Granulocyte Colony-Stimulating Factor (Filgrastim) Treatment Primes for Increased ex Vivo Inducible Prostanoid Release

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1 Grnuloyte Colony-Stimulting Ftor (Filgrstim) Tretment Primes for Inresed ex Vivo Induile Prostnoid Relese Sonj von Aulok, Ev-Mri Boneerg, Isel Diterih, nd Thoms Hrtung Biohemil Phrmology, University of Konstnz, Konstnz, Germny Reeived August 5, 2003; epted Novemer 7,2003 ABSTRACT We investigted whether nti-inflmmtory effets of tretment with grnuloyte olony-stimulting ftor (G-CSF, filgrstim) re medited vi prostglndin E 2 (PGE 2 ) indution. In doule-lind rossover study, 10 helthy volunteers reeived 300 I1-g of filgrstim or sline 1 week prt. This ws repeted fter orl dministrtion of 50 mg of fluriprofen 1 h efore injetion. The inrese in neutrophili grnuloytes initited y G-CSF ws ugmented signifintly y fluriprofen. Lipopolyshride-indued PGE 2 nd thromoxne (TxB 2 ) relese were inresed 8 h fter G-CSF tretment. This inrese ws ro- gted y fluriprofen. However, fluriprofen did not ffet G-CSF-medited derese in tumor nerosis ftor- or interferon-)' relese. Of the volunteers treted with G-CSF, eight reported side effets (hedhe nd one pin) ginst none in the sline group. When fluriprofen ws given efore injetion, one volunteer eh reported side effets in the G-CSF nd in the sline group. These dt show tht G-CSF primes for inresed PGE 2 nd TxB 2 relese. Cyiooxygense inhiition ounterts neither the hemtopoieti nor the nti-inflmmtory tivity of G-CSF ut redues side effets. The grnuloyte olony-stimulting ftor (G-CSF) hs een ville in reominnt form s filgrstim (Neupogen) or lenogrstim (Grnoyte) for over dede. It is used in the lini to restore neutrophil ounts nd prime neutrophil funtions to protet vulnerle ptients from infetion. G-CSF is pproved for tretment ofptients undergoing hemotherpy or lso ptients with ute neutropeni due to HIV (humn immunodefiieny virus) infetion. Aprt from inresing the prodution of neutrophili grnuloytes, ontinuous G-CSF dministrtion lso inreses monoyte nd lymphoyte prodution (Hrtung et l., 1999; von Aulok et l., 2000). Also, G-CSF hs nti-inflmmtory effets on monoytes, inresing the relese of ytokine ntgonists, i.e., IL-1 reeptor ntgonist nd the solule TNF reeptors; reduing the proinflmmtory ytokine response, i.e., TNF, IL-1{3, IL-12, nd onsequently the lymphokine IFN)'; nd inresing the relese ofil-6 nd IL-8, in response to immune stimuli ex vivo (Hrtung et l., 1995, 1999; Boneerg et l., 2000). These effets my e enefiil for immunoreonstitution ofptients with HIV fter initition of highly tive ntiretrovirl therpy (von Aulok nd Hr- This study ws supported y the Germn Reserh Counil Grnt 324/1 TP2. Artile, pulition dte, nd ittion informtion n e found t DOl: jpet tung, 2002). In vitro, G-CSF dereses LPS-stimulted trnsription oftnf, ut IL-1{3 relese is modulted on the level ofpro-il-1{3 proessing (Boneerg et l., 2000; Boneerg nd Hrtung, 2002). The side effets most ommonly reported under G-CSF tretment re hedhe nd mild one pin, symptoms resemling the onset of old. However, the mehnism responsile for these side effets hs not een investigted. Beuse prostglndin (PG)E 2 is onsidered the min meditor of pin, ut lso redues the prodution of TNF on the trnsriptionl level (Sptfor et l., 1991), we onsidered it likely ndidte to medite the nti-inflmmtory effets s well s the side effets of G-CSF. Mesurement of eiosnoid relese y lood in response to stimultion hs minly een used to