Differential effects of protein kinase C inhibitors on chemokine production in human synovial fibroblasts

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1 British Journl of Phrmcology (1996) 117, Stockton Press All rights reserved /96 $12. Differentil effects of protein kinse C inhibitors on chemokine production in humn synovil fibroblsts 'Nicol J. Jordn, Mlcolm L. Wtson, *Teizo Yoshimur & John Westwick Deprtment of Phrmcology, School of Phrmcy nd Phrmcology, University of Bth, Clverton Down, Bth, BA2 7AY nd *Ntionl Cncer Institute, Frederick Cncer Reserch nd Development Center, Frederick, Mrylnd, U.S.A. 1 Rheumtoid rthritis is ssocited with the ccumultion nd ctivtion of selected popultions of inflmmtory cells within the rthritic joint. One puttive signl for this process is the production, by resident cells, of group of inflmmtory meditors known s the chemokines. 2 The chemokines interleukin-8 (IL-8), monocyte chemotctic protein-1 (MCP-1) nd RANTES (regulted on ctivtion norml T-cell expressed nd presumbly secreted) re trget-cell specific chemottrctnts produced by synovil fibroblsts in response to stimultion with interleukin-l (IL-l) or tumour necrosis fctor (TNF). The signlling pthwys involved in their production re not well defined. We therefore used four different protein kinse C inhibitors to investigte the role of this kinse in the regultion of chemokine mrna nd protein expression in humn cultured synovil fibroblsts. 3 The non-selective PKC inhibitor, sturosporine (1-3 nm) significntly incresed the production of IL-l-induced IL-8 mrna nd protein. A specific PKC inhibitor, chelerythrine chloride (.1-3 gm), lso cused smll concentrtion-dependent increse in IL-8 mrna nd protein production. In contrst, 3-[1-[3-(midinothio)propyl]-3-indoly]-4-(1-methyl-3-indolyl)-1H-pyrrole-2,5-dione methnesulphonte (Ro ) nd 2[1-(3-dimethylminopropyl)-1H-indol-3-yl]-3-(lH-indol-3-yl)-mleimide (GF 1923X), two selective PKC inhibitors of the substituted bisindolylmleimide fmily hd concentrtion-dependent biphsic effect on IL-l or TNF-induced chemokine expression. At low concentrtions they cused stimultion in chemokine production, which ws especilly evident t the mrna level. At higher concentrtions both inhibited IL-l or TNF-induced chemokine mrna nd protein production. Ro ws 1 fold more potent thn GF 1923X, with n IC5 of ,M (men+s.e.men, n=4) for IL-l induced IL-8 production. Ro lso inhibited the expression of IL-l or TNF-induced MCP-1 nd RANTES mrna with similr potency. 4 The stimultory effect of sturosporine is discussed in reltion to the known poor selectivity of this inhibitor for PKC. It is proposed tht ctivtion of n isoform of PKC, possibly PKC epsilon or zet, which is inhibited by higher concentrtions of the bisinodolylmleimides, plys role in the regultion of chemokine expression induced by IL-l or TNF in synovil cells. 5 The inhibition of chemokine production by bisindolylmleimide compounds herlds novel pproch for future nti-inflmmtory therpies. Keywords: Protein kinse C inhibitors; chemokine; synovil fibroblsts; interleukin l; tumour necrosis fctor ; bisindolylmleimide; Ro ; GF 1923X; sturosporine; chelerythrine chloride Introduction The ccumultion nd ctivtion of selected popultions of inflmmtory cells in the synovil cvity ppers to ply pivotl role in the pthogenesis of rheumtoid rthritis (Plmer et l., 1986). This process requires series of coordinted signls leding to the locl genertion of chemotctic fctors. An importnt group of recently identified chemotctic proteins is the chemokine fmily (Lindley et l., 1993,b). Members of this fmily include interleukin-8 (IL-8), which is selectively chemotctic for neutrophils nd subset of lymphocytes (Yoshimur et l., 1987; Bcon et l., 199), monocyte chemotctic protein-i (MCP-1) which ttrcts monocyte derived mcrophges (Yoshimur et l., 1989) nd RANTES which ttrcts eosinophils nd CD4/CD45RO T-cells (Schll et l., 199; Kmeyoshi et l., 1992). The production of chemokines my be relevnt to the pthogenesis of rheumtoid rthritis, since in ptients with this disese, interleukin-8 (IL-8) nd MCP-1 hve been detected in synovil fluid nd synovil cells from these ptients hve been found to express mrna for IL- 8, MCP-1 nd RANTES (regulted on ctivtion norml T- cell expressed nd presumbly secreted) (Brennn et l., 199; 'Author for correspondence. Schll et l., 1991; Koch et l., 1992; Villiger et l., 1992). In cultured synovil cells the most importnt stimuli