws obtined by resuspending the cells in Tyrode solution contining geltin nd deoxyribonuclese (DNse) nd subjecting the cells to density centrifugtion on Metrizmide grdients. The wshed lvge cells were overlid onto density grdients which consisted of cushion of 2ml Mxidens upon which discontinuous grdient of 16, 18, 2, 22, 24, 26 nd 3% Metrizmide in Tyrode solution (supplemented with geltin nd DNse) hd been prepred. Ech step in the grdient hd volume of 2 ml nd mximum of 18 cells ws loded onto ech grdient. Centrifugtion ws performed t 12g for 45min t room temperture. The eosinophil-rich bnd t the 24-26% Metrizmide interfce ws crefully spirted., wshed in DPBS nd the number nd purity of cells determined by use of eosin/pontmine sky blue. Vibility ws determined by exclusion of trypn blue nd ws generlly >9%. Humn neutrophils were obtined from the blood of volunteers who hd tken no mediction within t lest one week prior to dontion. Eight volumes of citrte-nticogulted blood were treted with 1 volume of 6% dextrn (Mr 5,) nd 1 volume of norml sline. Erythrocytes were sedimented t 1 g for 3 min, fter which the plsm lyer ws crefully spirted, mixed with n equl volume of phosphte-buffered sline nd lyered s 5 ml liquots onto 5 ml of Mono-Poly resolving medium. After centrifugtion t 4 g for 3 min the polymorphonucler cell lyer ws spirted, the cells wshed in Krebs solution nd n liquot tken for differentil cell count by use of Wright's stin nd n ssessment of cell vibility with trypn blue. Histology At the end of the experiment segments were fixed for 48 h in 4% formldehyde solution. Smll pieces were then dissected nd embedded in Cryo-M-Bed prior to the preprtion of 1 lsm cryostt sections. Sections were stined with hemtoxylin nd eosin nd the ppernce of the irwy mucos ssessed by light microscopy (Zeiss Axiovert 1 or Leitz Lborlux 12). Dt presenttion Dt re shown s either the increse in intrluminl pressure bove the resting vlue of 6 cmh2o or s percentge of the mximl response to 1 mm ACh. The sensitivities of the segments to dventitilly dded ACh were compred by determining the EC5 vlues. The responses to intrluminl ACh rrely exceeded 3% of mximum nd consequently the sensitivities were compred by clculting concentrtions corresponding to EC2 of the mximum dventitil response. Results re expressed s the men ± s.e.men of n preprtions. The significnce of ny difference between mens ws evluted with Student's t test, with P <.5 being considered sttisticlly significnt. Tests for pired smples were used to compre dt obtined within individul segments (i.e. control versus subsequent mnipultion), wheres unpired tests were used to evlute dt between experiments. Mterils The following were purchsed from Sigm (Poole, Dorset): cetylcholine, ionophore A23187, polyrginine (Mr 11,6), polymyxin B sulphte, dimethysulphoxide, geltin (from bovine skin) nd DNse. DPBS nd Mono-Poly resolving medium were obtined from Flow Lbortories (Rickmnsworth, Herts). Metrizmide nd Mxidens were purchsed from Nycomed (Birminghm). Metofne (methoxyfluorne) ws obtined from C-Vet Ltd (Bury St Edmunds, Suffolk). Regents for the rdioimmunossy of leukotriene B4 were purchsed from NEN-Du Pont Reserch Products (Stevenge, Herts). Stins for microscopy nd ll other lbortory regents were purchsed from BDH (Poole, Dorset). Results Responses of the preprtion The responses of bovine bronchil segment mintined t resting trnsmurl pressure of 6 cmh2o re shown in Figure 2. Electricl field stimultion (EFS) t 6 V with durtion of.5 ms nd frequency of 2 Hz produced n increse in intrluminl pressure which relxed redily upon cesstion of the stimulus (Figure 2). Also illustrted in Figure 2 is typicl cumultive response to ACh tht hd been pplied to the dventitil surfce, wheres Figure 2b displys the significntly smller nd non-cumultive dose-response chrcteristics for ACh injected intrluminlly. For exmple, 1O mm ACh pplied dventitilly elicited contrction tht produced men increse in pressure of 33. ± 1.8 cmh2o (n = 45), wheres introduction of ACh into the lumen produced pressure chnge of only 12.1 ± 1.2 cmh2o (n = 45, Ps.1). Effect ofpolyrginine In these experiments, luminl or