estlish seletivity of NSAIDs (Glser et l., 1995; Brideu et l., 1996; Riendeu et l., 2001; Blin et l., 2002). Eiosnoids regulte vsond ronhotonus, indue pin nd fever, nd hve immunosuppressive or hemotti properties. Arhidoni id relesed from the ells' memrne in response to stimultion is onverted to PGH 2 y onstitutive ylooxygense-1 nd induile ylooxygense-2 (COX, prostglndin-endoperoxide synthse). PGH 2 is then further metolized y other enzymes to prostglndins, prostylin, nd thromoxnes. We hve stndrdized method to mesure eiosnoid relese in superntnts from in vitro-stimulted whole lood ABBREVIATIONS: G-CSF, grnuloyte olony-stimulting ftor; IL, interleukin; TNF, tumor nerosis ftor-; IFNy, interferon-i'; LPS, lipopolyshride; PG, prostglndin; NSAID, nonsteroidl nti-inflmmtory drug; COX, ylooxygense; Tx8 2, thromoxne 8 2 ; LT8 4, leukotriene 8 4 ; U46.619, 9,11-dideoxy-11,9-epoxymethnoprostglndin F2. 754

2 G-CSF Primes for Prostnoid Relese 755 inutions (von Aulok et t, 2003), whih n lso e used in the lini to investigte the role ofeiosnoid relese in the ourse of diseses nd to lrify orreltions etween ytokines nd eiosnoids mesured in the sme smples. This report lso desries the indution of eiosnoids y numerous other immune stimuli. For the present study, the well estlished stimulus LPS t high onentrtion ws hosen s stndrd to ssess eiosnoid relese pity. Here, we investigte the effets of G-CSF tretment on eiosnoid relese pity nd whether these eiosnoids ply role in the modultion ofytokine relese y G-CSF y omining COX inhiition nd G-CSF tretment. Mterils nd Methods Volunteer Popultion. The study protool ws reviewed nd pproved y the institutionl review ord of the University of Konstnz. All sujets gve written informed onsent efore study entry. Ten helthy Cusin mle volunteers, ged 22 to 32 yers (28 :': 1 yers) nd weighing 77 :': 2 kg, were enrolled. The volunteers were free from signs or symptoms ofute infetious disese nd hd no history of linilly signifint disese nd took no medition for 4 weeks efore the study. Study Design. The protool ws doule-lind nd pleo-ontrolled for G-CSF tretment ut unlinded for fluriprofen tretment. Ten helthy mle volunteers were rndomized to two tretment groups, whih were rossed over for G-CSF tretment. There ws I-week wshout in etween eh ofthe four tretments. inil grde isotoni sline or 300 JLg of linil grde, pyrogen-free filgrstim (Neupogen; Amgen, Munih, Germny) ws dministered s.. t 9:00 AM in week 1 nd rossed over in week 2. This ws repeted in weeks 3 nd 4 nd dditionlly every volunteer ws given one 50-mg tlet of fluriprofen (Froen; BASF Phrm, Aott, Switzerlnd) t 8:00 AM., i.e., 1 h efore the injetion (Tle 1). Blood smples were olleted immeditely efore tretment t 8:00 AM, 9:00 AM, nd 5:00 PM on every dy of tretment nd t 9:00 AM on the following dy. The volunteers hd the right to withdrw from the study drug nd study t ny time for ny reson. Volunteers would hve een removed from the study in se of signifint protool violtion, uneptle side effets, or unrelted medil illness. No one withdrew from the study or ws removed. Differentil Blood Counts. Differentil lood ounts were routinely performed on the lood of ll volunteers to rule out ute infetions nd to ontrol for hnges in leukoyte supopultions with Pentr60 (ABX Dignostis, Montpellier, Frne). Whole Blood Inutions. Stimuli, i.e., LPS from Slmonell ortus equi or stphylool enterotoxin B (Sigm Chemie, Deisenhofen, Germny) or solvent, were dded to 1600 JLI of RPMI 1640 