Possible new role for NF-κB in the resolution of inflammation

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1 Possile new role for NF-κB in the resolution of inflmmtion TOBY LAWRENCE, DEREK W. GILROY, PAUL R. COLVILLE-NASH & DEREK A. WILLOUGHBY Deprtment of Experimentl Pthology, Willim Hrvey Reserch Institute, Chrterhouse Squre, London EC1M 6BQ, UK Correspondence should e ddressed to T.L.; emil: t.lwrence@mds.qmw.c.uk Inflmmtion involves the sequentil ctivtion of signling pthwys leding to the production of oth pro- nd nti-inflmmtory meditors. Although much ttention hs focused on pro-inflmmtory pthwys tht initite inflmmtion, reltively little is known out the mechnisms tht switch off inflmmtion nd resolve the inflmmtory response. The trnscription fctor NF-κB is thought to hve centrl role in the induction of pro-inflmmtory gene expression nd hs ttrcted interest s new trget for the tretment of inflmmtory disese. We show here tht NF-κB ctivtion in leukocytes recruited during the onset of inflmmtion is ssocited with pro-inflmmtory gene expression, wheres such ctivtion during the resolution of inflmmtion is ssocited with the expression of nti-inflmmtory genes nd the induction of poptosis. Inhiition of NF-κB during the resolution of inflmmtion protrcts the inflmmtory response nd prevents poptosis. This suggests tht NF-κB hs n nti-inflmmtory role in vivo involving the regultion of inflmmtory resolution. The inflmmtory response involves the sequentil relese of meditors nd the recruitment of circulting leukocytes, which ecome ctivted t the inflmmtory site nd relese further meditors. This response is self-limiting nd resolves through the relese of endogenous nti-inflmmtory meditors nd the clernce of inflmmtory cells. The persistent ccumultion nd ctivtion of leukocytes is hllmrk of chronic inflmmtion. Current pproches to the tretment of inflmmtion rely on the inhiition of pro-inflmmtory meditor production nd of mechnisms tht initite the inflmmtory response. However, the mechnisms y which the inflmmtory response resolves might provide new trgets in the tretment of chronic inflmmtion. Studies in different experimentl models of resolving inflmmtion hve identified severl puttive mechnisms nd meditors of inflmmtory resolution. We hve shown tht cyclopentenone prostglndins (cypgs) my e endogenous nti-inflmmtory meditors nd promote the resolution of inflmmtion in vivo 1. Others hve shown temporl shift to the production of nti-inflmmtory lipoxins during the resolution of inflmmtion 2. In recent yers, poptosis hs een identified s n importnt mechnism for the resolution of inflmmtion in vivo 3,4. It hs een postulted tht defects in leukocyte poptosis re importnt in the pthogenesis of inflmmtory disese. In ddition, the selective induction of poptosis in leukocytes my offer new therpeutic pproch to inflmmtory disese. Nucler fctor-κb (NF-κB) is generic term for dimeric trnscription fctor formed y the hetero- or homodimeriztion of proteins from the rel fmily, reviewed recently 5. There re five rel proteins: RelA (p65), RelB nd crel, which contin trnsctivtion domins, nd p50 nd p52, which re expressed s the precursor proteins p105 (NF-κB1) nd p100 (NF-κB2), respectively. These precursors require post-trnsltionl processing nd do not contin trnsctivtion domins. NF-κB is thought to ply pivotl role in immune nd inflmmtory responses through the regultion of genes encoding pro-inflmmtory cytokines, dhesion molecules, chemokines, growth fctors nd inducile enzymes such s cyclooxygense 2 (COX2) nd inducile nitric oxide synthse (inos) NF-κB is usully kept inctive in the cytoplsm through ssocition with n endogenous inhiitor protein of the IκB (inhiitor of NF-κB) fmily. The consensus pthwy for NF-κB ctivtion in response to pro-inflmmtory stimuli such s tumor necrosis fctor-α (TNF-α) nd interleukin-1β (IL-1β) hs een extensively chrcterized 5. These cytokines ct through distinct signling pthwys tht converge on the ctivtion of n IκB kinse (IKK); the susequent phosphoryltion of IκB molecules trgets them for degrdtion y the proteosome. IKK consists of three suunits, α, β nd γ; IKK-β is responsile for NF-κB ctivtion in response to pro-inflmmtory stimuli. Previous studies of mice geneticlly deficient in components of the NF-κB pthwy hve shown tht Rel proteins re importnt in the recruitment of leukocytes in the innte immune response. RelA hs role in the non-hemtopoietic comprtment 11, wheres crel hs role in promoting hemtopoietic cell survivl nd production of meditors tht mintin the inflmmtory response Similr studies hve shown role for p50 in negtive regultion of mcrophge ctivtion 15,16. Rt crrgeenin-induced pleurisy is n estlished model of cute inflmmtion, chrcterized y sequentil relese of inflmmtory meditors nd recruitment of leukocytes tht peks t 24 h nd resolves y 48 h (ref. 1). The cellulr infiltrte is initilly dominted y neutrophil grnulocytes, with n incresing percentge of mononucler phgocytes s inflmmtion progresses, nd with these dominting during resolution. The onset of inflmmtion is ssocited with expression of COX2 nd inos nd production of the pro-inflmmtory meditors prostglndin E 2 (PGE 2 ) nd nitric oxide (NO) 17. The resolution of inflmmtion is ssocited with further increse in COX2 expression, without PGE 2 production or inos expression ut ccompnied y the production of the nti-inflmmtory cypg 15-deoxy- [12,14] PGJ 2 (15dPGJ 2 ) nd its precursor PGD 2 (ref. 1). Here we investigte NF-κB ctivtion in leukocytes during the NATURE MEDICINE VOLUME 7 NUMBER 12 DECEMBER

2 c d Fig. 1 NF-κB ctivtion in leukocytes during inflmmtion. d, NF-κB ctivtion in leukocytes fter the induction of rt crrgeenin pleurisy., Time course of NF-κB ctivtion s mesured y EMSA with protein extrcts from leukocytes collected from the pleurl cvity t the indicted time points. Filled rrow indictes NF-κB DNA inding complex., Control rections incuted with unleled consensus (S) or mutnt oligonucleotide (N). Supershift ssys for RelA, p50 nd crel in DNA inding complexes from 6 nd 48 h; filled rrow indictes shifted complexes. c, Western-lot nlysis of crel DNA inding ctivity t 6 nd 48 h. d, Western-lot nlysis of IκBα, inos, COX1 nd COX2 protein expression in leukocytes. e g, Inflmmtory prmeters nd NF-κB ctivtion in the mouse crrgeenin ir pouch. e, Time course of exudte formtion (open oxes) nd leukocyte ccumultion (closed oxes). Dt represented s men ± s.e.m. (n = 8 10). f, Time course of NF-κB ctivtion in leukocytes. Control rections incuted with unleled evolution of the inflmmtory response. We show NF-κB ctivtion ssocited with oth the onset nd the resolution of inflmmtion in oth rt crrgeenin pleurisy nd mouse crrgeenin ir pouch. During resolution, however, NF-κB ctivity is not ssocited with inos expression or the relese of pro-inflmmtory meditors ut is ssocited with the expression of endogenous nti-inflmmtory pthwys nd leukocyte poptosis. In ddition, inhiition of NF-κB during the resolution of inflmmtion in vivo protrcts the inflmmtory response nd prevents clernce of leukocytes. NF-κB ctivtion during the resolution of inflmmtion in vivo We mesured the DNA-inding ctivity