Inhibitory effect of p38 mitogen-activated protein kinase inhibitors on cytokine release from human macrophages

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1 British Journl of Phrmology (26) 149, & 26 Nture Pulishing Group All rights reserved /6 $3. RESEARCH PAPER Inhiitory effet of p38 mitogen-tivted protein kinse inhiitors on ytokine relese from humn mrophges SJ Smith 1, PS Fenwik 1, AG Niholson 2, F Kirshenum 3, TK Finney-Hywrd 1, LS Higgins 3, MA Giemyz 4, PJ Brnes 1 nd LE Donnelly 1 1 Airwy Disese Setion, Ntionl Hert nd Lung Institute, Imperil College London, London, UK; 2 Deprtment of Histopthology, Royl Brompton Hospitl, London, UK; 3 Sios In., CA, USA nd 4 Deprtment of Phrmology & Therpeutis, Institute of Immunity, Infetion nd Inflmmtion, University of Clgry, Clgry, Alert, Cnd Bkground nd purpose: Mrophges relese ytokines tht my ontriute to pulmonry inflmmtion in onditions suh s hroni ostrutive pulmonry disese. Thus, inhiition of mrophge ytokine prodution my hve therpeuti enefit. p38 MAPK my regulte ytokine prodution, therefore, the effet of two p38 MAPK inhiitors, SB23963 nd SD-282, on the relese of TNF-, GM-CSF nd IL-8 from humn mrophges ws investigted. Experimentl pproh: Cytokine relese ws mesured y ELISA. Immunolots nd mrna expression studies were performed to onfirm p38 MAPK isoform expression nd tivity. Mrophges were isolted from lung tissue of urrent smokers, ex-smokers nd emphysem ptients nd exposed to lipopolyshride. These ells then relesed ytokines in onentrtion-dependent mnner. Key results: SB23963 only inhiited TNF- relese (EC mm). Disese sttus hd no effet on the effiy of SB SD-282 inhiited oth TNF- nd GM-CSF relese from mrophges (EC nm nd mm respetively) ut hd no effet on IL-8 relese. In ontrst, oth inhiitors suppressed ytokine prodution in monoytes. Conlusions nd Implitions: The differentil effets of p38 MAPK inhiitors etween mrophges nd monoytes ould not e explined y differenes in p38 MAPK isoform expression or tivity. However, the stility of TNF- mrna ws signifintly inresed in mrophges ompred to monoytes. These dt suggest differentil involvement for p38 MAPK in mrophge ytokine prodution ompred with monoytes. These effets re not due to lk of p38 tivtion or p38 expression in mrophges ut my reflet differentil effets on the stility of ytokine mrna. British Journl of Phrmology (26) 149, doi:1.138/sj.jp.76885; pulished online 4 Septemer 26 Keywords: TNF-; MAPKs; mrna stility; mrophge; monoyte; humn Arevitions: COPD, hroni ostrutive pulmonry disese; ELISA, enzyme-linked immunosorent ssy; GM-CSF, grnuloyte-mrophge olony stimulting ftor; HSP, het shok protein; IL, interleukin; LPS, lipopolyshride; MAPK, mitogen-tivted protein kinse; TNF, tumour nerosis ftor Introdution Correspondene: Dr LE Donnelly, Airwy Disese Setion, Guy Sdding Building, Ntionl Hert & Lung Institute, Imperil College London, Dovehouse Street, London SW3 6LY, UK. E-mil: l.donnelly@imperil..uk Reeived 2 July 26; epted 1 August 26; pulished online 4 Septemer 26 Lung inflmmtion is n importnt feture of hroni ostrutive pulmonry disese (COPD) nd is ssoited with inresed numers of infiltrting mrophges, CD8 þ T lymphoytes nd neutrophils, together with elevted levels of ytokines, inluding tumour nerosis ftor (TNF)-, interleukin (IL)-8 nd grnuloyte mrophge-olony stimulting ftor (GM-CSF) (Brnes, 24). Moreover, mrophges n ount for mny of the pthophysiologil fetures ssoited with COPD (Brnes, 24). There re no phrmologil interventions ville urrently tht hve een shown to slow the progression of COPD or tht effetively suppress the underlying inflmmtion in the lung prenhym, inditing need for lterntive nti-inflmmtory strtegies. Severl new pprohes tht trget the inflmmtion ssoited with COPD re in linil development, inluding mitogen-tivted protein kinse (MAPK) inhiitors (Kumr et l., 23). Of the three min MAPK pthwys, p38 nd JNK MAPK re implited in hroni inflmmtion (Johnson nd Lpdt, 22). p38 MAPK inhiitors hve een shown to suppress lung inflmmtion in in vivo models of disese

2 394 p38 MAPK nd mrophges SJ Smith et l (Underwood et l., 2; Hddd et l., 21). For exmple, SB23963 redued neutrophil umultion in the lungs of rts following inhltion of endotoxin (Underwood et l., 2). In ddition, inhltion of ntisense oligonuleotide of p38 MAPK inhiited ronhil hyperretivity nd redued the numer of inflmmtory ells in ronholveolr lvge fluid in murine model of sthm (Dun et l., 25). p38 MAPK inhiitors lso suppress ytokine relese from inflmmtory ells in vitro (Underwood et l., 2; Kumr et l., 23). Hene this pthwy is onsidered therpeuti trget in inflmmtory respirtory diseses nd p38 MAPK inhiitors re urrently in Phse I nd II trils for COPD (Prous, 25). There re four p38 isoforms,, g nd d tht re enoded y four seprte genes, the expression of whih ppers to e tissue dependent. The reltive ontriution of eh of these isoforms to the inflmmtory response is urrently