for chemokine induction re the pro-inflmmtory cytokines ILl nd tumour necrosis fctor (TNF) (Wtson et l., 1988; DeMrco et l., 1991; Bedrd & Golds, 1993; Rthnswmi et l., 1993). Their signlling mechnisms re not well defined, thus identifiction of intrcellulr events involved in chemokine regultion my identify potentil trgets for the ction of novel nti-inflmmtory gents (Skltvl & Guesdon, 1992). Both IL-l nd TNF cuse intrcellulr proteins to be phosphorylted on serine/threonine residues which implies ctivtion/inctivtion of protein kinses nd phosphtses respectively (Guy et l., 1991). The relevnce of protein kinse C (PKC) ctivtion in these IL-l nd TNF-induced phosphoryltion events remins uncertin. PKC is generic term for fmily of serine/threonine kinses, which cn be further subdivided into relted groups (Dekker & Prker, 1994). The conventionl PKC's (, fi, fill, nd y) re ctivted by C2+ nd dicylglycerol (DAG) or phorbol esters. The novel PKC's (delt, epsilon, et nd thet) re C2+-independent but cn be ctivted by DAG nd phorbol esters. The typicl PKC's (zet, iot) re both C21- independent nd unresponsive to phorbol esters. A recently identified isoform, PKCyI, my be clssed in seprte group

2 1246 N.J. Jordn et l Effect of PKC inhibitors on chemokine production due to typicl structurl fetures (Johnnes et l., 1994). Attempts to estblish whether PKC plys role in IL-l nd TNFx signl trnsduction hve been contrdictory. This is lrgely the result of the use of poorly selective 'PKC inhibitors', such s sturosporine nd H7 nd the exogenously pplied ctivtor, phorbol 12 myristte 13 cette (PMA) (Wilkinson & Hllm, 1994). Recently, number of synthetic derivtives of sturosporine hve been developed, these compounds show improved selectivity for PKC (Toullec et l., 1991; Dvies et l., 1992; Bit et l., 1993). The substituted bisindolylmleimides 3-[1-[3- (midinothio)propyl]-3-indolyl]-4-(1-methyl-3-indolyl)-1h-pyrrole-2,5-dione methnesulphonte (Ro ) nd 2[1-(3- dimethylminopropyl)-1h-indol-3-yl]-3-(1h-indol-3-yl)-mleimide (GF 1923X), re similr to sturosporine in tht they inhibit ATP binding competitively but they re lso highly selective for PKC over C2+/clmodulin-dependent protein kinse nd phosphorylse kinse nd hve inhibitory ctivity ginst ll isoforms of PKC (Toullec et l., 1991; Mrtiny- Bron et l., 1993; Wilkinson & Hllm, 1994). Another specific PKC inhibitor used in this study is chelerythrine chloride, benzophennthridine lkloid which cts t the protein substrte binding site, hence does not block phorbol ester binding to PKC nd is non-competitive inhibitor of ATP binding (Herbert et l., 199). Chelerythrine chloride is lso selective for PKC over tyrosine protein kinses, denosine 3':5'-cyclic monophosphte (cyclic AMP)-dependent protein kinses nd C2+ /clmodulin-dependent protein kinses. We hve investigted the bility of PKC inhibitors to ffect TNFe nd IL-l-induced chemokine gene expression nd protein production in synovil fibroblsts. The results indicte tht bisindolylmleimide-sensitive protein kinse is involved in IL-l/TNF signlling nd tht these compounds hve potentil for use s nti-inflmmtory gents. Methods Synovil cell culture Humn synovil tissue ws obtined from ptients undergoing joint replcement s tretment for rheumtoid or osteorthritis (Bth nd Wessex Orthopedic Reserch Unit, Royl United Hospitl, Bth). Tissue ws minced nd subjected to sequentil digestion t 37 C with periodic shking in Dulbecco's modified Egle's medium (DMEM) contining 1 mg ml-' collgense (type 1) for 3 h followed by digestion in.25 mg ml-' trypsin/.1 mg ml-' EDTA for further hour (Dyer et l., 1976). Dispersed cells were recovered by centrifugtion nd resuspended in DMEM contining 1% HI- FBS, 1 u ml-' penicillin nd streptomycin nd.5 Mg ml-' fungizone, which ws the medium used for routine culture. Confluent cells were routinely pssged by detching in trypsin/edta. Cells were used between pssges 4-17 when the cultures contined predominntly fibroblst-like cells. The cultures of synovil cells used were from 12 different ptients. Mesurement of secreted IL-8, MCP-J nd RANTES protein by enzyme-linked immunosorbent ssy (ELISA) Synovil fibroblsts were grown to confluence in 24 well pltes, when there were pproximtely 1.6 x I4cells/well. The culture medium ws removed nd replced with fresh DMEM contining 5% HI-FBS. The cells were pretreted for 2-4 h with PKC inhibitors or vehicle t the concentrtions indicted in the figure legends, then TNF or IL-l