of NF-κB in leukocytes from rt crrgeenin pleurisy y the electrophoretic moility shift ssy (EMSA) over 48-hour time course. NF-κB ctivtion in this model ws iphsic (Fig. 1). DNA inding ctivity ws initilly detected t 6 h during the onset of inflmmtion e consensus (S) or mutnt oligonucleotide (N). Filled rrow indictes NFκB DNA inding complex. g, Western-lot nlysis of inos protein expression in leukocytes. nd ws coincident with inos protein expression nd the degrdtion of IκBα protein (Fig. 1d). NF-κB DNA inding ctivity then further incresed t 24 nd 48 h in the presence of IκBα protein nd the sence of inos expression. This ltephse ctivtion of NF-κB ws ssocited with the expression of COX2 (Fig. 1d). Control experiments using excess unleled consensus or mutnt κb oligonucleotides indicted tht the complexes detected t oth 6 nd 48 h were specific for the κb inding site (Fig. 1). The ddition of ntiodies to the p50 suunit of NF-κB showed strong shift in the moility of the complexes t oth 6 nd 48 h (Fig. 1). The ddition of RelA ntiserum produced weker shift in the moility of complexes t oth time points, indicting some DNA-inding of RelA-p50 heterodimers t oth 6 nd 48 h (Fig. 1). Addition of crel ntiserum seemed to reduce the intensity of the DNAinding ctivity t 6 h, ut with no visile shift in the moility of the complex; however, crel ntiserum hd no effect on f g c Fig. 2 Effects of PDTC on inflmmtory prmeters. nd, Effects of PDTC t 6 nd 48 h in crrgeenin pleurisy. Leukocytes nd exudte were collected t 6 h () nd 48 h () fter PDTC tretment. Dt is represented s men ± s.e.m. (n = 8 10). ***, P < 0.001, s determined y ANOVA with post hoc Bonferroni-corrected t-test. c nd d, Effects of PDTC on inflmmtory prmeters t 24 nd 72 h in crrgeenin ir pouch. Leukocytes nd exudte were collected t 24 (c) nd 72 h (d) fter PDTC tretment. Dt is represented s men ± s.e.m. (n = 8 10). *, P < 0.05; ***, P < 0.001, s determined y ANOVA with post hoc Bonferroni-corrected t-test. d 1292 NATURE MEDICINE VOLUME 7 NUMBER 12 DECEMBER 2001

3 Fig. 3 Inhiition of NF-kB or COX2 ctivity protrcts the inflmmtory response in vivo. Shown re effects on inflmmtory prmeters nd NF-kB ctivtion determined s descried in Figs. 1 nd 2 of By () MG132 () nd NS398 (c) given during the resolution of inflmmtion in rt crrgeenin pleurisy. Dt is represented s men ± s.e.m. (n = 8 10). *, P < 0.05; **, P < 0.01; ***, P < 0.001, s determined y ANOVA with post hoc Bonferroni-corrected t-test. c DNA-inding ctivity t 48 h (Fig. 1). Antiodies to RelB nd p52 did not produce ny chnge in the intensity of DNA inding or the moility of the complexes t 6 or 48 h (not shown). Western-lot nlysis of proteins inding n grose-conjugted κb response element showed crel inding ctivity in cell extrcts otined t 6 h ut not t 48 h (Fig. 1c). Western-lot nlysis did not detect RelB or p52 DNA-inding ctivity t either 6 or 48 h (not shown). This suggested shift from predominnce of crel-p50 complexes t 6 h to predominnce of p50 p50 complexes t 48 h. Effects of PDTC in rt crrgeenin pleurisy The NF-κB inhiitor pyrrolidine dithiocrmte (PDTC) ws nti-inflmmtory during the onset of the inflmmtory response when given prophylcticlly with crrgeenin t 300 mg/kg intrperitonelly (i.p.). The tretment reduced exudte formtion y 45% (P < 0.001) nd the numer of leukocytes y 26% (P < 0.001) s mesured 6 h fter crrgeenin chllenge (Fig. 2). When given therpeuticlly 24 h fter crrgeenin chllenge, however, PDTC protrcted the inflmmtory response, inhiiting the resolution of oth exudte nd leukocytes. Tretment with PDTC led