dventitil surfces of bovine bronchil segments were exposed to severl concentrtions of polyrginine. By itself, polyrginine ltered neither the resting tone of the bronchi, nor the responses to EFS. However, the responses of bronchil segments to ACh dded intrluminlly were significntly enhnced by prior exposure of the lumen to 1 ig ml-' polyrginine for 2 h (Figure 3). For exmple, the response to 1 mm ACh dded into the lumen ws incresed from 7.3 ± 1.6 to 28.9 ± 3.9 cmh2o (n = 4, P..1). This effect ws not seen if the polyrginine ws dded to the dventitil bthing medium (Figure 3). In contrst, the responses to ACh dded dventitilly were unchnged by exposure of either the luminl or dventitil surfces to polyrginine when using the sme tretment regime (Figure 3b, n = 4 for ech type of exposure). In the cse of EFS, the responses of bronchil segments were unchnged by polyrginine, even fter 1 h exposure to I mg ml' (21.7 ± 6.2 versus 22.2 ± 7.4 cmh2 in control nd treted tissues respectively, n = 4). However, it ws of interest tht the responses to depolriztion by KCI 2 - Ci E - 2 Hz b min Figure 2 Increses in the intrluminl pressure of bronchil segment contrcting isovolumiclly to electricl field stimultion (EFS) nd cetylcholine (ACh) from resting pressure of 6 cmh2o: () illustrtes the response to EFS nd cumultive responses to ACh dded to the dventitil surfce; (b) illustrtes single responses to ACh dministered intrluminlly.

4 556 T. OMARI et l. N IE ) QC U) b lo-4 1o-3.', were enhnced in similr wy to those of ACh (Figure 4). Tble 1 summrizes the effect of different tretments with polyrginine, s well s mechnicl injury, on the EC2 nd EC5 vlues which indicte the sensitivity to luminlly nd dventitilly pplied ACh respectively. Exposure to 1 lag ml-' polyrginine for 12 min did not lter the sensitivity to ACh t ll, wheres higher concentrtions in the lumen (1 ILg ml' nd 1 mg ml') enhnced the sensitivity to luminlly-pplied ACh by 11 nd 135 fold respectively. The EC5 vlues for ACh pplied to the outside of the tissue were unchnged by these tretments. Similrly, tissues tht hd been subjected to mechnicl trum of the epithelium were on verge 32 fold more sensitive to ACh dded into the lumen when compred with epithelium-intct segments for the sme lung. However, there ws no chnge in sensitivity to ACh dded to the dventitil side of mechniclly injured preprtions (Tble 1). Overll, there ws no significnt difference in the chnges in tissue sensitivity evoked by 1-2 polyrginine or mechnicl trum. Tht mechnicl injury ppered to be less effective thn polyrginine tretment is probbly reflection of the limittions of the different experimentl protocols used nd the wy in which control re N 3 I E ) ) o 2- )1 T T -1..= Figure 3 Effect of polyrginine on the responsiveness to cetylcholine (ACh). In (), ACh ws dded into the lumen. In (b), ACh ws dded to the dventitil surfce. Ech tissue served s its own control (-) before exposure of the lumen (-) or the dventitil surfce () to 1 gml-' polyrginine for 2h. Dt re men±s.e.men of 4 seprte experiments. *P <.5; **P<.1 with respect to pproprite control. 1 I In Out In Out KCI KCI Control PA I mg ml-' Figure 4 Effect of polyrginine (PA, 1 mg ml-', 1 h in the lumen) on the response to electricl field stimultion (EFS) nd depolriztion evoked by K+. Dt re men ± s.e.men from 4 seprte experiments. *P <.1 with respect to pproprite control. Tble 1 Clculted EC2 nd EC5 vlues for cetylcholine (ACh) dded to either the luminl or dventitil sides of bovine bronchil segments before nd fter exposure to polyrginine: lso included re vlues obtined from tissues where the epithelium hd been injured mechniclly nd their corresponding epithelium-intct controls from the sme lungs Tretment Polyrginine (1 fig ml1, 2 h) Polyrginine (1 ig ml', 2 h) Polyrginine (1 mg ml- ', 1 h) Epithelium-intct segments Mechniclly injured segments loglo EC2 ACh (luminl) ± ± ± ±.26** ± ±.33* ± ±.12t log,o EC5 ACh (dventitil) ± ± ± ± ± ± ± ±.2 Asterisks indicte significnt differences between pre- nd vlues, *P <.5; **P <.1. tsignificntly different from intct segments, P <.1. All dt re men ± s.e.men vlues from four experiments in ech cse.