were dded to the wells nd the incubtion ws continued for 2 h. Superntnts were collected nd stored t - 7 C until ssyed by ELISA. The cell monolyers were exmined microscopiclly to check for cell dmge. IL-8 protein ws mesured by ELISA s described previously (Cesk et l., 1989; Brown et l., 1991). A mouse monoclonl nti-il-8 ntibody ws used to cot the pltes (Nunc MxiSorp, Gibco BRL) nd polyclonl got nti-il-8 ntibody conjugted to lkline phosphtse ws used for detection. MCP-1 protein ws mesured in sndwich ELISA s previously described (Yoshimur et l., 1991). Briefly, pltes were coted with monoclonl MCP-l ntibody (4.5 Mg ml-' in sodium bicrbonte buffer ph9.6). The pltes were blocked with.2% bovine serum lbumin in Tris buffered sline (TBS, 42 mm Tris, 15 mm NCl ph7.3). Smples nd stndrds in TBS +.5% Tween were dded for 9 min t 37 C, the stndrd curve rnge ws from ng ml-1. Pltes were wshed nd incubted with polyclonl rbbit nti-mcp-l (9 min 37 C). Binding ws detected with lkline phosphtse lbelled nti-rbbit IgG followed by p-nitrophenylphosphte s the substrte. For the RANTES ELISA, pltes were coted overnight t 4 C with monoclonl nti-rantes ntibody (1 4ug ml-' in sodium bicrbonte buffer ph9.6). Pltes were wshed nd incubted for 2 h t 37 C with smples nd stndrds. The detecting ntibody ws polyclonl got IgG nti-rantes dded t 1 ug ml-' for 1 h t 37 C. Binding ws detected with lkline phosphtse conjugted rbbit nti-got with p- nitrophenylphosphte s the substrte. The stndrd curve of the ssy rnged from.2-2. ng ml-'. All ELISA estimtes were mde from duplictes. Northern blot nlysis Synovil fibroblsts were grown to confluence in 1 cm dimeter Petri dishes (.5-1 x 16 cells ml-'). The culture medium ws removed nd replced with 5 ml of fresh medium contining 5% HI-FBS. The cells were pretreted with PKC inhibitors for 1-4 h, then IL-l or TNF ws dded for the time shown in the figure legends. Initil time course studies reveled tht the optimum time for detection of IL-8 nd MCP-1 mrna ws 6 h wheres RANTES mrna ws detected following 24 h stimultion. Cells were lysed with 4 M gunidinium thiocynte extrction buffer (Chomczynski & Scchi, 1987). Totl cellulr RNA ws extrcted with phenol/ chloroform nd chloroform/isomyllcohol s described by Strieter et l. (1989). RNA ws ethnol precipitted, dissolved in wter nd the concentrtion mesured by bsorbnce t 26 nm. Approximtely 1 jg totl RNA ws loded per lne of 1% grose/formldehyde gel. Ethidium bromide ws included in ech smple, enbling equl loding to be ssessed by densitometric nlysis of 18S nd 28S ribosoml RNA. Results were corrected for differences in loding when necessry. RNA ws trnsferred overnight to nylon membrne (Boehringer Mnnheim) by cpillry blotting nd fixed by bking t 12 C for 2 min. The hybridiztion protocol ws essentilly s described in the DIG luminescent detection kit (Boehringer Mnnheim). Briefly, membrnes were prehybridized for 1 h t 42'C then hybridized overnight t the sme temperture with DIGlbelled oligonucleotide probes (1 ng ml-'). Bound probes were detected with nti-dig Fb conjugted to lkline phosphtse followed by chemiluminescent substrte (CSPD) (Boehringer Mnnheim). Bnds were visulized by exposure to X-ry film for 1-2 h nd quntified by scnning densitometry. Mterils All PCK inhibitors were prepred s concentrted stock solutions stored t -2 C nd diluted in culture medium just before use. Sturosporine ws stored s 1 mm solution in dimethylsulphoxide (DMSO) (Sigm, Poole, UK). Chelerythrine chloride ws stored s 1 mm stock solution in wter (Clbiochem, Nottinghm, UK). GF 1923X ws 5 mm stock solution in DMSO (Clbiochem). Ro ws gift from Dr J.S. Nixon (Roche, Welwyn Grden City, UK) nd ws prepred s 5 mm stock solution in DMSO. Phorbol 12 myristte 13 cette (PMA) ws from Sigm. Humn re-

3 N.J. Jordn et l combinnt IL-l (specific ctivity 5 x 17 u mg-') nd TNF (specific ctivity 6 x 1O' u mg-') were generous gifts from Glxo (Greenford, UK) nd Byer (Slough, UK) respectively. All cell culture regents nd het inctivted foetl bovine serum (HI-FBS) were from Gibco BRL (Pisley, Scotlnd). Linbro 24 well tissue culture pltes were from ICN (Thme, UK) nd Flcon petri dishes were from Becton Dickinson (Oxford, UK). The Digoxigenin (DIG) chemiluminescent detection kit for Northern blotting ws from Boehringer Mnnheim (Lewes, UK). 