unknown due to lk of speifi phrmologil tools; however, there is evidene tht they hve different sustrtes. For exmple, p38 nd p38 phosphorylte MAPKAPK2, ut this protein is not phosphorylted y p38g or p38d. Thiswouldsuggestthtdifferentp38 MAPK isoforms my hve differentil effets in inflmmtion. As nonsteroidl nti-inflmmtory therpy p38 MAPK inhiitors ould e of potentil use in COPD, s gluoortiosteroids re lrgely ineffetive in inhiiting the inflmmtory response oth in vivo nd in vitro (Burge et l., 2; Httotuw et l., 22; Russell et l., 22; Culpitt et l., 23). The im of this study ws to determine the importne of the p38 MAPK pthwy on ytokine relese from lung tissue mrophges with view to identifying novel trgets for phrmeutil intervention. Consequently, we exmined the effets of novel p38 MAPK inhiitor, SD-282, s this ompound is highly potent for seletively inhiiting p38 (EC nm) nd p38 (EC nm), without ffeting either p38g or p38d (Lim et l., 24). Furthermore, SD-282 hs een shown to redue mrna levels of proinflmmtory ytokines in murine model of ollgen-indued rthritis (Mediherl et l., 26). We desrie the phrmologil tivity of SD-282 in omprison with referene p38 MAPK inhiitor, SB23963 whih inhiits p38 nd p38 with equl poteny (EC 5 44 nm), without ffeting either p38g or p38d (Underwood et l., 2). Methods Sujets The Ethis Committee of the Brompton nd Hrefield NHS Trust nd NHLI pproved this study nd ll sujets gve informed written onsent. All sujets hd smoking history of 415 pk yers. Ex-smokers hd esed smoking for 46 months. The emphysem sujets were ll undergoing lung trnsplnts. There ws no differene in the ge of the three sujet groups (Tle 1). Isoltion nd ulture of humn mrophges Mrophges were isolted from mrosopilly norml lung tissue, otined from ptients undergoing surgil Tle 1 Clinil hrteristis of lung tissue donors Smokers Ex-smokers Emphysem lung trnsplnt Age (yers) Sex (M:F) 3:3 3:4 3:1 FEV 1 (% predited) NA FEV 1 /FVC rtio NA Smoking history (pk yers) NA Dt re presented s men7s.e. NA indites dt re not ville. resetion for either rinom or lung trnsplnt s desried previously (Smith et l., 24). The mrophge preprtions used were greter thn 95% purity, s determined y Kimur stining nd onfirmed y immunoytohemistry with lelling using n nti-cd68 ntiody. Cytokine mesurements Cell-free superntnt ws removed 2 h post-stimultion with lipopolyshride (LPS) (Slmonell enteriditis) nd TNF-, GM-CSF nd IL-8 mesured y enzyme-linked immunosornt ssy (ELISA) (R & D Systems Europe, Aingdon, UK) ording to the mnufturer s instrutions. The detetion limits of the IL-8 detetion ssy ws 31 pg ml 1 nd the detetion limit for the GM-CSF nd TNF- ssys were 15.5 pg ml 1. Western immunolot nlysis Cells were prepred for Western lotting s desried previously (Smith et l., 24). Cell lystes were prepred, insolule proteins were removed nd 2 mg of solule extrt were dentured nd sujeted to eletrophoresis on 1% (wt/vol) sodium dodeyl sulphte (SDS) polyrylmide gels. Cell viility Cell viility ws determined olourimetrilly y mesuring the redution of the tetrzolium slt, 3-[4,5-dimethylthizol- 2-yl]-2,5-diphenyl tetrzolium romide (MTT), to formzn. Monoyte isoltion Monoytes were isolted from peripherl lood using disontinuous Peroll grdients followed y dherene to tissue ulture plsti (Seldon et l., 1995). This method generted dherent monoyte preprtions with purity greter thn 97%, s determined y Kimur stining. Expression of p38 MAPK isoforms DNAs enoding,, g nd d p38 MAPK isoforms were mplified y polymerse hin retion (PCR) tehniques nd suloned into n untgged mmmlin expression vetor, pshuttle (BD Biosienes/Clonteh, CA, USA). Clones were sequened to onfirm their respetive essions: British Journl of Phrmology (26)

3 p38 MAPK nd mrophges SJ Smith et l 395 L35264, NM_2751, NM_2969 & NM_2754. Suonfluent HeL ells were trnsfeted with 2 mg of DNA per well using Lipofetmine 2 (Invitrogen, CA, USA). Cells were hrvested 48 h post-trnsfetion nd lysed in RIPA uffer (Upstte Bioteh, Milton Keynes, UK) supplemented with protese inhiitor Complete oktil (Rohe, Lewes, UK). Results Effet of LPS on the relese of ytokines from humn lung mrophges There ws no detetle sl relese of TNF- from mrophges hrvested from lung prenhym of sujets who were igrette smokers (n ¼ 6) (Figure 1), wheres 4 Rel-time PCR (Tqmn) Monoytes nd lung mrophges were treted with either 3 or 1 ng ml 1 LPS, for 4 h efore the ddition of 5 mgml 1 of tinomyin D (Sigm-Aldrih, Poole, UK). At vrious times points indited, ells were lysed for RNA extrtion. p38 MAPK inhiitors, these were dded simultneously with the tinomyin D nd the ells hrvested 6 min lter. RNA ws extrted using RNesy (Qigen, Crwley, UK) nd quntified spetrophotometrilly. Totl RNA (.5 mg) ws reverse trnsried s previously desried (Smith et l., 23). Reltime PCR ws performed using the equivlent of 1 ng RNA in 25 ml for TNF-, GM-CSF, IL-8 nd HPRT1 for 45 yles. Anlysis used