5 POLYARGININE AND BRONCHIAL RESPONSIVENESS 557 sponses were determined (see Methods). It is lso possible tht mechnicl injury filed to remove significnt mounts of the epithelium. Effect of inflmmtory cells Bronchil segments were exposed to ionophore A (51M, intrluminlly) to estblish whether A23187 could be used s mens of ctivting inflmmtory cells without interfering with the responsiveness of bronchil smooth muscle. The ionophore produced slow increse in bronchil tone tht reched 5% of the mximl response fter pproximtely 3 h (Figure 5). Despite the chnge in tone, the smooth muscle responsiveness to ACh pplied either intrluminlly or dventitilly ws unltered by 6 min exposure to 5 LM A23187 in the lumen (Figure 6). When bronchil segments were exposed to either stimulted or unstimulted guine-pig peritonel eosinophils (15.9 ±.1 x 16 mi1 for 6 min) there were no significnt effects on the responsiveness to ACh dministered intrluminlly when this ws compred with the corresponding control responses mde prior to the ddition of cells (Figure 7). This ws true irrespective of whether the eosinophils were used s unfrctionted peritonel lvge cells or whether they hd been purified by density grdient centrifugtion. However, when ACh ws pplied dventitilly fter exposure to eosinophils it ws noted tht there ws smll reduction in the mgnitude of response compred with the pre-eosinophil control vlues. In segments exposed to stimulted eosinophils this tendency ws sttisticlly significnt t ll but the highest dose of ACh (Figure 7). Pre- nd EC2 nd EC3 vlues derived from these dose-response curves re illustrted in Tble 2. In one series of studies we incubted bronchil segments with eosinophil popultions t 91.6 x 16 ml-' nd purity of 92.6% (with nd without ionophore stimultion) nd even under these conditions there ws no E c; :L Lc 3 Min Figure 5 A trcing of chnges in intrluminl pressure in bovine bronchil segment tht hd been exposed to 5 g4m A23187 (C-io) on the luminl side. 3 N E 2 1) CL ) cr A Figure 6 Concentrtion-response curve to cetylcholine (ACh) dded either luminlly () or dventitilly () under control conditions. Tissues were then exposed to SlM A23187 dded into the lumen () or into the dventitil bthing medium (-) for 6min before retesting tissue responsiveness. Dt re men ± s.e.men of six experiments. increse in muscle responsiveness (dt not shown). As judged by the relese of immunorective leukotriene B4, the eosinophils were ctivted for meditor relese by A23187, with net relese of 1.39 ±.29 ng LTB4 per 16 eosinophils being detected in the fluid returned from the bronchil lumen. Similr experiments were performed with humn neutrophils with 6 min exposures of bronchil segments to cell numbers rnging from 6-33 x 16 neutrophils ml-' t n verge purity of 88.6 ± 4.2%. In no cse ws the responsiveness of the tissues mterilly ltered by these tretments (dt not shown). These cells exhibited net relese of leukotriene B4, (LTB4), confirming their stimultion by A23187, with net 2.81 ± 2.27 ng LTB4 per 16 neutrophils being detected in the fluid recovered from the bronchil lumen. Histologicl effects Cryostt sections prepred from control bronchil segments or tissues tht hd been exposed to 1gml-' polyrginine for 6 min showed preservtion of reltively intct, helthy epithelium. At high mgnifiction, bundnt cili were evident on cells, nd focl intercellulr contct ws notble feture between suprbsl epithelil cells. In contrst, those segments exposed to higher concentrtions of polyrginine exhibited cytoplsmic shrinkge, loss of interepithelil dhesion nd evidence of exfolition (Figure 8). Interestingly, the res where exfolition ws noted showed retention of bsl cells nd cellulr debris on the mtrix substrtum. Bronchil segments tht hd been exposed to either unstimulted or ionophore-stimulted eosinophils nd neutrophils were lso exmined. In contrst to the effects of polyrginine, the exposure to inflmmtory cells ws chrcterized by little effect on the gross morphology of the irwy mucos. Discussion In this series of experiments we hve demonstrted tht exposure of the bronchil lumen to the bsic mino cid