medium; BioWhittker, Verviers, Belgium) supplemented with 100 IUlml peniillin, 100 JLg/ml streptomyin, nd 2.5 IU/ml heprin (Liquemin; F. Hoffmnn LRohe, Grenzh-Whylen, Germny) nd 400 JLI of heprinized whole lood in 2-ml polypropylene tues mppendorf, Hmurg, Germny). In some experiments, 1 to 100 ng/ml rhg-csf (Neupogen), 33 ng/ml to 1 JLg/ml LTB 4 (Sigm Chemie), 33 nm to 1 JLM of the stle thromoxne reeptor gonist U (Pesel, Frnkfurt, Germny), or 0.02 to 10 JLg/ml PGE 2 IUpjohn, Heppenheim, Germny) ws dded to the inutions s well. After inution t 37 C nd 5% CO 2 for 24 h, ells were sedimented y entrifugtion nd superntnts were frozen in liquots t -80 C until meditor mesurement. Meditor Mesurements. Enzyme-linked immunosorent ssy ws sed on ntiody pirs ginst TNF, IL-II3, nd IFNy (Endogen, Biozol, Ehing, Germny), nd IL-6 m&d, Wiesden, Germny). Binding of iotinylted ntiody ws quntified using streptvidin-peroxidse (Biosoure, Cmrillo, CA) nd the sustrte 3,3',5,5'-tetrmethylenzidine (Sigm Chemie). Reominnt ytokines serving s stndrds were gifts from Dr. S. Poole (Ntionl Institute for Biologil Stndrds nd Controls, Potters Br, UK) (TNF, IL-ll3) or from Genzyme (Ruesselsheim, Germny) (IL-6), nd Thome (Bierh, Germny) (hu IFNy). The enzyme immunossys for PGE 2 nd TxB 2 were purhsed from Cymn (SPI Europe, Gif sur Yvette Cedex, Frne). All ssys were performed ording to mnufturers' instrutions. Sttistis. Norml distriution of dt were estlished in log trnsformed dt. Repeted mesures one-wy nlysis of vrine ws performed followed y Bonferroni's multiple omprison test (options of the GrphPd InStt 3.00; GrphPd Softwre In., Sn Diego, CA). p < 0.05 ws onsidered signifint. Dt re mens :': S.E.M. of the numer of lood donors indited lulted per milliliter of lood, i.e., orreted for the dilution ftor of 5. Results We investigted whether G-CSF ffets LPS-indued eiosnoid formtion diretly in vitro. Addition of 100 ng/ml G-CSF to 10 J.Lg/ml LPS did not influene PGEz relese, lthough TNF relese ws signifintly ttenuted (Fig. 1). Preinution ofwhole lood with G-CSF for up to 4 h yielded similr results. The relese of LTB 4 ws lso not influened y ddition of G-CSF (dt not shown). We verified tht the ddition of PGE z to LPS-stimulted lood rogtes TNF relese (LPS plus solvent: 9 ± 4 ng/ml versus LPS plus 20 ng/ml PGEz: 0.3 ± 0.3 ng/ml lood, p < 0.05l. Similrly, LPS-indued IFNy relese ws suppressed in the presene of PGEz (LPS plus solvent 66 ± 26 ng/ml versus LPS plus 20 ng/ml PGEz: 3 ± 2 ng/ml lood,p < O.Oll, wheres there ws no signifint effet on either other ytokine mesured, i.e., IL-1,B (LPS 18 ± 8 ng/ml versus LPS plus PGEz 17 ± 6 ng/mll or IL-6 (LPS 3.4 ± 0.6 ng/ml versus LPS plus PGE z 3.0 ± 0.8 ng/mll. However, stimultion of whole lood with LPS in the presene ofltb 4 (up to 1 J.Lg/mll or the stle thromoxne gonist U (up to 1 J.Lg/mll hd no signifint effet on LPS-induile TNF, IL-1,B, IL-6, or IFNy (dt not shown). Thus, lthough G-CSF in vitro did not ffet eiosnoid relese, we showed effets of PGEz similr to those of G-CSF, impliting it s potentil meditor ofthe ttenuted TNF nd IFNy prodution oserved under G-CSF tretment. To determine whether G-CSF tretment indues endogenous PGEz formtion diretly, serum levels were mesured TABLE 1 Study design of the volunteer study Week 1 Week 2 Week 3 Week 4 Group 1 (n = 5) 2 (n = 5) Pleo Filgrstim Filgrstim Pleo Fluriprofen Pleo Fluriprofen Filgrstim Fluriprofen Filgrstim Fluriprofen Pleo Isotoni sline. 300 /Lg offilg:rstim s.. 50 mg of fluriprofen p.o.

