Veterinary Immunology and ImmunopathoIogy, 25 ( 1990 ) 209-217 Elsewer Scmnce Pubhshers B V, Amsterdam - - Prmted m The Netherlands

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Effects of P r o i n f l a m m a t o r y Mediators on Canine N e u t r o p h i l C h e m o t a x i s and A g g r e g a t i o n H STROM* and M K THOMSEN**

Department of Pharmacology, Leo Pharmaceuttcal Products, DK-2750 Ballerup (Denmark) (Accepted 7 December 1989)

ABSTRACT Strom, H and Thomsen, M K , 1990 Effects of promflammatory medmtors on canine neutrophfi chemotaxls and aggregatmn Vet Immunol Immunopathol, 25 209-217 The present study investigated m vitro the quahtatlve and quantitative neutrophll functionactivating propertms of the most important non-cytoklne participants in the acute inflammatory cell response Apart from the results obtained with leukotrmne (LT) D4, similar quahtatlve relatmnsh~ps were found for the several medmtors tested regardmg effects on neutrophfl mlgratmn and aggregatmn Thus, LTB4, PAF-acether and zymosan-actlvated serum all ach~ated both functions, while f-Met-Leu-Phe had no effect In all cases, the halt-mammal ehcltmg concentratmn (ECho) for mductmn of chemotams was much lower than for aggregatmm indicating that high and low receptor afflmty responses were being studmd, respectively LTD4 reduced modest aggregatmn, but was virtually w~thout effect on m~gratlon Using PAF-acether as stimulus, the mechanism of aggregatmn was studmd m more detail The results mdmate that LTB4, PAF-acether, the complement-spht products C5a/C5a desArg, and perhaps LTD4, may play a role as shmulants of neutrophfi functmns m inflammatory processes m wvo

INTRODUCTION

Polymorphonuclear granulocytes ( P M N ) play a v~tal role m host defence against mlcroorgamsms The temporal sequence revolved m activation of P M N can be divided into early events, 1 e aggregation, vascular adherence and chemotaxls, and late events occurring at ten-fold higher stimulant concentrahons, 1 e lysosomal enzyme and superomde amon secrehon (Showell et al, 1976) Finally, phagocytosm is a process lmtlated by the antibody- or C3bcoated microorganism Endogenous p r o m f l a m m a t o r y mediators generally have an effect on P M N *Present address Department of Small Ammal Chmc and Chmcal Practice, Royal Veterinary and Agricultural Umverslty, Bulowsvej 13, DK-1870 Fredenksberg C (Denmark) **To whom correspondence should be addressed

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© 1990 Elsewer Scmnce Pubhshers B V

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motlhty and will be referred to as chemotaxms, while their effect on the oxidative burst and degranulatlon is more variable Chemotaxms may be mediators of innate or adaptive ymmumty, or bacterml or tissue-derived substances Most emphasis has been placed on the complement-spht products C5a and C5a desArg, present m zymosan-actlvated serum (ZAS), and leukocyte membranederived hpld mediators such as leukotnenes and PAF-acether (Snyderman et al, 1969, Ford-Hutchinson et al, 1980) All have been shown to be mvolved m inflammation m VlVO,at least under experimental condltmns (Cochrane et al, 1959, Braquet et al, 1987) Formyl-Met-Leu-Phe (FMLP) produced by Eschenchm coh is generally used as a representative of bacterml chemotaxms (Schfffmann et al, 1975 ), but has been reported to be without effect on canine PMN (Redl et al, 1983) The early stages of PMN activation revolve responses that are complete at low receptor occupancy (Sklar et al, 1983) Consequently, studms on stimulus-response couphng necessitate the continuous assay of fast PMN functmns m order to obtain a quantitative estimate of the response before receptor-hgand eqmhbrmm is reached (Yuh and Snydermann, 1984) While chemotaxls is a well characterized early event, aggregatmn fulfils the above cnterm, since it revolves the contmuous measurement of medmtor-mduced changes m hght transmlssmn through a dense PMN suspensmn The functmnal slgmficance of PMN aggregatmn is less well studmd than that of chemotaxls, but must be considered to represent the m wtro correlate of the transmnt neutropenm observed following systemic mfusmn of chemotactm factors (Ford-Hutchinson and Evans, 1988) Even though studms on PMN filter assay chemotaxls and aggregatmn (Kroese et al, 1981, Redl et al, 1983, Thomsen and Ahnfelt-R~nne, 1989) have been undertaken m healthy dogs, there have been no quantitative comparisons between the migratory and aggregatory effects of varmus promflammatory medmtors It was the mm of this study to obtain canme dose-response relatmnshlps for a number putative partlopants m acute mflammatmn, ~e leukotrmne B4 (LTB4), LTD4 (constituent of slow-reacting substance of anaphylaxm (SRS-A)), PAF-acether, C5a/C5a desArg (obtained by activating serum with zymosan), and FMLP MATERIALS AND METHODS