6 558 T. OMARI et l N E Co C (A 4 b b io Figure 8 Sections of bronchil segments following () control tretment, or (b) exposure for 2 h to 1 jig ml-' polyrginine. The scle brs represent 2 jam in ech cse. Figure 7 The effect of exposure of bronchil segments to either unstimulted () or ionophore stimulted (b) eosinophils. Control concentrtion-response curves to cetylcholine (ACh) (solid symbols) were initilly undertken before ddition of the unfrctionted peritonel eosinophils to the bronchil lumen (15.9 ±.1 x 16 ml t n verge purity of %, n = 5). Responses fter exposure to eosinophils re denoted by open symbols. In ech experiment one tube of ech pir dissected from the lung received unstimulted eosinophils, the other received cells which hd been ctivted by the ionophore. Dt re men ± s.e.men of 5 experiments nd sterisks denote significnt differences from the pproprite control vlues. Tble 2 Clculted EC2 nd EC5 vlues for cetylcholine (ACh) dded luminlly or dventitilly before nd fter exposure of bronchil segments to guine-pig peritonel eosinophils (15.9 x 16 ml-') loglo EC2 ACh log,o EC5 ACh Tretment (luminl) (dventitil) Unstimulted eosinophils Stimulted. eosinophils ± ±.22-3.± ± ± ± ± ±.27 Dt re from five experiments in ech cse. polymer, polyrginine, results in reltively rpid ugmenttion of the responsiveness of the underlying smooth muscle to ACh nd KCI-induced depolriztions, but not to EFS. This enhncement ws not observed if the dventitil surfce of the preprtion ws similrly treted. A further feture of the 'sidedness' of this phenomenon ws tht only the responses to gonists introduced into the bronchil lumen were enhnced. Responses to gonists pplied dventitilly were unchnged, even in those bronchil segments which hd been treted with polyrginine in the bronchil lumen. This pttern of ltertion in the responsiveness of the bronchil smooth muscle ws similr to tht seen in segments in which the bronchil mucos hd been injured mechniclly (this study; Munkt et l., 1989; Sprrow & Mitchell, 1991; Omri & Sprrow, 1991) nd is consistent with the effects seen fter exposure of the mucos to proteinses (Omri et l., 1991b; Omri & Sprrow, 1992). Collectively, these similrities suggest tht polyrginine my be cusing disruption of the irwy muscos, view tht ws verified experimentlly by undertking histologicl evlution before nd fter tretment with vrious concentrtions of polyrginine. Tissues tht hd not been exposed to polyrginine, or those which hd experienced only low concentrtions, exhibited norml ppernce with n essentilly intct epithelium. In contrst, tissues incubted with concentrtions of polyrginine tht significntly ugmented the smooth muscle responsiveness were chrcterized by evidence of cytolytic dmge, cytoplsmic shrinkge nd frnk exfolition. It ws

7 POLYARGININE AND BRONCHIAL RESPONSIVENESS 559 of interest tht res of mucos tht hd been denuded of epithelil cells in this wy still retined significnt number of bsl cells. This pttern of tissue injury is thus in mny wys similr to tht produced by stimulted eosinophils in n in vitro model of mucosl injury (Herbert et l., 1991) which hs been suggested to represent convenient model of the mucosl lesion which chrcterizes lung diseses such s bronchil sthm (Herbert et l., 1991; Montefort et l., 1992). The retention of bsl cells in injury of the irwy mucos my rise becuse the desmosoml ttchment of columnr epithelil cells to bsl cells is more susceptible to clevge thn is the dhesion of bsl cells to the biomtrix (Montefort et l., 1992). As the irwy epithelium functions, in prt, s protective brrier it seems plusible tht one effect of polyrginine is to render the mucos more permeble to solutes s consequence of the cellulr injury nd loss of interepithelil tight junctions. In support of this view Herbert et l. (1991) demonstrted