to 7-fold increse in exudte volume nd 2.3-fold increse in leukocytes (P < 0.001) t 48 h (Fig. 2). NF-κB ctivtion nd PDTC in mouse crrgeenin ir pouch The oservtions in rt crrgeenin pleurisy were confirmed in different species nd model, the mouse crrgeenin ir pouch. The crrgeenin-induced ir pouch is nother well-estlished model of inflmmtion, chrcterized y formtion of fluid exudte nd leukocyte recruitment tht egin to resolve t 72 h (Fig. 1e). As for the rt model, the cellulr infiltrte is initilly dominted y neutrophils, with n incresing percentge of mononucler cells s inflmmtion progresses nd with these dominting during resolution. NF-κB ctivtion in leukocytes from the crrgeenin ir pouch followed iphsic pttern similr to tht in rt crrgeenin pleurisy. NF-κB ctivtion t 18 h (Fig. 1f) ws ssocited with the expression of inos protein in leukocytes (Fig. 1g), wheres NF-κB ctivity occurred during the resolution of inflmmtion in the sence of inos protein expression. PDTC (200 mg/kg i.p.) ws lso nti-inflmmtory during the onset of inflmmtion when given prophylcticlly, reducing exudte formtion y 40% nd cell ccumultion y 30% (P < 0.05) 24 h fter crrgeenin injection (Fig. 2c). PDTC given therpeuticlly 48 h fter crrgeenin inhiited the resolution of inflmmtion c Fig. 4 Effects of the NF-κB inhiitors PDTC nd MG132 on leukocyte poptosis nd TGF-β1 relese during the resolution of rt crrgeenin pleurisy. Apoptosis ws ssessed t 48 h fter tretment with PDTC nd MG132 y nnexin V FITC inding nd mesured y FACS nlysis. The level of poptosis is expressed s the percentge of cells in R1 nd R2 regions of the dot-plot distriution., Representtive dot plots (n = 5)., The sme dt represented s men ± s.e.m. (n = 5). c, TGF-β1 mesured fter tretment with PDTC (300 mg/kg) nd MG132 (200 µg) in cell-free inflmmtory exudtes t 48 h. Dt is represented s men ± s.e.m. (n = 10). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ANOVA with post hoc Bonferroni-corrected t-test. NATURE MEDICINE VOLUME 7 NUMBER 12 DECEMBER

4 Inhiition of NF-κB DNA inding protrcts inflmmtion To confirm the results otined with the rod-spectrum NF-κB inhiitor PDTC, we used two other inhiitors of NF-κB with distinct mechnisms nd routes of dministrtion: By nd the proteosome inhiitor MG132. Both inhiited the DNA-inding ctivity of NF-κB in leukocytes from the pleurl cvity t 48 h when given therpeuticlly 24 h fter crrgeenin. Reduced NF-κB DNA-inding ctivity ws ssocited with the inhiition of resolution. By (30 mg/kg i.p.) incresed fluid exudte 4.6-fold nd the numer of leukocytes 2.1-fold (P < 0.05) t 48 h (Fig. 3). MG132 (200 µg loclly) given t 24 h incresed exudte volume 3.8-fold nd the numer of leukocytes 2.1-fold (P < 0.001) t 48 h (Fig. 3). When given in the sence of crrgeenin, these inhiitors did not provoke ny mesurle inflmmtory response. The selective COX2 inhiitor NS398 (10 mg/kg) inhiited the resolution of inflmmtion t 48 h, incresing exudte volume 6.8-fold (P < 0.01) nd cell numer 2.6-fold (P < 0.05), ut this tretment did not decrese NF-κB DNA-inding ctivity in leukocytes (Fig. 3c). Fig. 5 Anlysis of cytokine nd poptosis relted gene expression in crrgeenin pleurisy. RNse protection ssys for cytokine- () nd poptosisrelted () gene expression with RNA isolted from cells t 6 nd 48 h in crrgeenin pleurisy. Representtive smples of t lest 3 independent experiments re shown. t 72 h, resulting in 1.7-fold increse in the numer of leukocytes (P < 0.001) compred to controls, lthough the volume of exudte ws only slightly incresed (Fig. 2d). NF-κB inhiition reduces leukocyte poptosis