Cell preparatmn PMN were isolated from EDTA-stablhzed venous blood by a method prevmusly described (Thomsen and Ahnfelt-R~nne, 1989) In each experiment, adequate buffer controls were included, and only dogs that had not partmlpated m any experiment during the prewous 2 weeks were used Cell recovery, purity and wablhty as jugded by the eosm Y dye exclusmn test were approx 50%, > 95% and > 95% respectively

EFFECTS OF PROINFLAMMATORY MEDIATORS ON CANINE NEUTROPHILS

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Chemotax~s A modlficatmn of the Boyden chamber techmque (Boyden, 1962) using acryhc bhnd well chambers (Molytex, Denmark) was used The upper chainber, containing the PMN adjusted to 2 × 106/ml and suspended m Hank's balanced salt solutmn (HBSS, Glbco, U S A ) ennched with 0 2% glucose and 2% bowne serum albumin (Sigma, U S A ) was separated from the chemoattractant chamber by a 3-/tm pore cellulose mtrate filter (Sartorms, F R G ) Randora mlgratmn (RM) was estimated with HBSS m the lower chamber, while chemotaxls was evaluated by filhng the lower chamber with HBSS-dfiuted ZAS (Maderazo and Woromck, 1978), LTB4, LTD4 (Ultrafine Chemicals, Great Britain), PAF-acether (Leo Pharmaceutical Products, Denmark) or FMLP (Sigma, U S A ) Incubatmn at 37°C for 45 mm was followed by fixatmn and stammg of the filters Experiments were carned out m duphcate, and mlgratmn was determined according to the leadmg front techmque (Zlgmond and Hlrsch, 1973), as previously described (Thomsen and Strom, 1989) Chemotactic responsiveness was evaluated as absolute mlgratmn (/1m/45 ram), chemotact~c dffferentml (CD d~rected mlgratmn minus random m~gratmn) or chemotactlc index (CI directed mlgratmn/random mlgratmn) Aggregatmn 200 ~tl PMN, adjusted to 5 × 107/ml, were placed m a sfllcomzed aggregometer cuvette and recalclfied with 5/~1 recaloficatlon medmm (40 m M CaCI2, 30 m M MgC12) Light transmission was adjusted to 10% and 90% with and w~thout the PMN suspension m the aggregometer respectively Aggregatmn was assayed using a Payton hght aggregometer (Payton Association Inc, U S A ) at 37°C with continuous stirring at 900 rpm Stimulants for aggregatmn were diluted m HBSS and added in a volume of 5/11 when the basehne was stable, and hght transm~ssmn was recorded for 5 mm Curves were exammed for number, t~me and height of peaks For the mvestlgatmn of the mechamsms mvolved m aggregatmn, brief premcubatmns m 0 25% ethanol or 10/~g/ml cytochalasm B were made prmr to addltmn of the stimulus S tatavt~cs Parametric stat~stlcs were apphcable to the techmque employed (Pedersen, 1987) Thus, Student's independent t-test was used to compare results Values are presented as the mean + s e m RESULTS

LTB4 and PAF-acether slgmficantly ( P < 0 05) reduced chemotaxls at all concentrations (Fig 1) Only LTB4 exhibited a normal dose-response curve, with a half-maximal e h o t m g concentration (ECso) of 5 × 10 - 9M ( CI ) or 10 - ~ M (CD) The maximum CI for ZAS, LTB4 and PAF-acether was 3 90, 3 94