tht polyrginine (1 mg ml-') significntly incresed the permebility of the bovine bronchil mucos to serum lbumin. The filure of lower concentrtion of polyrginine to produce significnt effect on the net unidirectionl flux of lbumin, compred to the present results in smooth muscle, probbly reltes to the fct tht Herbert nd collegues focused their ttention on mcromoleculr solute permebility rther thn highly diffusible low moleculr mss drugs. The irwy epithelium hs lso been suggested s source of poorly-chrcterized fctors which inhibit the responsiveness of the underlying smooth muscle (Brnes et l., 1985; Vnhoutte, 1988; Spin et l., 1992). It is therefore possible tht removl of such inhibitory fctors my contribute to the heightened responsiveness seen in the present experiments. Whilst the bovine bronchil epithelium does relese substnces, such s prostglndin E2 (PGE2), which hve relxnt effects on irwy smooth muscle (Herbert & Robinson, unpublished), we discount this s n explntion for our observtions in this study. Moreover, we lso rgue ginst the possibility tht polyrginine enhnces responsiveness by relesing bronchoconstrictor gents (e.g. histmine from mst cells). The principl evidence ginst these hypotheses is tht ddition of polyrginine to the bronchil lumen does not chnge the responses of the tissue to gonists dded dventitilly, even though the responses to intrluminl ACh re enhnced. Furthermore, in study of mst cell heterogeneity in bovine lung Hunt et l. (1991) demonstrted the refrctoriness of bovine mst cells to polybsic secretgogues. Thus, we fvour the view tht the principl effect of polyrginine is to disrupt the epithelil diffusion brrier. The behviour of the irwy epithelium s diffusion brrier cpble of modulting the ctions of phrmcologicl gonists hs been suggested on the bsis of other studies with bronchil segments (Smll et l., 199; Sprrow & Mitchell, 1991; Omri & Sprrow, 1992; Omri et l., 1993). One of our resons for studying polyrginine in the present experiments is tht it my be useful model of the rgininerich cytotoxic proteins of the eosinophil (Butterworth et l., 1979; Uchid et l., 199). Attention hs been focussed on the role of eosinophil-derived proteins, in prticulr the mjor bsic protein (MBP), in the pthogenesis of mucosl injury (Gleich et l., 1988). Deposits of MBP hve been found djcent to res of epithelil exfolition in the sthmtic lung (Filley et l., 1982) nd t reltively high concentrtions in vitro it is known to cuse disruption of nsl nd bronchil epithelium (Frigs et l., 198; Hstie et l., 1987; Ayrs et l., 1989; Motojim et l., 1989) nd modest increse in the responsiveness of irwy smooth muscle (Flvhn et l., 1988). Other studies hve demonstrted tht MBP cuses n epithelium-dependent increse in bronchil tone in the guine-pig nd monkey (White et l., 199; Gundel et l., 199) nd n ugmenttion of responses to gonists dministered intrvenously (Brofmn et l., 1989; White et l., 199). However, the mechnism(s) of these chnges re not understood. In our studies polyrginine hd no effect on the resting tone of the bronchil segments, nor did we observe ny chnge in the response of the tissue to ACh dded dventitilly. These dt suggest tht there my be importnt differences between the ction of MBP nd bsic mino cid polymers, implying tht the interction between MBP nd trget cells is more subtle thn tht defined by ctionic nture lone. Clerly, further work is necessry to estblish exctly how polyrginine, MBP nd other eosinophil proteins cuse such n pprently trgeted injury of epithelil cells. In view of the estblished toxicity of eosinophil grnule proteins nd other eosinophil-derived meditors towrds irwy epithelium it ws surprising to find tht guine-pig peritonel eosinophils tht hd been stimulted by A23187 produced only smll decrese in the responsiveness to ACh dded to the dventitil surfce of the preprtion. The bsis of this effect remins unknown. Reltively smll numbers of eosinophils were used in the