polymorphonuclear neutrophil release of granular

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1 Br. J. Pharma. (1985), 86, Phorbol myristate aetate enhanes human polymorphonulear neutrophil release of granular enzymes but inhibits hemokinesis J.R.S. Hoult & Sussan Nourshargh Department of Pharmaology, King's College, Strand, London WC2R 2LS 1 The effets of the o-arinogeni phorbol ester, phorbol myristate aetate (PMA), on N-formyl- Met-Leu-Phe (FMLP)-indued human polymorphonulear leukoyte hemokinesis and release of granular lysozyme and,-gluuronidase were ompared with those of the inative phorbol dideanoate (PDD). 2 Release of the enzymes was enhaned by PMA but was unaffeted by PDD whih also had no effet on hemokinesis. 3 In ontrast, FMLP-indued hemokinesis was ompletely suppressed by PMA in a dose-dependent fashion (ID5 = 3.5 nm). 4 PMA also inhibited the FMLP-indued inrease in ytoplasmi alium level, measured by the fluoresent indiator quin-2. 5 These and other results suggest that although the diaylglyerol/protein kinase C system is involved in the positive regulation of ertain neutrophil funtions (degranulation and superoxide generation), if it is very powerfully stimulated, as with PMA, it has inhibitory ations on other neutrophil properties suh as motility. Introdution Polymorphonulear leukoytes (PMNs) exhibit a variety of ellular responses when stimulated by soluble fators suh as hemotati agents and the alium ionophore A These responses inlude aggregation, generation of superoxide anion radials, release of granular proteins and enzymes and, at lower doses of stimulant, hemokinesis and hemotaxis. It is thought that phospholipase C-dependent phosphatidylinositol bisphosphate breakdown, ativation of protein kinase C by diaylglyerol and a rise in the level of intraellular alium all ontribute to these effets after surfae stimulation of the ells, although their relative ontributions to the overall response may vary aording to the nature of the stimulant. The o-arinogen phorbol myristate aetate (PMA) whih diretly ativates protein kinase C (Castagna et al., 1982) auses neutrophil aggregation, superoxide generation, enzyme seretion and phosphorylation of several polypeptides (see White et al., 1984) but does not inrease levels of intraellular alium (Sha'afi et al., 1983). However, it is not known what effets PMA may have on stimulated neutrophil loomotion (hemokinesis). This paper douments the full dose-response urves for N-formyl-Met-Leu-Phe (FMLP)-indued hemokinesis and release of granular lysozyme and P-gluuronidase enzymes from human PMNs and shows that PMA has a divergent effet on these responses, enhaning degranulation but inhibiting hemokinesis. Methods Suspensions of PMN from healthy non-mediated volunteers were prepared aording to Dougherty et al. (1984) and suspended at > 95% purity and viability in agle's minimum essential medium buffered at ph 7.4 with 2 mm HPS for hemokinesis assays, or in ph 7.4 phosphate buffered saline (NaCI 138 mm, Na2HPO4 8.1 mm, KH2PO4 1.5 mm, KCI 2.7 mm, gluose.1% w/v and ontaining.6 mm CaCI2 and 1. mm MgCl2) for enzyme seretion experiments. Chemokinesis was measured by the agarose mirodrop tehnique (Smith & Walker, 198) with approximately 5 x 15 ells per well and 1 jl volumes of test solution inubated for 4 h at 37TC. Seretion of lysozyme and P-gluuronidase was determined spetrophotometrially in supernatants The Mamillan Press Ltd 1985