the ddc T method, wherey the TNF-, GM-CSF nd IL-8 mrna levels were expressed reltive to the ontrol gene expression nd susequently nlysed y reltive quntifition (RQ) to mrna levels in unstimulted ells. The results were then normlized for LPS-stimulted ells (1% of ytokine expression). Sttistil nlyses Dt nd sttistil nlyses: dt points, nd vlues re presented s men7s.e. of n independent determintions using ells from different donors. Conentrtion response urves were nlysed y the GrphPd PRISM urve fitting progrm (GrphPd softwre, Sn Diego, CA, USA) nd EC 5 vlues were susequently interpolted from urves of estfit. Where pproprite, dt were nlysed sttistilly using Student s pired t-test or y ANOVA with the Dunn test. The null hypothesis ws rejeted when Po.5. TNF-α Relesed (pg ml -1 ) GM-CSF Relesed (pg ml -1 ) log [LPS (g ml -1 )] log [LPS (g ml -1 )] 3 Mterils The p38 MAPK inhiitor SB23963 ws gift from Glxo- SmithKline, Phildelphi, USA nd the inhiitor SD-282 ws gift from Sios In. (Fremont, CA, USA). The nti-p38 nd nti-p38d ntiodies were purhsed from Autogen Bioler (Clne, Wiltshire, UK). The nti-p38 ntiody ws purhsed from Zymed (Invitrogen, Pisley, UK). The nti-p38g ntiody ws purhsed from Upstte Bioteh (Chndlers Ford, Hmpshire, UK). The nti-p38 ntiody, the ntiphosphorylted p38, the nti-phosphorylted het shok protein (HSP)27 nd nti-totl HSP27 were purhsed from New Englnd Biols (Hertfordshire, UK). The gene expression ssys used for nlysis of TNF- (HS _m1), GM-CSF (HS171266_m1), IL-8 (HS _m1) nd the ontrol gene hypoxnthine phosphoriosyltrnsferse-1 (HPRT1) (HS _m1) y RT PCR were purhsed from Applied Biosystems (Wrrington, UK). IL-8 Relesed (ng ml -1 ) log [LPS (g ml -1 )] Figure 1 Effet of LPS on ytokine relese y lung mrophges. Mrophges from smokers (open irles) (n ¼ 4 6) nd emphysem ptients (solid irles) (n ¼ 3 4) were stimulted with inresing onentrtions of LPS nd superntnts hrvested fter 2 h. Susequently, the onentrtions of TNF- (), GM-CSF () nd IL-8 () were mesured y ELISA. Dt re presented s men7s.e. British Journl of Phrmology (26)

4 396 p38 MAPK nd mrophges SJ Smith et l mrophges from emphysem sujets relesed.27.1 ng ml 1 TNF- (n ¼ 4) (Figure 1). LPS inresed TNF- relese y mrophges from smokers nd emphysem sujets in onentrtion-dependent mnner with EC 5 vlues of nd ng ml 1, respetively. Lung mrophges from smokers nd sujets with emphysem relesed GM-CSF spontneously with levels of pg ml 1 (n ¼ 4) nd pg ml 1 (n ¼ 3), respetively (Figure 1). LPS enhned GM-CSF relese y mrophges from smokers nd emphysem sujets in onentrtion-dependent mnner with EC 5 vlues of nd ng ml 1, respetively. Lung mrophges from smokers nd sujets with emphysem, relesed ng ml 1 (n ¼ 5) nd ng ml 1 (n ¼ 4) of IL-8, respetively under sl onditions (Figure 1). LPS inresed IL-8 relese y mrophges from smokers nd emphysem sujets in onentrtion-dependent mnner with EC 5 vlues of nd ng ml 1, respetively. There ws no signifint differene in the sl relese or LPS-indued response of ny of these ytokines y mrophges from smokers ompred to emphysem sujets. Effet of SB23963 on LPS-stimulted ytokine relese To evlute the inhiitory tivity of p38 MAPK inhiitors on ytokine relese y lung mrophges, sumximl onentrtion of LPS (1 ng ml 1 ) ws seleted tht would llow mesurement of TNF-, GM-CSF nd IL-8 (Tle 2). SB23963 inhiited LPS-indued TNF- relese from mrophges from ll sujet groups in onentrtion-dependent mnner (Figure 2, Tle 3). There ws no signifint differene in the effet of SB23963 on LPS-indued TNF- relese etween ny of the sujet groups (Tle 3). The relese of either GM-CSF or IL-8 relese ws not mrkedly inhiited y SB23963 t ny of the onentrtions tested (.1 1 mm) (Figures 2 nd ) (Tle 3). Effet of SD-282 on LPS-stimulted ytokine relese from humn lung mrophges To investigte further whether the wek effet of SB23963 on LPS-indued GM-CSF nd IL-8 relese y lung mrophges ws due to lk of effiy of the drug or the lk of involvement of the p38 MAPK pthwy, the effet of struturlly dissimilr p38 MAPK inhiitor (SD-282) ws lso evluted in prllel with SB Tle 2 Cytokine relese from humn lung mrophges stimulted with 1 ng ml 1 LPS Cytokine Ex-smokers Current smokers Emphysem TNF GM-CSF IL Arevitions: GM-CSF, grnuloyte mrophge-olony stimulting ftor; IL, interleukin; TNF, tumour nerosis ftor. Dt re presented s men7s.e. ytokine relese in ng ml 1, n ¼ 3 8. SD-282 suppressed LPS-indued TNF- relese from humn lung mrophges (Figure 3). On molr sis, SD-282 ws 5-times more potent thn SB23963 (Po.2) (Tle 4). TNF-α Relesed (% Inhiition) GM-CSF Relesed (% Inhiition) IL-8 Relesed (% Inhiition) LPS ** log [SB23963 [M)] LPS log [SB23963 [M)] LPS log [SB23963 [M)] Figure 2 Effet of SB23963 on LPS-indued ytokine relese y lung mrophges. Mrophges from ex-smokers (open irles) (n ¼ 5), urrent-smokers (losed irles) (n ¼ 8) nd emphysem ptients (solid tringles) (n ¼ 4) were treted with inresing onentrtions of SB23963 for 3 min efore the ddition of LPS (1 ng ml 1 ). Cells were ultured nd superntnts were hrvested fter 2 h nd TNF- (), GM-CSF () nd IL-8 () were mesured y ELISA. Dt re presented s men7s.e. **Po.1 for emphysem smples ompred with ontrol, zz Po.1, zzz Po.1 for smoker smples ompred with ontrol nd w Po.5, ww Po.1 for exsmoker smples ompred with ontrol. British Journl of Phrmology (26)