nd TGF-β1 relese We ssessed poptosis in inflmmtory exudtes y nnexin V FITC inding s nlyzed y flow cytometry. Cell viility in ll tretment groups ws more thn 92%, s determined y propidium iodide (PI) exclusion. At 48 h in the pleurisy, ± 2% of leukocytes were poptotic, s ssessed y nnexin V inding to phosphtidylserine (Fig. 4). PDTC nd MG132 given t 24 h reduced the numer of poptotic leukocytes t 48 h to 4.74 ± 0.86% nd 6.5 ± 0.92%, respectively (Fig. 4). We otined very similr results with FACS-sed terminl deoxynucleotidyltrnsferse medited dutp nick end-leling (TUNEL) ssy (not shown). We lso mesured the nti-inflmmtory cytokine trnsforming growth fctor-β1 (TGF-β1) in cell-free pleurl exudtes y ELISA. Both PDTC nd MG132 reduced TGF-β1 relese t 48 h, y 50% (P < 0.001) nd 61% (P < 0.001), respectively (Fig. 4c). Inhiition of NF-κB reduces Bx, p53 nd TGF-β1 expression in vivo We used RNse protection ssys nd western-lot nlysis to nlyze gene expression in leukocytes. The profiles of cytokine- (Fig. 5) nd poptosis-relted (Fig. 5) gene expression in leukocytes from the pleurl cvity indicte tht the relted pro-inflmmtory cytokines lymphotoxin β (Ltβ) nd TNF-α nd the nti-poptotic protein Bcl2 were preferentilly expressed t 6 h fter crrgeenin injection, wheres the nti-inflmmtory cytokine TGF-β1 nd the pro-poptotic Bcl2 homolog Bx were expressed t 48 h. Prophylctic tretment with MG132 concurrently with crrgeenin inhiited IκBα degrdtion nd NF-κB ctivtion in leukocytes t 6 h (Fig. 6 nd ); this ws ssocited with reduced TNF-α nd Ltβ gene expression (Fig. 6c). However, therpeutic tretment with MG132 s descried ove inhiited NF-κB1 (p105) processing to p50 nd reduced TGF-β1, Bx nd p53 expression in leukocytes t 48 h (Fig. 6d nd e). d Fig. 6 Effects of MG132 tretment during inflmmtion. c, Effects of 200 µg MG132 on NF-κB ctivtion nd pro-inflmmtory gene expression t 6 h fter the induction of crrgeenin pleurisy. Shown re effects on NF-κB DNA-inding ctivity (), IκBα protein expression () nd TNF-α nd Ltβ gene expression (c; ssessed y RNse protection ssy with RNA from cells t 6 h). d nd e, Effects of 200 µg MG132 on p105 processing nd expression of p53, Bx nd TGF-β1 t 48 h fter crrgeenin injection. Shown re effects on processing of p105 to p50 (d) nd the expression of Bx nd p53 protein (ssessed y western-lot nlysis) nd Bx nd TGF-β1 gene expression (e; ssessed y RNse protection ssy with RNA from cells t 48 h). c e 1294 NATURE MEDICINE VOLUME 7 NUMBER 12 DECEMBER 2001

5 Discussion In the models descried here, onset of inflmmtion is chrcterized y the relese of pro-inflmmtory cytokines TNF-α nd IL-1β, which signl pro-inflmmtory gene expression through IKK-β (ref. 5). Resolution of inflmmtion is ssocited with production of COX2 derived nti-inflmmtory prostglndins PGD 2 nd 15dPGJ 2 (ref. 1) nd the nti-inflmmtory cytokine TGF-β1. Here we show NF-κB ctivtion coincident with inos expression during the onset of inflmmtion in oth rt crrgeenin pleurisy nd mouse crrgeenin ir pouch, in keeping with previous results 10. However, we did not detect inos expression during the resolution of inflmmtion despite the presence of sustntil NF-κB ctivtion. Although DNA-inding complexes t the onset of inflmmtion contined oth crel nd p50, crel-inding ctivity ws not detected during the resolution of inflmmtion. The crel-p50 heterodimer inds nd regultes the mouse inos promoter 18 nd mcrophges from crel knockout mice show impired inos gene expression 13. This my provide mechnistic explntion for the lck of inos expression in response to NF-κB ctivtion during