present studies nd it is conceivble tht the quntities of eosinophil-derived meditors relesed were indequte to ffect irwy function over the time course of the experiment. Interestingly, Jongejn (1991) did not observe ny chnge in the sensitivity of humn bronchil rings to methcholine fter exposure to grnulocytes (1-5 x 1' of which >9% were neutrophils) tht hd been ctivted by zymosn. However, bronchil rings incubted with lrger numbers of grnulocytes (1-2 x 16) exhibited decresed responsiveness. Similrly, McAlpine nd collegues (1988) found tht zymosn-ctivted neutrophils did not ffect the responsiveness of rbbit bronchi. The stimultion of the eosinophils nd neutrophils ws confirmed in our own experiments by demonstrting net relese of leukotriene B4 in the fluid recovered from the segments fter incubtion with the cells. The most likely sources of this leukotriene re the eosinophils nd neutrophils becuse, in contrst to the leukocytes bovine bronchil mucos is only poor source of this eicosnoid (Sun et l., 1989; Herbert, 1992). In contrst to the unexpected effect on bronchil responsiveness in this study, reltively smll numbers of guine-pig eosinophils nd humn neutrophils (3-1 x 16 ml-') hve been shown to increse the net unidirectionl flux of lbumin cross the bovine bronchil mucos by cusing the exfolition of epithelil cells (Herbert et l., 1991; Herbert, 1992). In the present work, histologicl exmintion of the bronchil segments tht hd been exposed to stimulted leukocytes showed considerbly less disruption thn those exposed to polyrginine. These findings re in mrked contrst to the tissue morphology reported previously fter exposure to eosinophils obtined nd stimulted by methods identicl to those used in the present study (Herbert et l., 1991). However, there re severl importnt differences which my ccount for the bsence of eosinophil-induced tissue injury. Firstly, in the current study the eosinophils were dded to the bronchil lumen nd not the dventitil side of the isolted epithelium s reported by Herbert et l. (1991). Secondly, no specil mesures were tken to promote eosinophil dhesion to the trget tissue within the bronchil lumen. This is in contrst to the experiments performed by Herbert et l. (1991) in which eosinophils were incubted in smll volume under conditions which promoted settling onto limited nd defined re of bronchil mucos. Moreover, these uthors presented specific evidence which indicted tht the eosinophil-dependent injury to the mucos ws contctdependent phenomenon (Herbert et l., 1991). In summry, these studies hve shown tht polyrginine is ble to ugment the responsiveness of bronchil smooth muscle to ACh in n effect which is dependent on the removl of the diffusion brrier presented by the irwy epithelium. Eosinophils stimulted by the ionophore A23187 were unble to reproduce this phenomenon, suggesting tht if eosinophils re responsible for the bronchil hyperrectivity seen in sthm then their bility to injure the irwy mucos

8 56 T. OMARI et l. is more chronic process thn tht produced in this in vitro system. Alterntively, certin spects of the interction between eosinophils nd the irwy epithelium my be different in certin types of clinicl sthm. In view of the findings reported by Herbert et l. (1991) one such difference References AYARS, G.H., ALTMAN, L.C., MCMANUS, M.M., AGOSTI, J.M., BAKER, C., LUCHTEL, D.L., LOEGERING, D.A. & GLEICH, G.J. (1989). Injurious effect of the eosinophil peroxide-hydrogen peroxide-hlide system nd mjor bsic protein on humn nsl epithelium in vitro. Am. Rev. Respir. Dis., 14, BARNES, P.J., CUSS, F.M. & PALMER, J.B. (1985). The effect of irwy epithelium on smooth muscle contrctility in bovine trche. Br. J. Phrmcol., 86, BEASLEY, R., ROCHE, W.R., ROBERTS, A.J. & HOLGATE, S.T. (1989). Cellulr events in the bronchi in mild sthm nd fter bronchil provoction. Am. Rev. Respir. Dis., 139, BROFMAN, J.D., WHITE, S,R., BLAKE, J.S., MUNOZ, N.M., GLEICH, C.J. & LEFF, A.R. (1989). Epithelil