5 p38 MAPK nd mrophges SJ Smith et l 397 Tle 3 Effet of SB23963 on LPS-stimulted ytokine relese from mrophges Ex-smokers Current smokers Emphysem Cytokine EC 5 (mm) % Inhiition t 1 mm EC 5 (mm) % Inhiition t 1 mm EC 5 (mm) % Inhiition t 1 mm TNF GM-CSF NI NI NI IL-8 NI NI NI.75. Arevitions: GM-CSF, grnuloyte mrophge-olony stimulting ftor; IL, interleukin; TNF, tumour nerosis ftor. Dt re presented s men7s.e. EC 5 vlues nd perentge inhiition t 1 mm, n ¼ 4 8. NI indites tht n EC 5 vlue ould not e generted from the urve. At the highest onentrtion studied (1 mm), the mximl inhiition of TNF- relese y SD-282 ws not signifintly different to tht of SB23963 (n ¼ 6) (Figure 3) (Tle 4). Consistent with the dt shown in Figure 2, SD-282 filed to ffet the relese of GM-CSF or IL-8 (Figure 3 nd ) exept t the highest onentrtion tested where there ws modest inhiitory effet (pproximtely 35%) on GM-CSF output (n ¼ 6) (Figure 3) (Tle 3). Effet of SD-282 nd SB23963 on LPS-indued p38 MAPK tivtion The wek effet of the p38 MAPK inhiitors in this system prompted n ssessment of the ility of LPS to tivte, nd SB23963 nd SD-282 to inhiit, the p38 MAPK signlling pthwys in mrophges. A frtion of the totl p38 MAPK pool ws phosphorylted onstitutively (tivted) in unstimulted mrophges (Figure 4), however, LPS stimultion (1 ng ml 1 ), fter lg of pproximtely 1 min, enhned the sl phosphoryltion of p38 MAPK in time-dependent mnner. By 3 min, the effet of LPS ws onentrtion-dependent with mximl phosphoryltion seen t pproximtely 1 mgml 1 LPS (Figure 4). Hving estlished tht the p38 MAPK pthwy is tivted y LPS tretment in these ells, the ility of SB23963 nd SD-282 to inhiit p38 tivity ws ssessed. The phosphoryltion of HSP27 ws used s mesure of p38 MAPK tivtion. LPS-indued phosphoryltion of HSP27, whih ws inhiited in onentrtion-dependent mnner y oth SD-282 nd SB23963 (Figure 5 nd ). In oth ses, t the highest onentrtion of p38 MAPK inhiitor tested (1 mm) the level of phospho-hsp27 ws similr to tht found in unstimulted ells. In ontrst, expression of totl- HSP27 ws unffeted y inution of the mrophges with LPS, SD-282 or SB23963 (Figure 5). Therefore, oth p38 MAPK inhiitors re effetive t loking the downstrem signlling events leding from p38 MAPK tivtion in humn lung mrophges. Effet of SD-282 nd SB23963 on LPS-stimulted ytokine relese from humn peripherl lood monoytes As the effets of oth p38 MAPK inhiitors on lung mrophges ws rther wek, ontrol system ws tested to ensure tht these drugs were effetive t inhiiting known p38 MAPK-dependent ytokine prodution. To this end, monoytes were exposed to LPS (3 ng ml 1 ) sumximl onentrtion required to evoke the relese of inflmmtory ytokines s reported previously (Mej et l., 2). LPSstimulted monoytes relesed TNF-, GM-CSF nd IL-8 t onentrtions of ng ml 1, pg ml 1 nd ng ml 1, respetively. Pretretment (3 min) of ells with SD-282 nd SB23963 inhiited LPSstimulted TNF- nd GM-CSF relese in onentrtiondependent mnner (Figure 6 nd ) (Tle 5). In ontrst to the output of TNF- nd GM-CSF, tht of IL-8 indued y LPS ws reltively resistnt to oth SD-282 nd SB23963 (Figure 6), however, SD-282 suppressed IL-8 relese from monoytes in onentrtion-dependent mnner (Figure 6). Expression of the p38 MAPK isoforms y humn lung mrophges nd monoytes As p38 MAPK inhiition ws effetive t reduing ytokine prodution y monoytes, the differentil effets of the inhiitors on the two ell types might e explined y vrition in p38 MAPK isoform expression etween monoytes nd mrophges. Therefore, the expression of the different isoforms of p38 MAPK ws exmined. Before nlyzing the mrophge nd monoytes lystes, the speifiity of ommerilly ville ntiodies used to detet the different p38 MAPK isoforms y Western lotting ws evluted using HeL ells tht hd een trnsfeted to express eh individul p38 MAPK isoform. The nti-p38 ntiody deteted n immunoretive protein t 38 kd, whih ws identil in size to ommerilly ville purified extrt of p38. The ntiody lso lelled immunoretive nds from the HeL ell lystes expressing p38, p38g nd p38d nd s some of these nds migrted t the sme moilities they therefore, ould not e exluded from the susequent nlyses (Figure 7). The ntiodies tht reognized p38 nd p38d exhiited greter speifiity for these proteins s they eh deteted only their speifi p38 MAPK isoform 38 kd nd no other immunoretive nds were oserved on these lots (Figure 7). The ntiody reognizing p38g deteted n immunoretive protein t 38 kd, however, it lso deteted n immunoretive nd in the p38 preprtion. There ws n dditionl immunoretive nd deteted t different moility to the 38 kd p38 isoform nd this ws exluded from the nlyses (Figure 7). Using these hrterized ntiodies, lung mrophges expressed p38 predominntly, ut lso expressed p38d. These ells did not express p38 or p38g s determined y Western lotting (Figure 7). The p38 MAPK isoforms British Journl of Phrmology (26)