2 534 J.R.S. HOULT & S. NOURSHARGH a 32- b 5 C U) U C_ 24 F 16-8 H 48 (A (a N ) OR U) a1) -i N C_ 4 _- 3 _ 2 _ 1_- OL Conentration (-log molar) Conentration (-log molar) d 2 r or (A (a N CD 151-1[- 5 *.._ C IQ- L Conentration (-log molar) IF j Conentration (-log molar) Figure 1 Human polymorphonulear leukoyte (PMN) responses and the effets of phorbol myristate aetate (PMA) and phorbol dideanoate (PDD). ( Chemokinesis (), seretion of lysozyme (A) and P-gluuronidase (V) as a funtion of N-formyl-Met-Leu-Phe (FMLP) onentration (results are mean values of 5, 4 and 4 separate experiments, respetively). (b and ) ffets of 1-8M PMA (A) or PDD (v) on seretion of lysozyme (b) and P-gluuronidase () ompared to ontrol responses () to various doses of FMLP (results are mean values from 3 separate experiments and the PMA/PDD were pre-inubated for 5 min). (d) ffets of varying doses of PMA (A) or PDD (v) on hemokinesis indued by 1-9M FMLP (3 separate experiments, phorbol esters added simultaneously). In (b) and () the effets of PMA in ausing release of4.3% and 2.8% ofellular lysozyme and P-gluuronidase, respetively, have been subtrated. Note that within experiments, enzyme release was assayed in dupliate tubes and hemokinesis in quadrupliate wells. Bars show s.e.mean and * indiates a value signifiantly different from ontrol by Student's unpaired t test, P <.5. obtained from ytohalasinb (5fjg ml- ')-treated ells, inubated with varying onentrations of FMLP for 5 min at 37TC and entrifuged; the substrates used were suspensions of Miroous lysodeiktius and 4- methylumbelliferyl-p-d-gluuronide, respetively (Yuli et al., 1982; Barrett & Heath, 1979). nzyme release is expressed as perentage of total ontent determined in detergent-lysed ells, after orreting for spontaneous release in vehile-treated blanks. Changes in intraellular alium levels were measured in

3 PHORBOL STRS INHIBIT PMN CHMOKINSIS 535 ells pretreated with quin-2-tetraaetoxymethyl ester (Lanaster Synthesis, U.K.) as desribed by White et al. (1983). Phorbol esters, enzyme substrates and FMLP were purhased from Sigma Chemial Company, U.K. Results FMLP dose-dependently inreased human PMN hemokinesis in the range 1"- to 1-9M, but higher onentrations progressively inhibited movement (Figure l. Release of granular enzymes was stimulated in the range 1-8 to 1-4M, peaking at 1-6M, a onentration that almost totally inhibited stimulated movement of the ells (Figure l. A higher proportion of total ellular lysozyme was released than of P-gluuronidase, refleting their origin from (predominantly) speifi and azurophil granules, respetively. Figure lb and show that over the higher range of FMLP onentrations needed to eliit release of ) o 2 min a C ) * C A A A* b Control + PMA Figure 2 ffet of phorbol myristate aetate (PMA) on N-formyl-Met-Leu-Phe (FMLP)-indued inrease in free intraellular alium as deteted by the quin-2 tehnique. The vertial line indiates fluoresene intensity in arbitrary units. In ( and (b) effets of 1-8M PMA added at A (or PMA vehile at A) are shown on the hanges subsequently indued by 1-7M ( or 1-8M (b) FMLP added at. In () resting ells preloaded with quin-2 were exposed at A to PMA vehile or at A to 1-8M PMA and the fluoresene was ontinuously monitored (exitation 339 nm, emission 492 nm) in a Perkin-lmer model MPF-4 reording spetrophotofluorimeter at 37 C with ontinuous stirring. Similar results to those shown in ( to () were obtained on at least 4 other oasions. In 23 different preparations of ells, the onentration of basal free intraellular alium was 138 ± 17 nm (s.e.mean) (f. White et al., 1983; Di Virgilio et al., 1984). granular enzymes (1-8 to 1-5M), PMA at 1-8M enhaned enzyme release. This was signifiant at FMLP onentrations of 1-7M and above. The analogue PDD (4 x-phorbol-12,13-dideanoate, whih does not ativate protein kinase C, Castagna et al., 1982) was not ative. However, in omplete ontrast, PMA dose-dependently inhibited hemokinesis indued by 1-9M FMLP, with an IC5 of 3.5 nm (Figure Id); again, PDD was inative. In separate experiments, 1-8M PMA (a onentration that enhanes enzyme release but very strongly inhibits hemokinesis) onsiderably redued the FMLP-indued inrease in free intraellular alium as deteted by the quin-2 method, and also aused a redution in the baseline level of free