3 756 von Aulok et l.... C N I w E *** 10.0 C' -I m " E s:l ";' w C) 3 l:l..::::i 200 E ; 100 :::J_ ID Q. 11'5 ':le PGE 2 lnf Fig. 1. G-CSF does not modulte LPS-indued PGE 2 relese in vitro. Whole lood (20%) from 10 donors ws stimulted with 10 JLg/ml LPS (hthed rs) or 10 JLg/ml LPS plus 100 ng/ml G-CSF (lk rs) overnight. PGE 2 nd TNF were mesured in the superntnt. Dt re mens ± S.E.M.; n., not signifint; ***, p < efore nd up to 24 h fter tretment with 480 /Lg of G-CSF (n = 6) or pleo (n = 4) in smples from study reported previously (Boneerg et l., 2000). Serum PGEz did not differ signifintly etween the pleo or G-CSF tretment groups either efore tretment or 2, 4, 8, or 24 h fter injetion (Fig. 2; men vlues in serum without tretment were 227 ± 17 pg/md. Therefore, oth in vitro nd in vivo results onur, inditing tht G-CSF tretment does not indue physiologilly relevnt endogenous systemi PGEz relese. However, when 10 volunteers were injeted with 300 /Lg of G-CSF, ex vivo LPS-induile PGEz nd TxBzlevels in whole lood inutions were inresed out 3- nd 2-fold, respetively, ove levels indued in lood from pleo-treted volunteers 8 h fter tretment. Twenty-four hours fter tretment, induile prostnoid levels were gin similr in oth groups (Fig. 3). In the sme smples, LPS-induile TNF relese lulted per monoyte ws hlved in lood from G-CSF-treted volunteers, wheres IFN'Y relese ws redued to one-third ofpleo vlues (Fig. 3). Inidentlly, lthough LPS-induile TNF relese did not vry signifintly over the ourse of dy, IFN'Y relese ws onsistently greter in lood drwn in the fternoon ompred with lood drwn in the morning dy 1 dy 2 dy 3 time of dy Fig. 2. G-CSF dministrtion does not lter the levels of serum PGE 2 Serum levels of PGE 2 were mesured in helthy volunteers treted with 480 JLg ofg-csf (filgrstim) (n = 6) or pleo (n = 4) t the time points indited. The study design ws reported erlier (Boneerg et l., 2000). Dt re normlized to pleo (set to 100% for eh time point) ± S.E.M. There were no signifint differenes etween the two groups t ny time point. z o 500.!: 8 1Il..- :g.e OO o 0 o ,l!. 100 *** -0- pleo: PGE 2 -+-G-CSF: PGE 2..J;}-- pleo: TxB 2... G-CSF: TxB 2 O+--r---.-_,_----r,,.---r_-r--r---.-_, r_ time post injetion [h] -0- pleo: TNF -+-G-CSF: TNF..J;}-- pleo: IFNy... G-CSF: IFNy O+---.-_,_-,.,-r_-r--.--r--r---., ** time post injetion [h] Fig. 3. G-CSF tretment modultes LPS-indued prostnoid nd ytokine relese. Prostnoid (TxB 2 nd PGE 2 ) nd ytokine (TNF nd IFNy lulted per 10 6 monoytes or lymphoytes, respetively) relese in response to 10 JLg/ml LPS from S. ortus equi. Times re given reltive to injetion of 300 JLg of G-CSF (filgrstim) or pleo. Initil vlues re set to 100%, vlues re mens ± S.E.M.; **, p < 0.01; ***, p < In the seond hlfofthe tretment study, one 50-mg tlet of fluriprofen ws given 1 h efore G-CSF or pleo injetion. Blood smples were tken immeditely efore dministrtion of fluriprofen nd immeditely efore dministrtion of G-CSF or pleo 1 h lter nd 8 h fter injetion of G-CSF or pleo, i.e., 9 h fter dministrtion of fluriprofen. Figure 4 onfirms tht fluriprofen is very effetive in reduing LPS-indued PGEz nd TxBz relese within 1 h of ingestion. The oservtion tht the vlues efore ny tretment (t = -1 h) in the group reeiving fluriprofen nd pleo were higher thn in the other groups is result of dy-to-dy vrition. This stresses further the strength ofthe effet, i.e., the differene is even greter when the prostnoid vlues indued efore tretment (t = -1 h) re ompred with those 1 h fter ingestion (t = 0 h) in tht group. This effet ws still in ple 9 h fter ingestion of the tlet, i.e., 8 h fter pleo or G-CSF injetion. Twenty-four hours fter ingestion of fluriprofen, LPS-induile PGEz levels were still deresed ompred with strting vlues, lthough TxBz levels hd returned to pretretment vlues. Although prostnoid relese ws ttenuted when fluriprofen ws given efore G-CSF injetion, TNF relese per monoyte nd IFN'Y relese per lymphoyte remined signifintly elow pleo vlues t levels equl to vlues ttined under G-CSF tretment without prior fluriprofen tretment (Fig. 5) even though initil LPS-induile IFN'Y relese t t = -1 hnd t = 0 h ws signifintly greter in