6 398 p38 MAPK nd mrophges SJ Smith et l 8 Tle 4 Comprison of SB23963 with SD-282 on LPS-stimulted ytokine relese from mrophges TNF-α Relesed (pg ml -1 ) LPS * *** *** ** *** log [Inhiitor (M)] SB23963 Cytokine EC 5 (nm) % Inhiition t 1 mm EC 5 (nm) SD-282 % Inhiition t 1 mm TNF GM-CSF NI IL-8 NI NI Arevitions: GM-CSF, grnuloyte mrophge-olony stimulting ftor; IL, interleukin; TNF, tumour nerosis ftor. Dt re presented s men7s.e. EC 5 vlues nd perentge inhiition t 1 mm, n ¼ 6 8. NI indites tht n EC 5 vlue ould not e generted from the urve. 35 Time (mins) GM-CSF Relesed (pg ml -1 ) ** 38 kd 38 kd [LPS (ng ml -1 )] pp38 Totl p38 LPS log [Inhiitor (M)] 38 kd 38 kd pp38 Totl p38 IL-8 Relesed (ng ml -1 ) Figure 4 Western lot nlysis of totl nd phosphorylted p38 MAPK expression. Lung mrophges were either treted with LPS (1 ng ml 1 ) for up to 6 min (pnel ) or treted with inresing onentrtions of LPS for 3 min (pnel ). Cell lystes were prepred, insolule proteins were removed nd 2 mg of solule extrt were dentured nd sujeted to eletrophoresis on 1% (wt/vol) SDS polyrylmide gels. Proteins were trnsferred to nitroellulose nd proed with either n nti-p38 ntiody or nti-phosphorylted p38 ntiody. The primry ntiodies were deteted with peroxidse-onjugted seondry ntiodies, nd lelled proteins were deteted y enhned hemiluminesene. The lot is representtive of four experiments nd pp38 indites phosphorylted p38. LPS log [Inhiitor (M)] Figure 3 Comprison of SD282 nd SB23963 on LPS-stimulted ytokine relese y lung mrophges. Mrophges were treted with inresing onentrtions of the p38 MAPK inhiitors SD-282 (solid irles) nd SB23963 (open irles) for 3 min efore stimultion with LPS (1 ng ml 1 ). Cells were ultured nd superntnts were hrvested fter 2 h nd TNF- (), GM-CSF () nd IL-8 () were mesured y ELISA. Dt re presented s men7s.e., n ¼ 6. *Po.5, **Po.1 nd ***Po.1 ompred with ontrol. expressed y peripherl lood monoytes followed the sme pttern of expression s oserved in the lung mrophges (Figure 7). The dt from the smples nlysed suggest tht mrophges express inresed levels of p38d ompred to monoytes. Moreover, there ws found to e signifintly higher levels of p38d mrna in lung mrophges ompred to monoytes (Figure 7). There were no differenes in the expression profile of the p38 MAPK isoforms from mrophges of smokers with norml lung funtion ompred to those from emphysem ptients (Figure 7d). Effet of SD-282 nd SB23963 on TNF-, GM-CSF nd IL-8 mrna stility y LPS-stimulted humn peripherl lood monoytes nd mrophges Another possiility tht my ount for the differentil effets of p38 MAPK inhiitors on ytokine prodution y British Journl of Phrmology (26)

7 p38 MAPK nd mrophges SJ Smith et l 399 LPS 27 kd 27 kd LPS 27 kd Log [SD-282 (M)] Log [SB23963 (M)] phsp27 Totl HSP27 phsp27 TNF-α Relesed (pg ml -1 ) LPS log [Inhiitor (M)] 27 kd Totl HSP27 1 Figure 5 Western lot nlysis of totl nd phosphorylted HSP27 expression in lung mrophges following tretment with p38 MAPK inhiitors. Lung mrophges were treted in the sene or presene of SD-282 () nd SB23963 () for 3 min efore the ddition of sumximl onentrtion of LPS (1 ng ml 1 ) for 3 min. Cell lystes were prepred, insolule proteins were removed nd 2 mg of solule extrt were dentured nd sujeted to eletrophoresis on 1% (wt/vol) SDS polyrylmide gels. Proteins were trnsferred to nitroellulose nd proed with either n nti- HSP27 ntiody or nti-phosphorylted HSP27 ntiody. The primry ntiodies were deteted with peroxidse-onjugted seondry ntiodies, nd lelled proteins were deteted y enhned hemiluminesene. The lot is representtive of five experiments nd phsp27 indites phosphorylted HSP27. GM-CSF Relesed (pg ml -1 ) LPS log [Inhiitor (M)] monoytes nd mrophges might e due to effets of p38 MAPK on stility of the mrna of the ytokine under investigtion. In order to investigte this possiility, the stility of TNF-, GM-CSF nd IL-8 mrna in humn monoytes nd mrophges ws determined following ddition of tinomyin D to the ells 4 h fter LPS stimultion. The stility of the ytokine mrna from LPSstimulted monoytes nd lung mrophges deresed, in time-dependent mnner with the stility of TNF- nd GM-CSF mrna from monoytes eing less stle thn tht derived from lung mrophges. After 4 h post-stimultion with LPS, monoyte TNF- mrna hd redued y 82% ompred with mrophges where expression ws deresed y only 37% (Figure 8). Following 4 h stimultion with LPS, there ws no differene etween monoyte nd mrophge GM-CSF or IL-8 mrna stility (Figures 8 nd ). The effetiveness nd poteny of the p38 MAPK inhiitors in suppressing LPS-indued ytokine