the resolution of inflmmtion in vivo. Signl-induced processing of p105 to p50 is dependent on inducile phosphoryltion nd proteosome-medited limited proteolysis, which is dependent in turn on the IKK complex 19. However, lthough the IKK complex is essentil for inducile p105 processing, IKK-β is redundnt in this pthwy 19. Activtion of NF-κB t 6 h in crrgeenin pleurisy ws ssocited with IκBα degrdtion s expected given tht IKK-β signls IκBα degrdtion, ut NF-κB ctivtion t 48 h ws detected in the presence of IκBα. Tking into considertion the high ffinity of IκBα for RelA- nd crel-contining complexes, this further suggests tht p50 p50 homodimers represent the predominnt NFκB ctivity t 48 h. Furthermore, cypgs produced during the resolution of inflmmtion 1 specificlly inhiit IKK-β in vitro This my indicte n IKK-β-independent pthwy of NF-κB ctivtion in vivo. Notly, recent report hs descried n IKK-αdependent pthwy for NF-κB ctivtion independent of IKK-β tht is ssocited with distinct profile of gene expression 23. The persistence of p50 p50 homodimers during the resolution of inflmmtion is perhps not surprising, considering the reported role of p50 p50 in the repression of pro-inflmmtory gene trnscription 15,16. Trnsgenic mice tht express p50 ut not the inhiitory C-terminus of the p105 molecule show incresed p50 p50 DNA-inding ctivity 16. These mice show reduced expression of cytokine genes in resident mcrophges nd impired leukocyte recruitment, suggesting p50-medited suppression of inflmmtion in vivo. These studies lso showed positive trnscriptionl effects in certin cell types, however, which my indicte role for cell-specific cofctors in dictting the outcome of NF-κB ctivtion in vivo. Both in vitro nd in vivo studies suggest tht p50 p50 my enhnce gene trnscription, prticulrly when complexed with the IκB-like protein Bcl3 (refs. 24,25). We were unle to detect Bcl3 in DNA-inding complexes t 48 h; however, this does not exclude the presence or sence of other trnscriptionl cofctors not of the Rel fmily tht might lter the trnscriptionl properties of NF-κB. This my e prticulrly relevnt to the expression of COX2 during the resolution of inflmmtion. RelA-p50 nd p50 p50 complexes ind to distinct κb response elements in the COX2 promoter 26. However, p50 p50 is importnt in COX2 trnscription through ssocition with trnscriptionl co-ctivtors of the C/EBP fmily 26,27. Inhiitors of the NF-κB pthwy hve nti-inflmmtory effects in vivo However, during the resolution of inflmmtion, these gents inhiited NF-κB ctivtion in leukocytes nd protrcted the inflmmtory response. Notly, the selective COX2 inhiitor NS398 did not reduce NF-κB DNA inding ctivity in leukocytes, which suggests tht cypgs my ct downstrem of NF-κB in the resolution of inflmmtion in vivo. There re concerns with the use of these inhiitors of NF-κB, which my hve effects independent of the NF-κB pthwy. However, use of inhiitors llows modultion of NF-κB t specific stges of the inflmmtory response, which is centrl to the findings reported here. It is importnt to note tht such inhiitors my prevent the proper resolution of inflmmtion in vivo. Unfortuntely, ville moleculr genetic tools do not llow modultion of the NF-κB pthwy t different stges of the inflmmtory response in vivo. It is to e hoped tht the development of such tools will help to define the role of the NF-κB pthwy in inflmmtion. Leukocyte poptosis nd susequent clernce y mcrophges is importnt for the resolution of inflmmtion 3,4 ; in ddition, the NF-κB pthwy regultes oth pro- nd ntipoptotic pthwys 31. We hve lso