5' Digoxigenin lbelled probes for IL-8, MCP-1 nd RANTES mrna were cocktils contining 3 ntisense 3-mer oligonucleotides (R&D Systems, Abingdon, UK). Antibodies for the IL-8 enzyme linked immunosorbent ssy (ELISA) were generous gift from Dr I.J.D. Lindley (Sndoz Forschungsinstitut, Vienn, Austri). Specific nti- RANTES ntibodies for the RANTES ELISA were from R&D Systems. All other chemicls were from Sigm or Fisons (Loughborough, UK). Sttistics Where pproprite, the effects of drug tretment compred to control were ssessed by two wy nlysis of vrince. Dunnett's test ws used for comprison of control to tretment groups of squre root trnsformed dt, where ech dtum unit ws the men protein level from 2 ELISA estimtes. Results Chemokine production by cultured humn synovil fibroblsts Unstimulted humn synovil fibroblsts obtined from ptients with either rheumtoid or osteo rthritis, which hd been kept in culture for 4 or more pssges, secreted <.5 ng ml-' IL-8 into the culture superntnt over 2 h period. There were differences in the mount of IL-8 secreted by the different cultures nd lthough cells from rheumtoid ptients produced more IL-8 initilly, fter 4 pssges ny differences did not correlte with the disese sttus of the ptient from whom the synovil tissue ws obtined. IL-8 secretion incresed in time nd concentrtion-dependent mnner when cells were stimulted with either TNF or IL-I (Figure 1). Little IL-8 could be detected in the culture superntnts fter 3 h of stimultion, but secretion incresed over the next 24 h. Chemokine secretion ws routinely mesured t 2 h. Stimultion with IL-l elicited twice s much IL-8 protein s compred with TNF tretment nd ws effective t 1 fold lower concentrtion. This incresed efficcy of ILl hs lso been shown in our lbortory in mesngil cells (Brown et l., 1991). The synovil cells shown in Figure lb responded to 2 h tretment with IL-l (3 ng ml-') nd TNF (3 ngml-') by secreting nd 28+5 ng ml-' IL-8 respectively. Stimultion with IL-l or TNF lso induced synovil fibroblsts to secrete the C-C chemokines MCP-1 nd RANTES. Stimultion for 2 h with IL-l (3 ng ml-') or TNF (3 ng ml-') induced 18+4 nd 19+4 ng ml-' of MCP-1 respectively (mens + s.e.men for 3 experiments). Cytokine stimultion over the sme time period induced little RANTES protein. Twenty hours stimultion with TNF (3 ng ml-') induced the secretion of ng ml-' RANTES (men+s.e.men for 3 experiments) nd IL-l (3 ng ml-') induced < 1 ng ml-' RANTES protein. This increse in IL-l nd TNF-induced protein ws preceded by n increse in mrna expression. MCP-1 nd IL- 8 mrna ws induced mximlly by IL-l t 4 to 6 h nd expression decresed by 24 h, wheres mrna induced by TNF remined mximlly expressed between 2 nd 24 h. RANTES gene expression differed from tht of the other chemokines, in tht pek levels of expression were not obtined before 24 h of stimultion with TNF (dt not shown). Effect of PKC inhibitors on chemokine producton E C C-W 2. -J 8i I I F'lrl-. -,.... it I.-Mm.-Mm Time (h) o TNF or IL-l (ng mlf1) o - Figure 1 IL-8 production by humn cultured synovil fibroblsts stimulted with IL-l or TNF. () Synovil fibroblsts were grown to confluence in 24 well dishes then treted with 3ngml-l TNF (solid columns) or.3ngmlf' IL-l (open columns). Superntnts were removed fter the times shown nd IL-8 ws mesured by ELISA. The results shown re mens of duplicte smples mesured in duplicte nd re expressed s ngml- IL-8 (men±s.d.). (b) Synovil fibroblsts were grown to confluence in 24 well dishes then treted with TNF (U) or IL-l (A) t the concentrtion shown. Superntnts were removed fter 2h nd IL-8 ws mesured by ELISA. The results shown re mens of duplicte smples mesured in duplicte nd re expressed s ngm1-f IL-8 (men±s.d.). IL-l ws poor inducer of RANTES mrna, even when stimultion ws extended beyond 24 h (Jordn et l., 1995). It hs been reported tht TNF-induced RANTES gene expression requires protein synthesis, which my explin the lte induction of this chemokine mrna by TNF (Rthnswmi et l., 1993). These uthors hve lso reported tht more RANTES protein ws produced in synovil fibroblsts stimulted with TNF compred to IL-l. Induction of IL-8 protein by the phorbol ester PMA 1247 The phorbol ester PMA, known ctivtor of PKC, ws lso ble to induce the production of IL-8 protein in synovil fibroblsts, lbeit t much lower level thn either IL-l or TNF. An optimum concentrtion of PMA (1 nm) induced ng ml-' IL-8 (men+s.d. for 2 experiments). When simultneously treted with TNF or IL-l nd PMA, synergistic increse in IL-8 secretion occurred which ws 2.4 fold greter thn tht produced by either gent lone (Tble 1).