prodution ws lower in lung mrophges thn in monoytes. Therefore, we exmined the effets of the p38 MAPK inhiitors on LPSindued TNF-, GM-CSF nd IL-8 mrna stility. The p38 MAPK inhiitors redued the stility of TNF-, GM-CSF nd IL-8 mrna in lung mrophges nd monoytes in onentrtion-dependent mnner, lthough there ws no effet on the expression of the ontrol gene HPRT1 (Tle 6 nd Figure 9 f). There ws no signifint differene in the EC 5 vlues for inhiition of ytokine mrna stility of IL-8 Relesed (ng ml -1 ) LPS log [Inhiitor (M)] Figure 6 Comprison of SD282 nd SB23963 on LPS-stimulted ytokine relese y monoytes. Monoytes were treted with inresing onentrtions of the p38 MAPK inhiitors SD-282 (solid irles) nd SB23963 (open irles) for 3 min efore stimultion with LPS (3 ng ml 1 ). Cells were ultured nd superntnts were hrvested fter 2 h nd TNF- (), GM-CSF () nd IL-8 () were mesured y ELISA. Dt re presented s men7s.e., n ¼ 5. either SD-282 or SB23963 etween monoytes nd mrophges (Tle 6), lthough SD-282 ws signifintly more potent ompred to SB23963 (Tle 6). At the highest onentrtion of inhiitors studied (1 mm), the stility of TNF- nd GM-CSF mrna ws redued in oth ell types y British Journl of Phrmology (26)

8 4 p38 MAPK nd mrophges SJ Smith et l Tle 5 Comprison of SB23963 with SD-282 on LPS-stimulted ytokine relese from peripherl lood monoytes α β γ δ SB23963 SD-282 Anti p38-α 38 kd Cytokine EC 5 (nm) % Inhiition t 1 mm EC 5 (nm) % Inhiition t 1 mm Anti p38-β 38 kd TNF GM-CSF IL-8 NI Anti p38-γ 38 kd Arevitions: GM-CSF, grnuloyte mrophge-olony stimulting ftor; IL, interleukin; TNF, tumour nerosis ftor. Dt re presented s men7s.e. EC 5 vlues nd perentge inhiition t 1 mm, n ¼ 5. NI indites tht n EC 5 vlue ould not e generted from the urve. 495% ompred to LPS-stimulted ells treted with tinomyin D lone (Figure 9 d). The p38 MAPK inhiitors only prtilly ffeted IL-8 mrna stility. The mximl inhiition vlues for suppression of IL-8 mrna stility from monoytes were nd % inhiition with SD-282 nd SB23963, respetively (Figure 9e). The mximl inhiition vlues for suppression of IL-8 mrna stility from lung mrophges were nd % inhiition with SD-282 nd SB23963, respetively (Figure 9e). Disussion nd onlusions The glol importne nd prevlene of COPD hve een inresing (Puwels nd Re, 24), nd it is predited to e the third most ommon use of deth y yer 22 (Lopez nd Murry, 1998). Sine COPD is hrterized y inflmmtion ssoited with inresed numers of mrophges (Jeffery, 1999), it is of importne to investigte the ontriution of inflmmtory meditors relesed from the ells. In this study, we found tht there ws no differene in the spontneous or LPS-indued relese of TNF-, GM-CSF nd IL-8 from the humn lung-derived mrophges etween smokers nd ptients with emphysem. Previously, it hs een reported tht lveolr mrophges from smokers relese inresed levels of TNF- under oth sl nd stimulted onditions when ompred with ells from nonsmokers (Gunell et l., 26). This study exmined the role of the p38 MAPK pthwy in the prodution of inflmmtory ytokines from lung mrophges using two struturlly dissimilr inhiitors. The p38 MAPK inhiitor SB23963 signifintly redued TNF- prodution from LPS-stimulted humn lung mrophges, ut not GM-CSF or IL-8, suggesting tht the mehnism of LPS stimultion for IL-8 nd GM-CSF is different from tht of TNF- nd is p38 MAPK independent. Furthermore, seond more potent p38 MAPK inhiitor, SD-282, lso hd differentil effet on inhiition of LPS-indued ytokine relese from humn lung mrophges. The wek effets of the inhiitors ould not e explined y lk of p38 MAPK tivtion or lk of effiy of inhiitors in these ells s phosphoryltion of HSP27 ws suppressed y 485%. Evidene for differentil effet of p38 MAPK on ytokine relese hs een shown y Westr et l. (24) who showed differentil effets of the d Anti-p38-α Anti-p38-β Anti-p38-γ Anti-p38-δ Anti-p38-α Anti p38-δ p38 delt mrna expression (rtio p38 delt / HPRT1) Mrophges monoytes *** 38 kd Monoytes lung mrophges Mrophges p38-α p38-α ontrol ES ES SM SM SM EMP EMP ontrol Figure 7 Western lot nlysis of p38 MAPK isoforms. The speifiities of the ntiodies used in this study were determined using HeL ell lystes ontining eh of the individul p38 MAPK isoforms expressed in HeL ells (). Blots re representtive of five experiments. Highly purified mrophges nd monoytes were lysed, insolule proteins were removed nd 2 mg of solule extrt were dentured nd sujet to eletrophoresis on 1% (wt/vol) SDS polyrylmide gels. Proteins were trnsferred to nitroellulose nd proed with nti-p38 isoform ntiodies. The primry ntiodies were deteted with peroxidse-onjugted seondry ntiodies, nd lelled proteins were deteted y enhned hemiluminesene (). Blots re representtive of four experiments. To determine the levels of p38d mrna Totl RNA ws extrted from monoytes (solid tringles) (n ¼ 9) nd lung mrophges (solid irles) (n ¼ 9) nd reverse trnsried using Avin myelolstosis virus (AMV) reverse trnsriptse. Tqmn rel-time PCR nlysis ws performed for p38d nd HPRT1 () ***Po.1. Western lot nlysis of p38- MAPK expression in humn lung mrophges from urrent smokers (SM), ex-smokers (ES) nd ptients with emphysem (EMP) (pnel d). Blots re representtive of 3 experiments. p38 MAPK inhiitor RWJ on the suppression of TNF-, IL-8 nd IL-6 from LPS-stimulted 7-dy monoyte-derived mrophges. We hve extended these oservtions y British Journl of Phrmology (26)