ugmenttion of trchelis contrction cused by mjor bsic protein of eosinophils. J. Appl. Physiol., 66, BUTTERWORTH, A.E., WASSOM, D.L., GLEICH, G.J., LOEGF.RING, D.A. & DAVID, J.R. (1979). Dmge to shistosomul of schistosom mnsoni induced directly by eosinophil mjor bsic protein. J. Immunol., 122, CHAPMAN, I.D., BOUBEKEUR, K. & MORLEY, J. (1991). Airwy eosinophili nd irwy hyperresponsiveness prsle other sequentil events in the guine pig. In New Drugs Fo.r Asthm. ed. Morley, J. & Anderson, G. Bsle: Birkhuser-Verlg. COCKROFT, D.W. (1989). Airwy hyperresponsiveness: therpeutic implictions. Ann. Allergy, 59, DJUKANOVIC, R., WILSON, J.W., BRITTEN, K.M., WILSON, S.J., WALLS, A.F., ROCHE, W.R., HOWARTH, P.H. & 1QLGATE, S.T. (199). Quntittion of mst cells nd eosinophils in the bronchil mucos of symptomtic topic sthmtics nd helthy control subjects using immunohistochemistry. Am. Rev. Respir. Dis., 142, DJUKANOVIC, R., WILSON, J.W., BRITTEN, K.M., WILSON, S.J., WALLS, A.F., ROCHE, W.R., HOWARTH, P.H. & HOLGATE, S.T. (1992). Effect of n inhled corticosteroid on irwy inflmmtion symptoms in sthm. Am. Rev. Respir. Dis., 145, ELWOOD, W., LOTVALL, J.O., BARNES, P.J. & CHUNG, K.F. (1992). Effect of dexmethsone nd cyclosporin A on llergen-induced irwy hyperresponsiveness nd inflmmtory cell responses in sensitized brown norwy rts. Am. Rev. Respir. Dis., 145, EMPEY, D.W., LAITINEN, L.A., JACOBS, L., GOLD, W.M. & NADEL, J.A. (1977). Mechnisms of bronchil hyperrectivity in norml subjects fter upper respirtory trct infections. Am. Rev. Respir. Dis., 113, FILLEY, W.V., HOLLEY, K.E., KEPHART, G.M. & GLEICH, G.J. (1982). Identifiction by immunofluorescence of eosinophil grnule mjor bsic protein in lung tissue of ptients with bronchil sthm. Lncet, ii, FLAVAHAN, N.A., SLIFMAN, N.R., GLEICH, G.J. & VANHOUTTE, P.M. (1988). Humn eosinophil mjor bsic protein cuses hyperrectivity of respirtory smooth muscle. Role of the epithelium. Am. Rev. Respir. Dis., 138, FRIGAS, E., LOEGERING, D.A. & GLEICH, G.J. (198). Cytotoxic effects of the guine pig eosinophil mjor bsic protein on trchel epithelium. Lb Invest., 42, GLEICH, G.J. (199). The eosinophil nd bronchil sthm: current understnding. J. Allergy Clin. Immunol., 85, GLEICH, G.J., FLAVAHAN, N.A., FUJISAWA, T. & VANHOUTTE, P.M. (1988). The eosinophil s meditor of dmge to respirtory epithelium: model for bronchil hyperrectivity. J. Allergy Clin. Immunol., 81, GUNDEL, R.H., LETTS, L.G. & GLEICH, G.J. (1991). Humn eosinophil mjor bsic protein induces irwy constriction nd irwy hyperresponsiveness in primtes. J. Clin. Invest., 87, HASTIE, A.T., LOEGERING, D.A., GLEICH, G.J. & KUEPPERS, G. (1987). The effect of purified eosinophilic mjor bsic protein on mmmlin ciliry ctivity. Am. Rev. Respir. Dis., 135, my involve the nture of the physicl contct between eosinophils nd the mucosl microenvironment. We thnk the British Council, The Medicl Reserch Council nd The British Lung Foundtion for finncil support. Thnks re lso due to Dr Crolyn Herbert for helpful discussion. HERBERT, C.A. (1992). The effects of leukocytes, locl hormones nd selected peptides on the permebility of the bronchil mucos to lbumin in vitro. Ph.D. Thesis. University of Southmpton. HERBERT, C.A., EDWARDS, D., BOOT, J.R. & ROBINSON, C. (1991). In vitro modultion of the eosinophil-dependent enhncement of the permebility of the bronchil mucos. Br. J. Phrmcol., 14, JENNINGS, L.C., BARNS, G. & DAWSON, K.P. (1987). The ssocition of viruses with cute sthm. N.Z. Med. J., 1, JONGEJAN, R.C. (1991). Responsiveness of isolted humn irwys. Modultion by inflmmtory cells, meditors nd physicl stimuli. Ph.D. Thesis. Ersmus University, Rotterdm, The Netherlnds. LAM, S., LERICHE, J., PHILLIPS, D. & CHAN-YEUNG, M. (1987). Cellulr nd protein chnges in bronchil lvge fluid fter lte sthmtic rection in ptients with red cedr sthm. J. Allergy Clin. Immunol., 8, MARSH, W.R., IRVIN, C.G., MURPHY, K.R., BEHRENS, B.L. & LARSEN, G.L. (1985). Increses