4 1248 Tble 1 IL-8 secretion by cultured fibroblsts treted with PMA PMA (nm) TNF (3 ng ml-i TNF (3 ng ml-')+pma (1 nm) IL-1 (3 ng ml-) IL-I (3 ng mrl)+pma (1 nm) humn N.J. Jordn et t synovil IL-8 (ng ml-).35.8 ± ± ± ± ± ± Synovil fibroblsts were grown to confluence in 24 well pltes, then stimulted with the indicted concentrtions of PMA, either lone or in combintion with TNF (3 ng ml-) or IL-l (3 ng ml-) for 2 h. IL-8 protein ws mesured by ELISA. The results re mens ± s.d. (ng ml'). from 2 experiments in which ech smple ws mesured in duplicte. Effect of PKC inhibitors on chemokine production 25-1 o 2 o 15 O-I 5 c X6 1 5, 18S-* b II l l Sturosporine (nm) +IL-1 (3 ng mlf1) I I 3 1 The PKC inhibitors sturosporine nd chelerythrine chloride enhnce IL-le induced IL-8 expression Sturosporine dded to unstimulted synovil fibroblsts did not ffect bsl secretion of IL-8. This non-selective PKC inhibitor cused n increse in IL-l-induced IL-8 protein production over concentrtion-rnge (1-1 nm) previously shown to be effective t inhibiting PKC in fibroblsts (Tylor et l., 199). As shown in Figure 2, concentrtion of 3 nm sturosporine cused 2 fold increse in IL-ln induced IL-8 production (P <.5). Sturosporine lso cused concentrtion-dependent bell shped increse in IL-8 mrna expression. In the experiment shown in Figure 2b, 3 nm sturosporine incresed IL-I-induced IL-8 mrna expression 4 fold. At concentrtions > 3 nm sturosporine ws cytotoxic. Chelerythrine chloride lso cused concentrtion-dependent increse in IL-I-induced IL-8 production (Figure 3). A 3 gm concentrtion of chelerythrine chloride incresed IL-8 expression 1.5 fold (P <.1). The increse in mrna expression ws even more pronounced with 1 gm chelerythrine cusing 2 fold increse in IL-8 mrna expression (Figure 3b). A 1 gm concentrtion of chelerythrine chloride ws cytotoxic. Effect of bisindolylmleimide PKC inhibitors on IL-il nd TNF-induced chemokine production We investigted the effects of two closely relted specific PKC inhibitors of the bisindolylmleimide fmily, GF 1923X nd Ro on IL-l nd TNF induced chemokine production. Neither compound ffected bsl levels of chemokine production. As shown in Figure 4, GF 1923X hd concentrtion-dependent biphsic effect on the secretion of IL-8 protein. At low concentrtions, < 3 gm, it cused smll but not sttisticlly significnt increse in IL-l-induced IL-8 protein secretion. Incresing the concentrtion of GF 1923X to 1 gm cused significnt inhibition of IL-8 protein nd lmost complete inhibition resulted t 3 gm. Hence GF 1923X hd n IC5 for IL-ln-induced IL-8 production of gm (men + s.e.men for 3 experiments). GF 1923X lso inhibited the ccumultion of IL-I-induced IL-8 mrna t concentrtions bove 1 gm (Figure 4b). The inhibition of both IL-l nd TNF-induced IL-8 nd MCP-1 nd TNF-induced RANTES protein by Ro ws concentrtion-dependent. Unlike GF 1923X, lower concentrtions of Ro did not increse chemokine E 2 Go 28S_- 18S Sturosporine (nm) +IL-1 (3 ng ml-1) Figure 2 Effect of sturosporine on IL-I-induced IL-8 mrna nd protein production. () Cells were pretreted for 2 h with sturosporine t the indicted concentrtions. IL-I (3 ng ml- 1) ws dded nd the incubtion continued for 2 h. Superntnts were removed nd IL-8 protein ws mesured by ELISA. The results re mens+ s.e.men for 3 experiments, which hve been expressed s percentge of the IL-I control, which ws 86 +5ng ml' (men + s.e.men). *P<.5 compred to IL-l control. (b) Cells were pretreted for lh with medium contining sturosporine t the concentrtions shown, then IL- I (3 ng ml - 1) ws dded for further 6 h. Totl RNA ws extrcted nd IL-8 mrna ws nlysed by Northern blotting. Reltive mounts of IL-8 mrna were quntified by scnning densitometry nd equl loding ws confirmed by ethidium bromide stining of the 18S nd 28S rrna shown in the lower pnel. The dt shown re from single experiment nd re representtive of 2 similr experiments. protein secretion. The IC5 for the inhibition of IL-I-induced IL-8 ws Mm (men + s.e.men for 4 experiments) nd 1.33 ±.33 gm (men + s.e.men for 3 experiments) for TNFn-induced IL-8, with lmost totl inhibition of ll of the chemokines being cused by 1 gm Ro (Figure 5). Thus Ro ws pproximtely 1 fold more potent thn GF 1923X. This inhibition ws not due to cytotoxicity, s ssessed by observtion of the cell morphology nd trypn blue vibility stining.