4 G-CSF Primes for Prostnoid Relese = 150 E W Dpleo DG-CSF _ f1uriprofen + pleo f1uriprofen + G-CSF ## -1 h 0 h 8 h time reltive to G-CSF or pleo injetion *** 75 'iii' g U. 0 E <D o 25 o 60 CJpleo CJG-CSF _f1uriprofen + pleo m f1uriprofen + G-CSF :.! e u. Co en :e ::;, I;: ### ; w_ -1h Oh 8h time reltive to injetion = 300.E re 200 I- 100 O..u...u h 0 h 8 h time reltive to G-CSF or pleo injetion G-CSFor fluriprofen pleo IO l_oo_dt+ _IOO_d!.. -1h Oh 8h Fig. 4. Fluriprofen tretment prevents G-CSF-dependent priming for prostnoid relese. PGEz () nd TxBz () relese in response to 10 flg/ml LPS from S. ortus equi., tretment sheme. Vlues re mens ± S.E.M.; #, p < 0.05; ##, p < 0.01; ###, p < reltive to pleo without fluriprofen; ***, p < reltive to G-CSF without fluriprofen. the group reeiving fluriprofen nd G-CSF ompred with the other groups. Interestingly, the omintion of fluriprofen with G-CSF tretment did not ountert G-CSF-indued leukoytosis, ut insted tully dded signifintly to the effet. The differentil lood ell ounts reveled tht fluriprofen hd further inresed the reruitment of neutrophili grnuloytes, inditing tht the prostnoids ply role in ontrolling neutrophil prodution negtively (Fig. 6). A rryover effet from the first G-CSF tretment on the leukoyte ounts ws exluded y the differentil lood ell ount. However, it nnot e ruled out tht the greter effiy of the seond G-CSF injetion 2 weeks fter the first tretment ould e due to lrger mrrow pool of neutrophils. On the other hnd, we hve no inditions for suh effets from previous study in whih volunteers reeived G-CSF tretment on weekly sis (von Aulok et l., 2000). Volunteers were sked out side effets on the dy fter every tretment. Eight of 10 volunteers reported hedhe or one pin fter G-CSF tretment ginst zero omplints from the pleo-treted volunteers. However, when volunteers reeived fluriprofen efore G-CSF injetion, one vol- (ij' II! g 40 : ;>-:: $ z Co e u.!!:: 130 Co en '" :e 0 ::;, q::: C h Oh 8h time reltive to injetion fluriprofen G-CSFor pleo l0j.+ lood,. lood!.. -1 h Oh 8h Fig. 5. Fluriprofen tretment does not ffet modultion of ytokine relese y G-CSF tretment. TNF () nd IFNy () relese in response to 10 flg/ml LPS from S. ortus equi., tretment sheme. Vlues re mens ± S.E.M.; #, p < 0.05; ###, p < reltive to pleo without fluriprofen; ***, p < reltive to G-CSF without fluriprofen. unteer reported side effets in oth the pleo nd the G-CSF-treted group. It seems tht the side effets ommonly ssoited with G-CSF tretment re medited y eiosnoids nd n e prevented effetively y ylooxygense inhiition without interfering negtively with neutrophil reruitment or with the nti-inflmmtory effets of G-CSF tretment. Disussion The results presented in this rtile demonstrte tht there is signifint disrepny etween the effets of G CSF in vitro in omprison to ex vivo with regrd to the modultion of prostnoid relese. Inution of whole lood with G-CSF in vitro dereses LPS-stimulted relese of TNF or IFNy s n lso e seen in lood from G-CSFtreted volunteers even 24 h fter tretment. However, whole lood preinuted with G-CSF in vitro ws not le to ring out the priming for eiosnoid relese tht ould e oserved in ex vivo-stimulted lood from G-CSF-treted volunteers. Apprently, the ells reruited y G-CSF from the one mrrow differ in their hrteristis ompred with the more mture popultion found in the lood of un-