in irwy rectivity to histmine nd inflmmtory cells in broncholveolr lvge fter the lte sthmtic response in n niml model. Am. Rev. Respir. Dis., 131, MCALPINE, L.G., MCGRATH, J.C., THOMSON, N.C. & WILSON, E.P. (1988). Effect of ctivted humn neutrophils on histmine response of rbbit secondry bronchi smooth muscle in vitro. Br. J. Phrmcol,, 95, 526P. MONTEFORT, S., HERBERT, C.A., ROBINSON, C. & HOLGATE, S.T. (1992). The bronchil epithelium s trget for inflmmtory ttck in sthm. Clin. Exp. Allergy, 22, MOTOJIMA, S., FRIGAS, E., LOEGERING, D.A. & GLEICH, G.J. (1989). Toxicity of eosinophil ctionic proteins for guine pig trchel epithelium in vitro. Am. Rev. Respir. Dis., 139, MUNAKATA, M., HUANG, I., MITZNER, W. & MENKES, H. (1989). The protective role of the epithelium in the guine pig. J. Appl. Physiol., 66, MURPHY, K.R., WILSON, M.C., IRVIN, C.G., GLEZEN, G.S., MARSH, W.R., HASLETT, C., HENSON, P.M. & LARSEN, G.L. (1986). The requirement for polymorphonucler leukocytes in the lte sthmtic response nd heightened irwys rectivity in n niml model. Am. Rev. Respir. Dis., 134, OHASHI, Y., MOTOJIMA, S., FUKUDA, T. & MAKINO, S. (1992). Airwy hyperresponsiveness, incresed intrcellulr spces of bronchil epithelium, nd incresed infiltrtion of eosinophils nd lymphocytes in bronchil mucos in sthm. Am. Rev. Respir. Dis., 145, OMARI, T.I., HERBERT, C.A., SPARROW, M.P., CHURCH, M.K., HOL- GATE, S.T. & ROBINSON, C. (1991). Effects of polyrginine on bronchil mucosl permebility nd its reltionship to smooth muscle contrction in isovolumic bronchil segments. Br. J. Phrmcol., 12, 179P. OMARI, T.I., MITCHELL, H.W. & SPARROW, M.P. (1991b). Epithelil dmge nd responsiveness of bronchil segments. Clin. Exp. Phrmcol. Physiol., 18 (Suppl.) 47. OMARI, T.I. & SPARROW, M.P. (1992). Epithelil disruption by proteses ugments the responsiveness of porcine bronchil segments. Clin. Exp. Phrmcol. Physiol., 19, OMARI, T.I., SPARROW, M.P. & MITCHELL, H.M. (1993). Responsiveness of humn isolted bronchil segments nd its reltionship to epithelil loss. Br. J. Clin. Phrmcol., 35, (in press). SANJAR, S., AOKI, S., KRISTERSSON, A., SMITH, D. & MORLEY, J. (199). Antigen chllenge induces pulmonry irwy eosinophil ccumultion nd irwy hyperrectivity in sensitized guine pigs: the effect of ntisthm drugs. Br. J. Phrmcol., 99, SMALL, R.C., GOOD, D.M., DIXON, J.S. & KENNEDY, I. (199). The effects of epithelium removl on the ctions of cholinomimetic drugs in opened segments nd perfused tubulr preprtions of guine pig trche. Br. J. Phrmcol., 1,

9 POLYARGININE AND BRONCHIAL RESPONSIVENESS 561 SPARROW, M.P. & MITCHELL, H.W. (1991). Modultion by the epithelium of the extent of bronchil nrrowing produced by substnces perfused through the lumen. Br. J. Phrmcol., 13, SPINA, D., FERNANDES, L.B., PREUSS, J.M.H., HAY, D.W.P., MUC- CITELLI, R.M., PAGE, C.P. & GOLDIE, R.G. (1992). Evidence tht epithelium-dependent relxtion of vsculr smooth muscle detected by co-xil biossys is not ttributble to hypoxi. Br. J. Phrmcol., 15, SUN, F.F., CZUK, C.I. & TAYLOR, B.M. (1989). Archidonic cid metbolism in guine pig eosinophils: synthesis of thromboxne B2 nd leukotriene B4 in response to soluble or prticulte ctivtion. J. Leukocyt. Biol., 46, UCHIDA, D.A., LARSEN, G.L. & IRVIN, C.G. (199). Ctionic chrge my be responsible for the increse in irwys responsiveness ssocited with mjor bsic protein. Am. Rev. Respir. Dis., 141, A174. VANHOUTTE, P.M. (1988). Epithelium-derived relxing fctor: myth or relity? Thorx, 43, WARDLAW, A.J., DUNETTE, S., GLEICH, G.J., COLLINS, J.V. & KAY, A.B. (1988). Eosinophils nd mst cells in broncholveolr lvge in subjects with mild sthm. Am. Rev. Respir. Dis., 137, WHITE, S.R., OHNO, S., MUNOZ, N.M., GLEICH, G.J., ABRAHAMS, C., SOLWAY, J. & LEFF, A.R. (199). Epithelium-dependent contrction of irwy smooth muscle cused by eosinophil MBP. Am. J. Physiol., 259, L294-L33. (Received November 18, 1992 Revised Jnury 28, 1993 Accepted Februry 18, 1993)