intraellular alium (Figure 2). Disussion Our finding that PMA inhibits FMLP-indued human PMN hemokinesis suggests that neutrophil motility is modulated in a negative way by the diaylglyerol/ protein kinase C system. Additional evidene for this is: (1) that PDD did not alter hemokinesis (Figure Id); (2) that another ative o-arinogeni phorbol ester (phorbol-12,13-dibutyrate) had similar effets to PMA; (3) that I-oleoyl-2-aetyl glyerol also inhibited hemokinesis; (4) that a very similar profile of effets was observed if leukotriene B4 was used to stimulate the neutrophils (points 2 to 4:S. Nourshargh and J.R.S. Hoult, unpublished experiments), and (5) that Gallin et al. (1978) showed that PMA at onentrations greater than 1.6 nm inhibited the ability of human neutrophils (previously allowed to degranulate and washed) to move hemotatially in a gradient of partially purified C5a. This inhibitory effet of PMA on hemokinesis is in sharp ontrast to its previously reognised ability to enhane the effets of neutrophil stimulants on other neutrophil funtions suh as superoxide generation and enzyme seretion ourring at higher stimulant doses (e.g., Gallin et al., 1978; Kajikawa et al., 1983; O'Flaherty et al., 1984; Dale & Penfield, 1984; Blakwell et al., 1985) and shown in Figure lb and. It should be noted that PMA is a very powerful stimulant of protein kinase C (e.g. Castagna et al., 1982) and thus it may not mimi aurately the physiologial ontext in whih the kinase is stimulated, sine this is brought about by the transient generation of diaylglyerol subsequent to the breakdown of inositol phospholipids (Nishizuka, 1984). Furthermore, there is a reent report whih ontradits the general onlusion from the studies ited above, in that PMA at onentrations of 16 nm or more preinubated with rabbit neutrophils for 1 to 5 min inhibited their subsequent ability to release lysozomal and azurophil

4 536 J.R.S. HOULT & S. NOURSHARGH enzymes in response to FMLP, C5a and leukotriene B4 (Naahe et al., 1985). The reasons for the disrepany between these results with rabbit neutrophils and the other (human) studies have not yet been established, although Naahe et al. did find that A23187-indued degranulation was enhaned by the addition of PMA. Nevertheless, like Naahe et al. (1985), we also found that PMA redues the magnitude of the quin-2- detetable inrease in intraellular alium that ours after triggering the PMNs with a hemokineti dose of FMLP. This may be beause PMA, and by extension protein kinase C, is responsible for ativating a alium-extruding pump mehanism, as shown in guineapig neutrophil plasma membranes by Lagast et al. (1984). These events an be aommodated in a simple model in whih ativation of neutrophil movement is assoiated with mobilisation of intraellular alium, needed for triggering ell motility by ativating alium-dependent gelsolin and the alium/almodulindependent phosphorylation of myosin (Southwik & Stossel, 1983), and inhibition of movement is regulated by the diaylglyerol/protein kinase C system. It is relevant that low doses of FMLP are known to mobilise intraellular alium (see for example White et al., 1983), whereas the doses needed to ause phospholipase C-dependent generation of diaylglyerol are higher and orrelate with those produing degranulation (Dougherty et al., 1984). Coupling of FMLP to different subellular transdution mehanisms may also be dependent upon the fat that there are at least two affinity states of the FMLP reeptor whih may be interonvertible (Yuli et al., 1982). Finally, it has been shown that PMA an trigger superoxide generation, exoytosis and protein phosphorylation at very low levels of intraellular alium, suggesting that the ation is not indispensable for these neutrophil funtions, provided that it is not needed for steps proximal to protein kinase C ativation (Di Virgilio et al., 1984). In