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-LOG CONCENTRATION (M) Fig 1 Effect of LTB4, LTD4, F M L P and PAF on neutrophll mlgratmn Mlgratmn towards each medmtor was studmd m four dogs m the range 10 -9 M to 10 -6 M *Only three values were used Values are expressed as mean +_s e m Random mxgratmn was 45 4- 3 p m / 4 5 mln

and 3 44 pm/45 mln respectively, and the maximum CD 138, 125 and 112 #m/ 45 mm There were no stgmficant differences between results for ZAS and LTB4, while PAF-mduced chemotaxis was significantly (P < 0 05) lower FMLP elicited no response at any concentration tested (Fig 1) whereas LTD4 at 10- 7M to 10- 6 M caused slgmficantly greater PMN migration (P < 0 05) than RM, with maximum CI and CD values of 1.38 and 18 respectively Thus, C5a/ C5a desArg, LTB4 and PAF-acether, but not FMLP and LTD4, reduced directed migration of canine PMN m a dose-dependent and potent manner Results obtained m aggregometry resembled the chemotax~s data in that LTB4, ZAS and PAF-acether were potent stimulants (Fig 2) ZAS and LTB4 had a higher efficacy than PAF-acether, and ZAS reduced a significantly higher response ( P < 0 05) than that obtained with LTB4 Marked deactivation at high concentrations was only present for ZAS (Fig 2), whereas only LTB4, and m particular PAF-acether, gave rise to blphas~c curves at htgh concentrattons LTD4 induced modest, dose-dependent aggregatmn FMLP had no effect The leukotrlenes and ZAS caused aggregation smmedmtely upon addltmn, with a peak w~thm 20-30 s, whereas PAF-acether-mduced aggregatmn peaked slightly later Aggregatmn lmtlated by LTBt was characterized by rapid

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EFFECTS OF PROINFLAMMATORY MEDIATORS ON CANINE NEUTROPHILS

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214

H STROMANDM K THOMSEN

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reverstbxhty, gtvmg rose to sharp aggregation peaks P r e t r e a t m e n t of P M N w~th 0.25% ethanol prmr to addttlon of 10 -7 M P A F - a c e t h e r resulted m an essentially monophasm curve whtch peaked later than usual and had a greater amphtude P M N p r e m c u b a t e d with cytochalasm B exhibited a meager early response to 10 -7 M PAF-acether, b u t w~th a lag t~me of approx 1 5 m m a continuously mcreasmg late response was detected The aggregatton expertments show that LTB4, P A F - a c e t h e r and C5a/C5a desArg (present m ZAS) are all stimulants of canme P M N aggregation, b u t only the first two reduce second-wave aggregation DISCUSSION

Only compounds exhibiting clear-cut stlmulatory effects on P M N m vitro may be considered to be candidates for P M N - m d u c e d inflammation In the present study, LTB4, P A F - a c e t h e r and C5a/C5a desArg from ZAS all reduced chemotaxts and aggregation of canine P M N , wtth a potency and efficacy whtch supports their putative m vlvo roles The order of efficacy w~th regard to both assays was ZAS >t LTB4 >~PAF-acether>>LTD4, F M L P was completely reef-