shown here tht inhiition of NF-κB inhiits poptosis during the resolution of inflmmtion in vivo. Expression of genes involved in poptosis, including the pro-poptotic Bcl-2 homolog Bx nd the pro-poptotic trnscription fctor p53 (refs. 32,33), ws modulted y NF-κB inhiitors in crrgeenin pleurisy. Inhiition of the processing of p105 to p50 nd of NF-κB DNA inding ctivity y the proteosome inhiitor MG132 reduced the expression of Bx nd p53 in leukocytes t 48 h. It is noteworthy tht Bx expression is detected in leukocytes during the resolution of inflmmtion. Cytokine-medited repression of Bx might cuse the persistence of grnulocytes in inflmmtory diseses through delyed poptosis 34. The pro-poptotic ction of p53 expression in cells of myeloid linege is lso suppressed y pro-inflmmtory cytokines 33, nd p53 nd Bx expression re induced during LPSinduced mcrophge poptosis in vitro 35. In contrst, TNF-α induced poptosis, which is dependent on IKK-β ctivtion nd IκBα degrdtion 36, is not ssocited with expression of Bx nd p Bx is thought to e trget gene for p53 rther thn for NF-κB (refs. 32,33), lthough the Bx promoter contins oth κb nd p53 response elements 37. NF-κB is known to regulte p53 expression, however; in ddition, p50 p50 homodimers positively regulte the p53 promoter It is possile tht p53 nd NF-κB (p50 p50) cooperte to regulte Bx expression nd poptosis. Previous studies suggest tht TGF-β1 relese during the resolution of inflmmtion my occur in response to phgocytosis of poptotic neutrophils y mcrophges We show tht NFκB inhiitors reduce TGF-β1 expression nd relese y leukocytes during the resolution of inflmmtion. The NF-κB pthwy my regulte leukocyte poptosis nd the susequent releseof TGF-β1 during the resolution of inflmmtion in vivo. 15dPGJ 2 induces poptosis in ctivted mouse mcrophges in vitro while inhiiting inos protein expression 44. Furthermore, 15dPGJ 2 induces poptosis nd suppresses djuvnt-induced rthritis in rts 45. CyPGs relesed during the resolution of inflmmtion 1 my suppress n nti-poptotic IKK-β/NF-κB pthwy, therey promoting leukocyte poptosis nd the resolution of inflmmtion. We suggest new role for NF-κB in the resolution of inflmmtion through the regultion of leukocyte poptosis. Current NATURE MEDICINE VOLUME 7 NUMBER 12 DECEMBER

6 studies on the role of NF-κB nd cypgs in the resolution of inflmmtion should yield new venues in inflmmtion reserch. The identifiction of new nti-inflmmtory pthwys is n exciting prospect. Their elucidtion will no dout provide potentil new trgets for the tretment of inflmmtory disese. Methods Crrgeenin-induced pleurisy. Mle Wistr rts (150 ± 20 g in ody weight; Tuck nd Sons, Bttlesridge, UK) were treted with 0.15 ml of 1% (w/v) λ-crrgeenin (Sigm-Aldrich, Poole, UK) injected into the pleurl cvity nd inflmmtory exudtes were collected s descried 1. All niml experimenttion ws done ccording to the pproprite regultions for the cre nd use of nimls. Mouse crrgeenin ir pouch. Three-milliliter ir pouches were rised on the dorsl surfce of femle outred Swiss lino mice (28 ± 2 g in ody weight; Tuck nd Sons) under light hlothne nesthesi, nd further 1.5 ml of ir ws injected 4 d lter. At dy 7, 0.5 ml of 1% (w/v) λ-crrgeenin ws injected into the pouch. Inflmmtory exudtes were collected s descried ove. Drug tretments. PDTC in physiologicl sline nd By (Alexis Corportion, Nottinghm, UK) in 20% (v/v) PEG 400 with 5% (w/v) BSA in physiologicl sline were dministered i.p. concurrently with crrgeenin or 24 h fter crrgeenin in the pleurisy nd 48 h fter crrgeenin in the ir pouch. NS398 (Alexis) ws