5 N.J. Jordn et l Effect of PKC inhibitors on chemokine production CD L- 4- NO 4-J 4 C.. -._ CL b Discussion The controversil role of PKC in IL-ll nd TNF signlling pthwys my hve risen from the former ssumption tht PKC ws single enzyme which required C2" nd DAG for its ctivtion; n effect which could be mimicked experimentlly by the phorbol esters. However, it is now evident tht phorbol esters ctivte only some isoforms of PKC nd cn hve dditionl non-pkc-relted effects (Wilkinson & Hllm, 1994). Although PMA cn mimic mny IL-lt nd TNF- 18SA- ui z D 51 E 25' X,. - w ') Chelerythrine chloride (>M) IL-1 (3 ng ml-1) 18S-* lo 16 z r_ -;L GF 1923X (gm) IL-1 (3 ng ml-1) b 28S-* o _- 18S-fr NT Chelerythrine chloride (gm) 1L-1 (3 no mlh1) Figure 3 Effect of chelerythrine chloride on IL-lt-induced IL-8 mrna nd protein production. () Cells were pretreted for 2-4h with chelerythrine chloride t the indicted concentrtions, before the ddition of IL-lt (3 ngml- 1) for further 2h. Superntnts were removed nd IL-8 protein ws mesured by ELISA. The results shown re mens+ s.e.men of 3 seprte experiments, expressed s percentge of the IL-l control, which ws 4+4ngml-' (men+ s.e.men). *P <.5, **P <.1 compred to the IL-l control. (b) Cells were pretreted for 1 h with chelerythrine chloride t the indicted concentrtions, then IL-l (3ngml-1) ws dded for further 6h. NT=no tretment. Totl RNA ws extrcted nd IL-8 mrna nlysed by Northern blotting. Reltive mounts of IL-8 mrna were quntified by scnning lser densitometry nd equl loding ws confirmed by ethidium bromide stining of the 18S nd 28S rrna shown in the lower pnel. The dt shown re from single experiment nd re representtive of 2 similr experiments. 28S_- 18S-W N GF 1923X (ILM) +IL-1 (3 ng ml-l) Figure 4 Effect of GF 1923X on IL-l-induced IL-8 mrna nd protein production. () Cells were pretreted for 4h with the indicted concentrtions of GF 1923X. IL-l (3ngml-') ws dded nd the incubtion continued for 2h. IL-8 in the superntnts ws mesured by ELISA. The results shown re mens +s.e.men for three seprte experiments, expressed s percentge of the IL-l control, which ws 41+11ngml-' (men+s.e.men). *P<.5; **P<.1 compred to IL-1 control. (b) Cells were pretreted for 1 h with GF 1923X t the indicted concentrtions before the ddition of IL-lt (3 ngml- 1) for 6h. Totl cellulr RNA ws extrcted nd IL-8 mrna nlysed by Northern blotting. Reltive mounts of IL-8 mrna were quntified by scnning densitometry nd corrected for ny smll differences in loding s ssessed by densitometric nlysis of the 18S nd 28S rrna shown in the lower pnel. The results shown re from representtive experiment. 3 Ro hd concentrtion-dependent biphsic effect on the ccumultion of IL-l or TNF-induced IL-8, MCP-1 nd RANTES mrna (Figure 6). Concentrtions of Ro up to.3 gm enhnced the expression of chemokine mrna, but bove 1 gm inhibition ws observed. Reduced expression of the chemokine mrna did not ffect the integrity of the 18S nd 28S rrna or the expression of /3- ctin mrna (not shown).