9 p38 MAPK nd mrophges SJ Smith et l 41 RQ vlues TNF-α (% LPS mins) RQ vlues GM-CSF (% LPS mins) RQ vlues IL-8 (% LPS mins) * ** Time (min) Time (min) Time (min) Figure 8 Comprison of TNF- mrna stility etween monoytes nd lung mrophges. Humn lung mrophges (open irles) nd humn peripherl lood monoytes (solid irles) were stimulted with LPS (1 nd 3 ng ml 1, respetively) for 4 h efore the ddition of 5 mgml 1 tinomyin D (t ¼ ). Cells were hrvested t the times indited fter the ddition of tinomyin D. Totl RNA ws extrted nd reverse trnsried using AMV reverse trnsriptse. Tqmn reltime PCR nlysis ws performed nd the ddc T vlues for TNF- nd HPRT1 were normlized to reltive ontriution of 1. for untreted ells. Dt re presented s the men7s.e., n ¼ 4 for expression of TNF- (), GM-CSF () nd IL-8 mrna () reltive to tht of HPRT1 mrna (ontrol gene). Dt re expressed s perentge of ytokine/hprt1 mrna expression t t ¼. *Po.5 nd **Po.1 ompred with ontrol. * exmining the effets of two different p38 MAPK inhiitors on relese of ytokines implited in lung inflmmtion y mrophges from ptients with emphysem ompred to ontrol sujets. It hd previously een found tht the seletive p38 MAPK inhiitor SB23963 exhiited differentil effets on LPSindued ytokine prodution from humn peripherl lood monoytes (Underwood et l., 2). This ompound inhiited LPS-indued IL-1 nd TNF- relese potently, GM- CSF less so nd ws unle to inhiit G-CSF relese (Underwood et l., 2). In this study, we hve shown tht high onentrtions of SD-282, ut not SB23963, suppressed GM-CSF relese from LPS-stimulted humn lung mrophges. In previous study, LPS-stimulted GM-CSF prodution from humn lveolr mrophges ws lso not inhiited y the p38 MAPK inhiitor SB2358 (Koh et l., 24). However, s the potenies of these ompounds re less thn tht of SD-282, with IC 5 vlues of 56 nd 44 nm, respetively, for SB2358 nd SB23963, this my ount for the lk of oserved inhiition on GM-CSF relese. The p38 MAPK inhiitor SD-282 suppressed IL-8 relese from peripherl lood monoytes, ut not from lung mrophges. Moreover, the EC 5 vlues for the suppression of IL-8 in peripherl lood monoytes were higher thn those for inhiition of GM-CSF nd TNF-. Similr findings hve een reported y Westr et l. (24) who showed tht the IC 5 vlues for inhiition of IL-8 relese ws higher thn tht required to suppress TNF- nd IL-1. In the present study, we hve shown tht SB23963 suppressed LPSindued TNF- nd GM-CSF relese from monoytes with omprle IC 5 vlues nd mximl inhiition to the findings of Underwood et l. (2). This dt thus vlidtes our experimentl system for the evlution of the novel inhiitor SD-282. Neither p38 MAPK inhiitor is reported to lter the tivity of either p38d or p38g (Underwood et l., 2), moreover, SD-282 does not use inhiition of the other MAPKs ERK2 nd JNK when tested t 5 mm (Lim et l., 24). In this study, we hve shown tht humn lung mrophges nd monoytes express oth p38 nd p38d protein onfirming the studies of Hle et l. (1999). Therefore, oth inhiitors hve the pity to suppress the p38 MAPK pthwy in oth monoytes nd mrophges. Indeed, p38 MAPK tivity is diminished in mrophges in the presene of oth inhiitors. Therefore, the reltive insensitivity of ytokine relese y p38 MAPK inhiitors is not simply due to lk of p38 MAPK inhiition in mrophges ut my e relted to differentil regultion of ytokine trnsription nd trnsltion in these ells. Alterntively, it is possile tht the undne of p38d in mrophges might lso ount for the diminished effet of these inhiitors. We hve shown tht, in mrophges isolted from humn lung tissue, the stility of TNF- mrna is signifintly greter thn in humn monoytes. This dt is in ordne with the findings of MKenzie et l. (22). In ddition, the stility of mrna for other proinflmmtory ytokines, suh s IL-1, is lso inresed in lveolr mrophges ompred to monoytes (Herzyk et l., 1992). The reson why the regultion of TNF- mrna is ltered in British Journl of Phrmology (26)