5 758 von Aulok et l Q.S1 =" eu.. l!?,.q-ci) ' 20 -et? ==.g(!) T"'" :2- Q. Q) 10 Ul :[ '" 1.0 O o:s E p1eo... G-CSF -o-fluriprofen + pleo -fr-fluriprofen + G-CSF O-f--,---,---r---r--r-.--,--,---,---,---,---r--,- time reltive to injetion [h] O+-..,.--,-..,...-,----r----r--,---,r ,.--,- time reltive to injetion [11] O.O+--,---.--r--r--r-,--.,-..,.--,---r---r--r--,r- time reltive to injetion [11] Fig. 6. Fluriprofen tretment ugments G-CSF-indueed grnulopoiesis. Whole lood eel! eount (), differentil neutrophili. grnuloeyte eount (), nd monoeyte eount (e) in lood from volunteers. One 50-mg tlet of fluriprofen ws dministered t t = 1 hnd 300 flg of G-CSF (filgrstim) or pleo ws injeted t t = 0 h s indieted. All omintions of pleo versus G-CSF t 8 nd 24 h were highly signifintly different (p < 0.001J. Vlues re mens ± S.E.M.; *,p < 0.05; **,p < 0.01 reltive to G-CSF without fluriprofen. treted donors. It seems unlikely tht the inresed prostnoid levels indued in lood from G-CSF-treted volunteers on stimultion merely reflet inresed popultions of the produing ells, euse the priming n only e oserved 8 h fter G-CSF tretment nd seline vlues re hieved 24 h fter tretment, wheres the ell ounts still signifintly inresed during this time period. This oservtion poignntly illustrtes tht not ll effets of G-CSF n e modeled in vitro. Although the ex vivo effets of G-CSF on TNF formtion ould lso e modeled in vitro, this did not trnslte to its effets on eiosnoids. This suggests tht G-CSF tretment reruits different popultion of peripherl leukoytes. The lk of effet of G-CSF on the serum levels of PGE 2 suggests tht the nture of the effet of G-CSF tretment is limited to priming for inresed response to inflmmtory stimuli, nd not to diret tivtion of ells. Even t higher dose thn given in the min study, there ws no effet on PGE 2 levels; in ft, the tendeny towrd lower PGE 2 levels ompred with the pleo group nd to the vlues efore G-CSF tretment does not give rise to expettions tht this would e different with even lower doses of G-CSF. It is not ler whih ells in the lood re the min produers of prostnoids. TxB 2 nd PGE 2 n e relesed y oth monoytes nd neutrophils (Nihols et I., 1987; Doerflet' et I., 1989; Nusing et I., 1990; Zheng et I., 1990; Juergens et I., 1992; Sreks et I., 1993; Ptrignni et I., 1994; Aiiki nd Cook, 1997), lthough this ws not found for oth popultions y ll investigtors. Furthermore, pltelets, whih were lso present in the whole lood inutions, express the highest mounts ofthromoxne synthse (Nusing et I., 1990) nd n relese TxB 2, not only in response to ogultion, whih ws prevented here with heprin, ut lso in response to inflmmtory stimultion (Sreks et I., 1993; Rele et I., 1996). In ontrst, intrellulr ytokine stining of LPS-stimulted whole lood showed tht TNF is produed exlusively y monoytes CBoneerg nd Hrtung, 2002) in this experimentl setup. Similrly, IFN'Y is lerly produed only y lymphoytes (Okmur et I., 1998; Boneerg et I., 2000), llowing reltion of the relese of these ytokines to the produing ell numers nd ounting for the hnges in differentil lood ell ounts due to the tretment. PGE 2 ws le to inhiit LPS-indued TNF s wehs IFN'Y relese in vitro, mking it ndidte meditor of the nti-inflmmtory effets of G-CSF in vivo. The strong inrese in LPS-indued PGE 2 nd TxB 2 indution under G CSF tretment ws lredy relized 8 h fter tretment. However, effetive inhiition ofprostnoid prodution y dditionl