6 125 N.J. Jordn et l Effect of PKC inhibitors on chemokine production o 12 CT o 1 C 6 4 O. o 2 C 8 -M 12- b Ro (gm) 1 T T --R 85 6 ~ E M)4 C 3 z Ro (gm) Ro (Jgm) Figure 5 Effect of the PKC inhibitor Ro on TNF nd ILl-induced chemokine protein production. Cells were pretreted for 4h with the indicted concentrtions of Ro The incubtion ws continued for further 2 h following the ddition of 3 ngml-' IL-l (open columns) or 3ngml-' TNF (solid columns). The culture superntnts were nlysed by ELISA for IL-8 (), MCP-l (b) nd RANTES protein (c). The results shown re mens +s.e.men of t lest 3 experiments. IL-8 nd MCP-l protein hs been expressed s percentge of the IL-l control. IL-l induced 51+6ngml-l IL-8 nd 18+4ngml-V MCP-l. TNF induced 28+3ngml-V IL-8 nd 19+4ngml-l MCP-l. IL-l induced RANTES ws <1 ngm'-1 (not shown). *P<.5; **P<.1 compred to IL-l or TNF controls. induced responses including the ccumultion of MCP-1 mrna in synovil fibroblsts (Villiger et l., 1992) nd IL-8 in skin fibroblsts (Zhng et l., 1992), it does so with different time course nd mgnitude to their induction by cytokines, suggesting tht the ctivtion of phorbol ester-sensitive isoform of PKC is not the only signlling pthwy ctivted by IL-l or TNF. This is lso supported by our finding tht PMA-stimulted synovil fibroblsts produced much less IL-8 thn fibroblsts stimulted with IL-l nd tht co-stimultion with these 2 stimuli synergisticlly enhnced IL-8 secretion. IL-l binding to its receptor does not ctivte the clssicl inositol phosphte nd clcium pthwy normlly ssocited with DAG production nd PKC ctivtion. This too hs been tken s evidence tht cytokine signlling does not involve PKC. HoweverDAG cnbe generted in thebsence ofc2+ by tretment of fibroblsts with IL-l. This DAG ppers to be produced from n lterntive pthwy involving the ctivtion of phosphtidyl choline-specific phospholipse C (Rosoff et l., 1988). It hs been shown tht DAG from this source, lthough unble to ctivte PKC in fibroblsts (H & Exton, 1993), cn ctivte recently identified isoform of PKC, designted PKC epsilon (Dekker & Prker, 1994). Fibroblsts lso express the isoform PKC zet (H & Exton, 1993), n isoform which is not ctivted by phorbol esters (Wys et l., 1992). Studies with dominnt negtive mutnts hve shown tht the induction of NFkB by TNF in fibroblsts requires PKC zet (Diz-Meco et l., 1993). Since NFkB is trnscription fctor importnt in IL-8 (Muikd et l., 199) nd MCP-1 (Ued et l., 1994) gene expression, the PKC zet isoform my lso be involved in IL-l/TNF-induced chemokine expression. We hve used PKC inhibitors to investigte the role of PKC ctivtion in IL-l/TNF-induced chemokine expression. Our results show tht while sturosporine nd chelerythrine chloride enhnce IL-l-induced chemokine mrna production, the bisindolylmleimides Ro nd GF 1923X hve biphsic effect. Lower concentrtions cuse n increse in chemokine mrna expression nd higher concentrtions cuse significnt inhibition in protein nd mrna expression. These differences my relte to vritions in the selectivity of these inhibitors. Sturosporine is known to be poorly selective for PKC. In ddition to brin PKC, which is inhibited with n IC5 of 22 nm (Wilkinson et l., 1993), sturosporine lso inhibits protein kinse A (PKA), phosphorylse kinse nd myosin light chin kinse, with IC5 vlues of 12 nm,.5 nm nd 55 nm respectively (Brdshw et l., 1993). Both the bisindolylmleimides GF 1923X nd Ro re selective for PKC over PKA nd phosphorylse kinse (Toullec et l., 1991; Dvies et l., 1992). However, lthough the ction of chelerythrine chloride nd the bisindolylmleimides is currently thought to be restricted to PKC isoforms, the reltive newness of these compounds mens tht they hve not been tested ginst wide enough vriety of protein kinses to ensure tht they re bsolutely specific for PKC. The possibility must be considered tht presently unknown kinses my be the trgets responsible for the results obtined in this study. For exmple, chelerythrine is currently climed to be selective for PKC (Herbert et l., 199), but the nture of its inhibition, which is vi the ctlytic domin which is homologous with other protein kinses (Von Stebut et l., 1994) my render it potentilly less selective. Indeed, chelerythrine hs been observed to hve prdoxicl stimultory effect on the phosphoryltion of 2 kd protein (Lombrdini, 1995). Differentil effects of the inhibitors ginst the different isoforms of PKC my lso be the cuse of some of the results tht we hve reported. Ro nd GF 1923X re ctive ginst most of the PKC isoforms. Both re very potent inhibitors of purified cell free PKC with IC5 vlues of 5 nd 8 nm for Ro nd GF 1923X respectively. The two compounds hve similr ctivity ginst PKCB with n IC5of 24 nd 18 nm. They differ in their bility to inhibit PKC