EFFECTS OF PROINFLAMMATORY MEDIATORS ON CANINE NEUTROPHILS

215

fective The atypical dose-response curve obtained with PAF-acether m the chemotax:s assay could be due to PAF-acether priming PMN to be more responsive to non-specific stimuli at minute concentrations, as recently reported (Vercellottl et al, 1988) Sedgwlck et al (1987) stuched equine PMN locomotion induced by ZAS, LTB4 and FMLP, and found the same ranking With human PMN, greater aggregation has - - as with canine PMN m this study - been obtained with ZAS compared to LTB4 (Ringertz et al, 1985) The margmal chemotactm response obtained with the SRS-A leukotrlene, LTD4, resembles observations of LTC4 m human PMN (Goetzl, and Pickett, 1980) LTB4 and PAF-acether in the range 10 -7 to 10 -6 M induced what was originally referred to as second-wave aggregation, 1 e a late increase in light transmission, peaking after approximately 1.5-2 mm. Rather than representing aggregation, subcellular events associated with lysosomal enzyme and superoxlde anion secretion have been proposed as the cause of this phenomenon (Yuh and Snyderman, 1984) This proposal is supported with respect to canine PMN by the present data on the temporal and dose-related characteristics of the response, since secretory events occur later and require higher concentrations of the hgands involved Furthermore, cytochalasm B, which potentiates secretion while inhibiting cell motility (Bennett et al., 1980), accentuated the late response but attenuated the early response Ahphatic alcohols, e g. ethanol, are reported to have opposite effects (Yuh et al., 1982), and in the present work indeed had reverse effects on light transmission, 1 e enchancmg and diminishmg early and late responses respectively. Complement-split products are known to be weak secretory stimulants (Vercellottl et al, 1988), m accordance with which no late response to ZAS was apparent However, the amount of C5a/ C5a desArg m our ZAS could be below the threshold concentration necessary for reducing secretion Deactivation at high stimulant concentrations was clearly present only for ZAS (data for chemotaxis not shown), which is in agreement with findings m other species (Chenoweth et al, 1980) With LTB4, a characteristic rapidly reversible early response was seen m aggregometry, as reported previously for human PMN (Rmgertz et al, 1985) In a clinical context, substantial evidence of the involvement of PMN chemotaxls towards different factors m a variety of inflammatory conditions has accumulated (Klebanoff and Clark, 1978), and circumstantial evidence of a role for C5a and LTB4 in inducing PMN aggregation m acute myocardial infarction and shock lung has been presented (Jacob, 1981) Present and previous findings make it probable that these substances, as well as PAF-acether, are important mediators of inflammatory conchtlons relevant to the dog The present findings do not indicate a chrect and simple mechamsm through which LTD4 influences PMN functions to a significant extent Likewise, FMLP, as previously reported (Redl et al, 1983), does not activate canine PMN The lack of receptors for formyl-Met peptldes in canine PMN does mean that the

216

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canine host orgamsm depends on endogenous chemoattractants, such as C5a/ C5a desArg, when mobxhzmg P M N to s~tes of refection In conclusmn, canine P M N clearly exhibit a functmnal profile t h a t encourages the study of these cells not only in disease but also In health, serving as a non-mvaslve tool m the preclxmcal development of, e g, novel antlmflammatory drugs on a cellular level ACKNOWLEDGEMENTS

We are indebted to Dr Ole Haagen Nielsen, Department of Gastroenterology, Herlev Umverslty Hospital, Denmark, for having assisted m originally setting up the chemotaxls assay The skillful techmcal assistance of Ms S Baumgarten, A B Hansen and B Nmlsen is gratefully acknowledged REFERENCES Bennett, J P , Cockcroft, S and Gomperts, B D , 1980 Use of cytochalasm B to dlstmgmsh between early and late events m neutrophll activation Blochem Blophys Acta, 601 584-591 Boyden, S , 1962 The chemotactlc effect of mixtures of antibody and antigen on polymorphonuclear leucocytes J Exp Med, 115 453-466 Braquet, P , Touqul, L , Shen, Y and Vargaftlg, B B , 1987 Perspectives m platelet-actlvatlng factor research Pharmacol Rev, 39 97-139 Chenoweth, D E , Lane, T A, Rowe, J G and Hugh, T E , 1980 Quantltat~ve comparisons of neutrophll chemotaxls m four ammal specms Chn Immunol Immunopathol, 15 525-535 Cochrane, C G, Welgle, W 0 and Dixon, F J , 1959 The role of polymorphonuclear leukocytes m the m~tmtmn and cessation of the Arthus vascuhtls ] Exp Med, 110 481-494 Ford-Hutchinson, A W , Bray, M A, Dolg, M V, Shlpley, M E and Smith, M J H V, 1980 Leukotrmne B a potent chemokmetlc and aggregating substance released from polymorphonuclear leukocytes Nature, 286 264-265 Ford-Hutchinson, A W and Evans, J F , 1988 Neutrophll aggregation and chemokmesls assays In G D~ Sabato (Editor), Chemotaxls and lnflammatmn, Methods m enzymology, Vol 162 Academic Press, New York, NY, pp 72-75 Goetzl E J and Pmkett, W C, 1980 The human leukocyte chemotactlc actlwty of complex hydroxy-emosatetraeomcaclds (HETEs) J Immunol, 125 1789-1795 Jacob, H S, 1981 The role of activated complement and granulocytes m shock states and myocardial lnfarctmn J Lab Chn Med, 98 645-653 Klebanoff, S J and Clark, R A, 1978 The neutrophfl functmn and chmcal disorders North Holland, Amsterdam, pp 73-161 Kroese, F G M , Wfilemse, A and Slappendel, R J , 1981 Granulocyte ~unctlon tests in canine lnfectmus diseases methods and prehmmary chmcal results Vet Immunol Immunopathol, 2 455-466 Maderazo, E G and Woromck, C L, 1978 A modffmd mlcropore filter assay of human granulocyte leukotaxls In J I Galhn and P G Qme (Editors), Leukocyte chemotaxls Raven Press, New York, NY, pp 43-55 Pedersen, M M , 1987 Comparison of two methods for measurement of chemotaxls of neutrophfi polymorphonuclear leukocytes m vitro Acta Pathol Mlcrobml Immunol Scand ~C ), 95 189193 Redl H , Flynn P J , Lamche, H , Schmsser, A, Schlag, G and Hammerschmldt, D E , 1983