dministrtion of fluriprofen did not restore LPSindued proinflmmtory ytokine relese. The similrity in the effets of PGE 2 nd G-CSF in reduing the relese of TNF nd IFN'Y prompted the hypothesis of n indution of PGE 2 y G-CSF. However, the nti-inflmmtory effets of G-CSF were not ttenuted y NSAID in vitro (Fig. 1) or ex vivo (Fig. 41. These results prove the hypothesis, tht the nti-inflmmtory effets ofg-csf require prostnoid medition, flse. Insted, the redution in the relese of proinflmmtory ytokines seems to e diret effet of G-CSF on monoytes, whih rry funtionl G-CSF reeptor CBoneerg et I., 2000). Although fluriprofen dministrtion lone hd no signifint effets on the whole lood ell ount, it signifintly ugmented the hemtopoieti effets of G-CSF y reruiting dditionl neutrophils. This oservtion sustntites previous oservtions in rodent ells ttriuting grnuloyte olony-inhiitory tivity to PGE 2 (Gentile nd Pelus, 1988; Sntngelo et I., 2000). A mehnism explining this oservtion hs not yet een put forwrd; likely explntion is tht the endogenously formed prostnoid regultes hemtopoiesis negtively. It lso remins to e tested whether the omintion of G-CSF with NSAIDs ould further inrese grnulopoiesis in i! 4 led I "

6 G-CSF Primes for Prostnoid Relese 759 leukopeni ptients. Pin mngement is ommonly prt of the tretment of these ptients; therefore, this question might e nswered y retrospetive nlysis of linil dt. It seems tht PGE 2 my ntgonize the grnulopoieti effets ofg-csf to prevent exessive neutrophil reruitment. Although we found no differene in the PGE 2 serum levels of G-CSF-treted volunteers fter G-CSF dministrtion, the time points were possily hosen too erly nd too lte euse the reported side effets usully ourred within 8 to 16 h of dministrtion. However, serum smples from former G-CSF tretment study with higher resolution lso showed no elevtion of serum PGE 2. On the other hnd, PGE 2 relese in vivo might not e systemi nd might only work lolly within the one mrrow. However, the type of side effets reported, i.e., hedhe nd one pin, whih re onsistently reported under G-CSF tretment, do support the hypothesis tht PGE 2 my e indued diretly in vivo y high onentrtions ofg-csf s negtive feedk. The effetiveness ofprior fluriprofen dministrtion in preventing the side effets ssoited with G-CSF dministrtion further supports this onept. Together, these dt show tht G-CSF primes for inresed PGE 2 nd TxB 2 relese ex vivo. Cylooxygense inhiition ounterts neither the hemtopoieti nor the ntiinflmmtory tivity of G-CSF ut prevents ssoited side effets. Aknowledgments We thnk Stefn Fennrih, Corinn Hermnn, Psl Renner, Susnne Deininger, nd Lrs Hreng for prompt help. Referenes Aiiki M nd Cook JA (997) Ulinsttin, humn trypsin inhiitor, inhiits endotoxin-indued thromoxne B2 prodution in humn monoytes. Crit Cre Med 25: Blin H, Boileu C, Lpique F, Nedele E, Loeuille D, Guillume C, Guher A, Jendel C, Netter P, nd Jouzeu JY (2002) Limittion ofthe in vitro whole lood ssy for prediting the COX seletivity of NSAIDs in linil use. Br J in Phrmol 53: Boneerg EM, Hreng L, Gntner F, Wendel A, nd Hrtung T (2000) Humn monoytes express funtionl reeptors for grnuloyte olony-stimulting ftor tht medite suppression ofmonokines nd interferon-gmm. Blood 95: Boneerg EM nd Hrtung T (2002) Grnuloyte olony-stimulting ftor ttenutes LPS-stimulted IL-Iet relese vi suppressed proessing of proil-let, wheres TNF-lph relese is inhiited on the level of protnf-lph formtion. Eur J lmmunol 32: Brideu C, Krgmn S, Liu S, Dllo AL, Ehrih EW, Rodger lw, nd Chn CC (1996) A humn whole lood ssy for linil evlution ofiohemil effiy of ylooxygense inhiitors. lnflmm Res 45: Doerfler