epsilon which Ro inhibits with n IC5 of 24 nm nd GF 1923X inhibits with n IC5 of 132 nm (Mrtiny-Bron et l., 1993; Wilkinson et l., 1993). Ro lso ppers to be much more ctive ginst frctions of pituitry tht contin PKC zet (IC nm) (Ison et l., 1993) when compred to GF 1923X which inhibits isolted PKC zet with n IC5 of 58 nm (Mrtiny-Bron et l., 1993). The enhnced chemokine production induced by sturosporine is probbly result of the poor selectivity of this inhibitor. Sturosporine hs previously been shown to double the secretion of IL-8 protein in the U937 monocytic cell line (Pless & Westwick, 199) nd enhnce prostglndin E2 (PGE2) production in synovil fibroblsts (Tylor et l., 199). This incresed PGE2 my then upregulte cyclic AMP with concomitnt protein kinse A (PKA) ctivtion. Since the IL-8 gene contins binding sites for the trnscription fctor AP-1, which cn be ctivted by PKA, (Muikd et l., 199) this could be n indirect mechnism by which sturosporine increses IL-8 expression in synovil fibroblsts. The significnt inhibition of both IL-l nd TNF-induced chemokine expression by the bisindolylmleimide PKC inhibitors, GF 1923X nd Ro , strongly suggests tht

7 N.J. Jordn et l Effect of PKC inhibitors on chemokine production +IL-1 (3 ng ml11) +TNF (3 ng ml-1) Ro (glm) IL-8 mrna MCP-1 mrna RANTES mrna Figure 6 Effect of Ro on IL-ll nd TNF-induced chemokine mrna expression. Synovil fibroblsts were pretreted for 1 h with Ro t concentrtions between.1-1 glm s shown, then IL-l (3 ngml- 1) or TNF (3 ngml- 1) ws dded. Totl cellulr RNA ws extrcted fter 6 h for IL-8 nd MCP-1 nd fter 24 h for RANTES nd mrna ws nlysed by Northern blotting. The tble shows the results obtined by densitometric nlysis of the Northern blots. Corrections were mde for ny smll differences in loding s determined from densitometric nlysis of the ethidium bromide stined 18S nd 28S rrna (not shown). The results shown re from representtive experiments. ND =not determined. ctivtion of n isoform of PKC does ply n importnt role in chemokine regultion. It is possible tht different gonists my preferentilly ctivte only one specific subtype of PKC, which in turn regultes the expression of specific genes. Since the bisindolylmleimides only inhibited chemokine expression when used t reltively high concentrtions, the PKC isoforms inhibited less potently by these compounds my hve been those tht plyed n importnt role in inducing chemokine gene expression in response to IL-l nd TNF. It is eqully possible tht gene expression could be negtively regulted by different isoform of PKC. If this were so it would provide n explntion for the observed stimultion of IL-8 mrna ccumultion which could be induced by low concentrtions of Ro , GF 1923X nd sturosporine. Since these inhibitors re ll most potent ginst PKC nd PKCJ3, these isoforms my ctully function s inhibitors in IL-l nd TNF-induced chemokine expression. Our results hve shown tht Ro is t lest 1 fold more potent thn GF 1923X in inhibiting IL-8 expression in synovil fibroblsts. Since it hs been shown tht the PKC isoforms epsilon nd zet, both of which re present in fibroblsts, re inhibited more potently by Ro thn GF 1923X, we propose tht one or both of these PKC isoforms my be importnt in IL-l-induced chemokine expression. Recently the potentil of specific PKC inhibitors to be used therpeuticlly s selective immunomodultors hs become pprent (Bit et l., 1993; Brdshw et l., 1993). Conditions which my benefit from tretment with PKC inhibitors include the utoimmune diseses rheumtoid rthritis, multiple sclerosis nd dibetes mellitus (Brdshw et l., 1993). A bisindolylmleimide compound closely relted to Ro hs recently been shown to inhibit selectively the secondry T- cell-medited response in developing djuvnt rthritis in rts (Bit et l., 1993). Our results support signlling pthwy in which n isoform of PKC, possibly PKC epsilon or zet, which is inhibited by the bisindolylmleimide compounds, plys n importnt role in ctivting chemokine gene expression in response to ILl nd TNFcx. We propose tht one of the wys tht bisindolylmleimide PKC inhibitors my reduce inflmmtion is by reducing the expression of the chemokines MCP-1, IL-8 nd RANTES which ttrct subsets of leukocytes responsible for inititing the ugmenting inflmmtory processes. We hve found no significnt difference in the potency of Ro ginst ny of the chemokines investigted, nd therefore suggest tht this compound could be of use in ny disese in which chemokine ctivity is implicted. We thnk Mrs J. Leithed for technicl help nd the Bth nd Wessex Orthopedic Reserch Unit for providing humn synovil tissue. This work ws supported by the Arthritis nd Rheumtism Council (UK) nd the Wellcome Trust (UK).

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