10 42 p38 MAPK nd mrophges SJ Smith et l TNF-α / HPRT1 mrna expression (% LPS Control) LPS monoytes log [Inhiitor(M)] TNF-α / HPRT1 mrna expression (% LPS Control) d mrophges LPS log [Inhiitor(M)] GM-CSF/HPRT1 mrna expression (% LPS Control) e IL-8/HPRT1 mrna expression (% LPS Control) LPS log [Inhiitor(M)] LPS log [Inhiitor(M)] GM-CSF/HPRT1 mrna expression (% LPS Control) f IL-8/HPRT1 mrna expression (% LPS Control) LPS log [Inhiitor(M)] LPS log [Inhiitor(M)] Figure 9 Comprison of SD-282 nd SB23963 effets on ytokine mrna stility etween monoytes nd lung mrophges. The effet of p38 MAPK inhiitors SD-282 (solid irles) nd SB23963 (open irles) on LPS-indued TNF-, GM-CSF nd IL-8 mrna stility t 6 min post ddition of 5 mgml 1 tinomyin D. Totl RNA ws extrted nd reverse trnsried using AMV reverse trnsriptse. Tqmn rel-time PCR nlysis ws performed nd the ddc T vlues for TNF- nd HPRT1. The effet of the MAPK inhiitors ws determined for monoytes (, nd e) nd lung mrophges (, d nd f). Dt re expressed s men7s.e. ytokine/hprt1 mrna expression, represented s perentge of the LPS ontrol (1%), n ¼ 4. Tle 6 Comprison of SB23963 with SD-282 on ytokine mrna stility of peripherl lood monoytes nd lung mrophges SB23963 SD-282 Cytokine Monoytes EC 5 (nm) Mrophges EC 5 (nm) Monoytes EC 5 (nm) Mrophges EC 5 (nm) TNF GM-CSF IL Arevitions: GM-CSF, grnuloyte mrophge-olony stimulting ftor; IL, interleukin; TNF, tumour nerosis ftor. Dt re presented s men7s.e. EC 5 vlues n ¼ 4. British Journl of Phrmology (26)

11 p38 MAPK nd mrophges SJ Smith et l 43 the mrophge ompred to the monoyte is unler, ut my llow mrophges, s resident tissue ells, to filitte more sustined prodution of ytokines nd hene prolong the inflmmtory response. We hve found tht there is no differene in the effiy of the two p38 MAPK inhiitors for deresing TNF-, GM-CSF or IL-8 mrna stility with mrginl differenes in the effiy of the two p38 MAPK inhiitors on protein relese. However, oth p38 MAPK inhiitors suppressed TNF- nd GM-CSF mrna stility t high onentrtions, ut hd only 5% inhiitory effet on TNF- protein relese nd only wek effet on GM-CSF protein relese. Tken together, the dt suggest tht the effets of the p38 MAPK inhiitors is prtly medited y their effets on mrna stility, ut tht p38 MAPK-independent pthwys medite TNF- nd GM-CSF protein relese. Other studies hve shown in humn monoytes tht p38 MAPK inhiitors do not regulte the trnsltion for the expression of either TNF- or COX-2 (Den et l., 1999). Interestingly, the EC 5 vlues for degrdtion of TNF- mrna re pproximtely 1-fold lower thn those found for the inhiition of TNF- protein relese from lung mrophges, whih suggest tht other p38 MAPK-dependent mehnisms, suh s posttrnsltionl modifition, my e involved in ytokine relese from mrophges. Inflmmtion in COPD is ssoited with n inrese in ytokines, inluding TNF-, GM-CSF nd IL-8. Moreover, there re no urrent therpies for COPD tht suppress the inflmmtory response. We report here tht p38 MAPK inhiitors trgeting the p38 nd p38 isoforms were wekly effetive in suppressing LPS-indued ytokine prodution from lung-derived mrophges ut the effet ws independent of donor sttus. These findings suggest tht p38 MAPK inhiitors my e of little enefit in reduing TNF-, GM-CSF or IL-8 ssoited lung inflmmtion ssoited with COPD ut the development of moleules tht trget oth p38 nd p38d MAPK isoforms my hve dded vlue in trgeting mrophge-driven inflmmtion. Aknowledgements We thnk Mr Peter Goldstrw (Deprtment of Crdiothori Surgery, Royl Brompton Hospitl) for generously providing humn lung speimens for mrophge isoltion. Conflit of interest The uthors stte no onflit of interest. Referenes Brnes PJ (24). Meditors of hroni ostrutive pulmonry disese. Phrmol Rev 56: Brnes PJ (24). Mrophges s orhestrtors of COPD. J COPD 1: 5 7. Burge PS, Clverley PM, Jones PW, Spener S, Anderson JA, Mslen TK (2). Rndomised, doule lind, pleo ontrolled study of flutisone propionte in ptients with moderte to severe hroni ostrutive pulmonry disese: the ISOLDE tril. BMJ 32: Culpitt SV, Rogers DF, Shh P, De Mtos C, Russell RE, Donnelly LE et l. (23). Impired inhiition y dexmethsone of ytokine relese y lveolr mrophges from ptients with hroni ostrutive pulmonry disese. Am J Respir Crit Cre Med 167: Den JL, Brook M, Clrk AR, Skltvl J (1999). p38 mitogentivted protein kinse regultes ylooxygense-2 mrna stility nd trnsription in lipopolyshride-treted humn monoytes. J Biol Chem 274: Dun W, Chn JH, MKy K, Crosy JR, Choo HH, Leung BP et l. (25). Inhled p38lph mitogen-tivted protein kinse ntisense oligonuleotide ttenutes sthm in mie. Am J Respir Crit Cre Med 171: Gunell G, Brdelli C, Amoruso A, Vino I, Blo P, Brunelleshi S (26). Mrophge-stimulting protein differently ffets humn lveolr mrophges from smoker nd non-smoker ptients: evlution of respirtory urst, ytokine relese nd NF-kppB pthwy. Br J Phrmol 148: Hddd EB, Birrell M, MCluskie K, Ling A, Weer SE, Foster ML et l. (21). Role of p38 MAP kinse in LPS-indued irwy inflmmtion in the rt. Br J Phrmol 132: Hle KK, Trollinger D, Rihnek M, Mnthey CL (1999). Differentil expression nd tivtion of p38 mitogen-tivted protein kinse lph, et, gmm, nd delt in inflmmtory ell lineges. J Immunol 162: Httotuw KL, Gizyki MJ, Ansri TW, Jeffery PK, Brnes NC (22). The effets of inhled flutisone on irwy inflmmtion in hroni ostrutive pulmonry disese: doule-lind, pleoontrolled iopsy study. 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