dministered orlly in gum trgcnth (1% (w/v) in tp wter) t 24 h nd every 6 h therefter. MG132 (Alexis) in 10 µl DMSO ws injected into the pleurl cvity concurrently or 24 h fter crrgeenin. Control groups received the pproprite vehicle. Electrophoretic moility-shift ssy (EMSA). Leukocytes from pleurl or irpouch exudtes were lysed in uffer contining 20 mm HEPES (ph 7.9), 350 mm KCl, 1 mm MgCl 2, 0.5 mm EDTA, 20% (v/v) glycerol, 0.6% (v/v) Nonidet P-40 nd 5 mm DTT. This included cocktil of protese inhiitors consisting of PMSF (0.5 mm), leupeptin (0.5 µg/ml), protese inhiitor (0.5 µg/ml), trypsin inhiitor (1 µg/ml), protinin (0.5 µg/ml) nd esttin (40 µg/ml). NF-κB consensus (5 -AGTTGAGGGGACTTTCCCAGGC-3 ) nd mutnt oligonucleotides (5 -AGTTGAGGCGACTTT CCCAGGC-3 ) were purchsed from Snt Cruz Biotechnology (Snt Cruz, Cliforni). Oligonucleotides were end leled with [γ- 32 P]ATP (ICN Biochemicls, Oxfordshire, UK) using T4 polynucleotide kinse (Promeg, Southmpton, UK). Binding rections were prepred with 20 µg of protein extrct in inding uffer (5 mm MgCl 2, 2.5 mm EDTA, 2.5 mm DTT, 250 mm NCl, 50 mm Tris-HCl, 0.5 µg poly di dc (Becton Dickinson, Oxford, UK), 2.5% (v/v) glycerol nd 2% (v/v) Ficoll) to finl volume of 20 µl. Supershift rections included 1 5 µg of ntiserum to either RelA (P65), p50, p52, c-rel or RelB. Consensus nd mutnt competitor oligonucleotides (1.75 pmol) were dded to pproprite controls. Rections were incuted on ice for 10 min, pmol of 32 P-leled oligonucleotide ws dded nd the rections were incuted on ice for further 20 min. Binding rections were electrophoresed on 5% nondenturing polycrylmide gels nd nlyzed y utordiogrphy. Western-lot nlysis. Protein extrcts were diluted 1:1 in loding uffer (125 mm Tris-HCl (ph 7.2), 4% (w/v) SDS, 20% (v/v) glycerol, 10% (v/v) 2-mercptoethnol, 2 mm EDTA nd 1 mg/ml Coomssie lue) nd oiled for 5 min. The extrcts were then seprted (20-µg liquots) on 7.5% (inos, p100-p52, p105-50), 10% (p53, COX1, COX2, crel, RelB, p65) or 15% (IκBα, Bx) polycrylmide gels nd trnsferred to nitrocellulose memrnes. Primry ntiodies nd horserdish-peroxidse conjugted secondry ntiodies were purchsed from Snt Cruz Biotechnology. Blots were developed with chemiluminescent Luminol regent (Snt Cruz Biotechnology). DNA inding ctivity of Rel proteins ws mesured in cell extrcts y western lotting. DNA inding proteins were precipitted from µg of protein extrct with grose-conjugted κb oligonucleotides (Snt Cruz Biotechnology) in the presence of 1 µg/ml poly di dc nd western lotting crried out s ove. Assessment of leukocyte poptosis. Apoptosis ws determined using n nnexin V FITC poptosis detection kit nd viility ws ssessed y propidium iodide exclusion, done ccording to the mnufcturer s instructions (Sigm-Aldrich). Leled cells were seprted nd quntified y flow cytometry with FACScn (Becton Dickinson) nd the dt ws nlyzed using CELLQuest softwre. TGF-β1 determintion. TGF-β1 ws ssyed in cell-free inflmmtory exudtes y commercil ELISA (Quntkine TGF-β1 sndwich ELISA, R&D Systems Europe, Aingdon, UK) ccording to the mnufcturer s instructions. RNse protection ssys. Totl cellulr RNA ws isolted with Trizol regent, nd 20 µg of RNA ws used for multiproe RNse protection ssys with Rioqunt templte sets (BD Phrmingen) done ccording to the mnufcturer s instructions. RECEIVED 4 SEPTEMBER; ACCEPTED 31 OCTOBER Gilroy, D.W. et l. Inducile cyclooxygense my hve nti-inflmmtory properties. Nture Med. 5, (1999). 2. Levy, B.D., Clish, C.B., Schmidt, B., Gronert, K. & Serhn, C.N. 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