EFFECTS OF PROINFLAMMATORY MEDIATORS ON CANINE NEUTROPHILS

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Aggregation, chemotaxls and chemiluminescence ofcanme granulocytes Inflammation, 7 6780 Rmgertz, B , Palmblad, J and Lmdgren, J ~,, 1985 Stimulus-specific neutroph~l aggregation Evaluation of possible mechamsms for the stimulus-response apparatus J Lab Chn Med, 106 132-140 Schlffman, E , Corcoran, B A and Wahl, S M , 1975 N-Formylmethlonyl peptldes as chemoattractants for leukocytes Proc Natl Acad Scl U S A , 72 1059-106 Sedgwlck, A D , Lees, P , Dawson, J and May, S A , 1987 Cellular aspects of inflammation Vet Rec, 120 529-535 Showell, H J , Freer, R J , Zlgmond, S H , Schlffmann, E , Aswamkumar, S , Corcoran, B A and Becker, E L , 1976 The structure-activity relation ofsynthetm peptldes as chemotactm factors and reducers oflysosomal enzyme secretion for neutrophlls J Exp Med, 143 1154-1169 Sklar, L A , Jesaltls, A J , Painter, R G and Cochrane, C G , 1983 Quantitative analysis of the relatmnshlp between receptor occupancy and cellular response in human neutrophlls Blophys J , 41 132a Snyderman, R , Shin, H S , Phillips, J K , Gerwurtz, H and Mergenhagen, S E , 1969 A neutrophi1 chemotactm factor derived from Cs upon interaction of guinea p~g serum with endotoxln J Immunol, 103 413-422 Thomsen, M K and Ahnfelt-Ronne, I , 1989 Inhibition by the LTD4 antagonist, SR 2640, and effects of LTD4 on canine polymorphonuclear leukocyte function Blochem Pharmacol, 38 2291-2295 Thomsen, M K and Strom, H , 1989 Blologmal varuatmn in random and leukotrmne B4-d~rected mlgratmn of canine neutrophfls Vet Immunol Immunopathol, 21 219-224 Vercellottl, G M , Ym, H Q, Gustafson, K S , Nelson, R D and Jacob, H S , 1988 Platelet-actlvatmg factor primes neutrophfl responses to agonlsts role m promoting neutrophfl-mediated endothehaldamage Blood, 7 1100-1107 Yuh, I A , Tomonaga, A and Snyderman, R , 1982 Chemoattractant receptor functions m human polymorphonuclear leukocytes are d~vergently altered by membrane fluldlzers Proc Natl Acad Scl U S A , 79 5906-5910 Yuh, I and Snyderman, R , 1984 Rapid changes in light scattering from human polymorphonuclear leukocytes exposed to chemoattractants J Clln Invest, 73 1408-1418 Z~gmond, S H and Hlrsch, J G , 1973 Leukocyte locomotion and chemotaxls J Exp Med, 137 387-410