summary, FMLP transdution mehanisms differ in relation to hemokinesis and granular exoytosis, suh that ativation of protein kinase C (in these experiments ahieved by adding PMA but not PDD) will inhibit movement. It has not esaped our notie that this would provide a logial mehanism for regulating human PMN funtion in response to a hemotati gradient. We thank the M.R.C. for a training studentship for S.N. Referenes BARRTT, A.J. & HATH, M.F. (1979). Lysozomal enzymes. In Lysozomes: a laboratory handbook, 2nd edition. ed. Dingle, A.T. pp Amsterdam: North-Holland. BLACKWLL, G.J., BONSR, R.W., DAWSON, J. & GARLAND, L.G. (1985). Stimulation and inhibition of seretion by phorbol myristate aetate in different ell types. Biohem. biophys. Res. Commun., 127, CASTAGNA, M., TAKAI, Y., KAIBUCHI, K., SANO, K., KIKK- AWA, U. & NISHIZUKA, Y. (1982). Diret ativation of alium-ativated, phospholipid-dependent protein kinase by tumour promoting phorbol esters. J. biol. Chem., 257, DAL, M.M. & PNFILD, A. (1984). Synergism between phorbol ester and A23187 in superoxide prodution by neutrophils. FBS Lett., 175, DI VIRGILIO, F., LW, D.P. & POZZAN, T. (1984). Protein kinase C ativation of physiologial proesses in human neutrophils at vanishingly small ytosoli Ca2+ levels. Nature, 31, DOUGHRTY, R.W., GODFRY, P.P., HOYL, P.C., PUTNY, J.W. & FRR, R.J. (1984). Seretagogue-indued phosphoinositide metabolism in human leukoytes. Biohem. J., 222, GALLIN, JI., WRIGHT, D.G. & SCHIFFMANN,. (1978). Role of seretory events in modulating human neutrophil hemotaxis. J. lin. Invest., 62, KAJIKAWA, N., KAIBUCHI, K., MATSUBARA, T., KIKK- AWA, U., TAKAI, Y. & NISHIZUKA, Y. (1983). A possible role of protein kinase C in signal-indued lysozomal enzyme release. Biohem. biophys. Res. Commun., 116, LAGAST, H., POZZAN, T., WALDVOGL, F.A. & LW, P.D. (1984). Phorbol myristate aetate stimulates ATP-dependent alium transport by the plasma membrane of neutrophils. J. lin. Invest., 73, NACCACH, P.H., MOLSKI, T.F.P., BORGAT, P., WHIT, J.R. & SHA'AFI, R.I. (1985). Phorbol esters inhibit the f Met-Leu-Phe- and leukotriene B4-stimulated alium mobilisation and enzyme seretion in rabbit neutrophils. J. biol. Chem., 26, NISHIZUKA, Y. (1984). The role of protein kinase C in ell surfae signal transdution and tumour promotion. Nature, 38, O'FLAHRTY, J.T. SCHMITT, J.D., MCALL, C.. & WYTL, R.L. (1984). Diaylglyerols enhane human neutrophil degranulation responses: relevany to a multiple mediator hypothesis of ell funtion. Biohem. biophys. Res. Commun., 123, SHA'AFI, R.I., WHIT, J.R., MOLSKI, T.F.P., SHFYK, J., VOLPI, M., NACCACH, P.H. & FINSTIN, M.B. (1983). Phorbol- 12-myristate- 13-aetate ativates rabbit neutrophils without an apparent rise in the level of intraellular free alium. Biohem. biophys. Res. Commun., 114, SMITH, M.J.H. & WALKR, J.R. (198). The effets of some antirheumati drugs on an in vitro model of human polymorphonulear leukoyte hemokinesis. Br. J. Pharma., 69,

5 PHORBOL STRS INHIBIT PMN CHMOKINSIS 537 SOUTHWICK, F.S. & STOSSL, T.P. (1983). Contratile proteins in leukoyte funtion. Seminars in Haematology, 2, WHIT, J.R., NACCACH, P.H., MOLSKI, T.F.P., BORGAT, P. & SHA'AFI, R.I. (1983). Diret demonstration of inreased intraellular onentration of free alium in rabbit and human neutrophils following stimulation by hemotati fator. Biohem. biophys. Res. Commun., 113, WHIT, J.R., HUANG, C.K., HILL, J.M., NACCACH, P.H., BCKR,.L. & SHA'AFI, R.I. (1984). ffet of phorbol 12-myristate-13-aetate and its analogue 4 a-phorbol 12,13-dideanoate on protein phosphorylation and lysozomal enzyme release in rabbit neutrophils. J. biol. Chem., 259, YULI, I., TOMONAGA, A. & SNYDRMAN, R. (1982). Chemoattratant reeptor funtions in human polymorphonulear leukoytes are divergently altered by membrane fluidizers. Pro. nain. Aad. Si. U.S.A., 79, (Reeived July 1, Aepted July 22, 1985.)