ME, Dnner RL, Shelhmer JH, nd Prrillo JE (1989) Bterillipopoly- shrides prime humn ne.utrophils for enhned prodution of leukotriene B4. J in lnvestig 83: Gentile PS nd Pelus LM (1988) In vivo modultion ofmyelopoiesis y prostglndin E2. IV. Prostglndin E2 indution ofmyelopoieti inhiitory tivity. J Immunol 141: Glser K, Sung ML, O'Neill K, Belfst M, Hrtmn D, Crlson R, Kreft A, Kurk D, Hsio CL, nd Weihmn B (1995) Etodol seletively inhiits humn prostglndin GIB synthse 2 (PGHS-2) versus humn PGHS-1. Eur J Phrmol 281: Hrtung T, Doke WD, Gntner F, Krieger G, Suer A, Stevens P, Yolk HD, nd Wendel A (1995) Effet of grnuloyte olony-stimulting ftor tretment on ex vivo lood ytokine response in humn volunteers. Blood 85: Brtung T, Doeke WD, Bundshuh D, Foote MA, Gntner F, Hermnn C, Lenz A, Milwee S, Rih B, Simon B, et l. (1999) Effet of filgrstim tretment on inflmmtory ytokines nd Iymphoyte funtions. in Phrml Ther 66: Juergens UR, Christinsen SC, Stevenson DD, nd Zurw BL (1992) Arhidoni id metolism in monoytes of spirin-sensitive sthmti ptients efore nd fter orl spirin hllenge. J Allergy in Immunol 90: Nihols FC, Shenkein HA, nd Rutherford RB (987) Prostglndin E2, prostglndin El nd thromoxne B2 relese from humn monoytes treted with C3 or teril lipopolyshride. Biohim Biophys At 927: Nusing R, Lesh R, nd Ullrih V (19901 Immunohistohemil loliztion ofthromoxne synthse in humn tissues. Eiosnoids 3: Okmur H, Kshiwmur S, Tsutsui H, Yoshimoto T, nd Nknishi K (1998) Regultion of interferon-gmm prodution y nd IL-18. Curr Opin lmmunol 10: Ptrignni P, Pnr MR, Greo A, Fuso 0, Ntoli C, IoeIli S, Cipollone F, Gni A, Creminon C, Mlouf J, et l. (1994) Biohemil nd phrmologil hrteriztion of the ylooxygense tivity of humn lood prostglndin endoperoxide synthses. J Phrmnl Exp Ther 271: Rele M, Brne RC, Fryds S, Anoginkis G, TrkteIlis A, Dimitridou D, Vlis D, Plido FC, De Fzio P, Porre E, et l. (1996) Humn reominnt interleukin-l et indues thromoxne A2 relese in polymorphonuler leukoytes, mrophges nd pltelets: effet of reeptor ntgonist. Mol Cell Biohem 159: Riendeu D, Perivl MD, Brideu C, Chrleson S, Due D, Ethier D. Flgueyret JP, Friesen RW, Gordon R, Greig G, et l. (2001) Etorioxi (MK-0663): prelinil profile nd omprison with other gents tht seletively inhiit ylooxygense-2. J Phrmol Exp Ther 296: Sreks V, Riutt A, Muh I, Alnko J, nd Vptlo H (1993) Niotine nd otinine modulte eiosnoid prodution in humn leukoytes nd pltelet rih plsm. Eur J Phrml 248: Sntngelo S, Shoup M, Gmelli RI., nd Shnkr R (2000) Prostglndin E2 reeptor ntgonist (SC-19220) tretment restores the lne to one mrrow myelopoiesis fter urn sepsis. J Trum 48: ; disussion, Sptfor M, Chippr G, D'Amio D, Volpes D, Melis M, Pe E, nd Merendino AM (1991) Prostglndin E2 down-regultes the expression of tumor nerosis lph gene y humn lood monoytes. Adv Prostglndin Thromoxne Leukot Res 21B: von Aulok S, Boneerg EM, nd Hriung T (2000) Intermittent G-CSF (filgrstim) tretment nnot indue lymphoytosis in volunteers. in Phrmol Ther 68: 104. von Aulok Snd Hrtung T (2002) Potentil for immune reonstitution through G-CSF tretment of HIV ptients. Arh lmmunol Ther Exp 50: von Aulok S, Hermnn C, nd Hrtung T (2003) Determintion of the eiosnoid response to inflmmtory stimuli in whole lood nd its phrmologil modultion ex vivo. J lmmunol Methods 277: Zheng H, Crowley JJ, Chn JC, Hoffmnn H, Htherill,TR, Ishizk A, nd Rffin TA (1990) Attenution oftumor nerosis ftor-mdued endothelil ell ytotoxiity nd neutrophil hemiluminesene. Am Rev Respir Dis 142: Address orrespondene to: Dr. Thoms Hrtung, Biohemil Phrmology, University of Konstnz, p.. Box M655, Konstnz, Germny. E-mil: thoms.hrtung@uni-konstnz.de