After beats by dre solo hd injection of il8 or groto

And growth chpbtsbydreuk related oncogene beats by dre online shop groagr in neutrophil infiltration Top of pAgeABStrActinterleukin8(Il8:Cxcl8)And Growth relAted oncogene(Gro:Cxcl1)Are memBerS of the cxc chemokineS.In the preSent Study, we explored the functionAl diStinction Between theSe cxc chemokineS in the regulAtion of neutrophil infiltrAtion.Injection of either rABBit Il8 or Gro(10 g each)Into rABBit knee jointS reSulted in A mASSive neutrophil infiltrAtion in the jointS.At their peAk time point(6 hourS), the numBer of neutrophilS induced By Il8 wAS more thAn thAt induced By Gro.EAch chemokine induced the other chemokine in the jointS.Tnf Activity wAS induced in the jointS After AdminiStrAtion of Gro, But not Il8.TreAtment with AntiGro mAB And/or AntiTnf mAB fAiled to inhiBit Il8induced neutrophil infiltrAtion.In contrASt, either AntiIl8 igg or AntiTnf mAB decreASed Groinduced reSponSe, And the inhiBition wAS further enhAnced By coAdminiStrAtion of theSe AntiBodieS.ThuS, it AppeArS thAt Il8 ActS directly, whereAS Gro ActS indirectly, in pArt, on neutrophil infiltrAtion.The diStinct difference in Tnf production Between Il8 And Gro wAS further inveStigAted.In vitro, Gro induced Tnf Activity in cultured SynoviAl cellS, the cellS producing Tnf in the jointS After Groinjection.However, Il8 fAiled to produce Tnf Activity from the cellS, Although equivAlent levelS of the mrnA expreSSion were induced By Il8 AS compAred with Gro.When recomBinAnt rABBit Tnf wAS incuBAted with SynoviAl fluidS oBtAined At2 hourS After Il8 injection, the reSultAnt Tnf Activity wAS SignificAntly decreASed, An event thAt wAS completely reStored By A Serine proteASe inhiBitor, phenylmethylSulphonyl fluoride(PmSf).Furthermore, Tnf Activity wAS unveiled in the jointS when Il8 wAS intrAArticulArly injected with pmSf.TheSe dAtA SuggeSt thAt Tnf iS degrAded By Serine proteASe(S)In the cAse of Il8.TAken together, the dAtA cleArly demonstrAte the functionAl distinction Between Il8 And Gro, which mAy influence the inflAmmAtory responses.Keywords:15,16,25Top of pAgeIntroductionNeutrophil is the first cell populAtion to migrAte into inflAmmAtory foci.MediAtors regulAting neutrophil recruitment hAve Been extensively studied, And AccumulAting evidence suggests thAt the cxc chemokines plAy criticAl roles in this process(BAggiolini et Al,1995;Rollins,1997).The cxc chemokines Are A structurAlly relAted And functionAlly redundAnt fAmily of proteins with specific leukocyte chemotActic Activity thAt cAn Be mAinly divided into two suBsets BAsed on the presence or ABsence of specific Amino Acid residues, gluleuArg(Elr) (BAggiolini et Al,1997;Zlotnik And yoshie,2000).The elr cxc chemokines AttrAct neutrophils, whereAs nonelr cxc chemokines Are chemotActic for t lymphocytes And nk cells.Interleukin8(Il8:Cxcl8)And GrowthrelAted oncogene(Gro:Cxcl1)Are memBers of elr cxc chemokines.These chemokines hAve Been detected in A vAriety of clinicAl diseAses thAt Are chArActerized By A mAssive focAl neutrophil infiltrAtion, including Acute respirAtory distress syndrome, BActeriAl pneumoniA, BActeriAl meningitis, And rheumAtoid Arthritis(Kunkel et Al,1995;Luster,1998).We hAve recently shown thAt il8 And gro Are essentiAl in the recruitment of neutrophils At the site of inflAmmAtion.EquivAlent levels of il8 And gro were detected in AnimAl models of inflAmmAtion where neutrAlizAtion of il8Or growith their specific AntiBodies resulted in decreAsed numBer of infiltrAting neutrophils(Fukumoto et Al,1998;MAtsukAwA et Al,1998,1999;Mo et Al,1999,2000).The inhiBition wAs further enhAnced when Both il8 And gro were neutrAlized(MAtsukAwA et Al,1999).In vitro, il8 And gro hAve Been shown to Be equAlly chemotActic for neutrophils(BAlentien et Al,1990).Thus, it AppeArs thAt il8 And gro Are equipotent neutrophil chemoAttrActAnts.Questions remAin ABout the relAtionship And the functionAl distinction Between these cxc chemokines.To Address these points, we injected either rABBit il8Or grointo rABBit knee joints And AnAlyzed the suBsequent inflAmmAtory responses in the joints.Our dAtA suggest suBstAntiAl differences Between these closely relAted cxc chemokines, especiAlly with respect to endogenously produced cytokines And their pArticipAtion in the recruitment of neutrophils.Top of pAgeresultskinetics of il8 or groinduced leukocyte infiltrAtioninjection of either il8Or grointo rABBit knee joints induced A mAssive neutrophil infiltrAtion into the joints.In either cAse, the numBer of neutrophils peAked At6 hours, decreAsed grAduAlly, And suBsided At 36 hours After the injection(Fig.1).When compAred At their peAk(6 hours After the injection), the numBer of infiltrAting neutrophils induced By IL8 wAs more thAn thAt induced By GRO(P 94% mAcrophAges)Induced By eAch chemokine wAs much less thAn thAt of neutrophils.The AccumulAtion of mononucleAr cells wAs first noted At6 hours And peAked At24 hours After the injection(Fig.1).At indicAted intervAls After intrAArticulAr injection of il8(Left pAnel)Or gro(Right pAnel) (10 g each), the numBers of infiltrAting neutrophils(And mononucleAr cells()In the joints were recorded.VAlues represent the meAn se of 9 estimAtions from sepArAte joints.The dotted line shows the BAckground for vehicletreAted joints.Full Figure And legend(44K)Cytokine production After beats by dre solo hd injection of il8 or groto exAmine if il8 And gro could induce other cytokines, levels of endogenously produced cytokines were meAsured After the injection of these chemokines.IntrAArticulAr injection of il8 resulted in An increAse in the synoviAl level of gro. The Gro level peAked At2 hourS, decreASed grAduAlly, And then increASed AgAin At12 hourS After the injection (Fig.2). Injection of Gro induced the production of Il8, the level of which peAked At2 And12 hourS After the injection (Fig.2). Il8Or gro(10 g each)WAs injected into knee joints.At indicAted intervAls After the injection, synoviAl fluids were hArvested And concentrAtions of endogenous gro or il8 in the joints were meAsured, respectively.VAlues represent the meAn se of 9 estimAtions from sepArAte joints.Below detection level.Full Figure And legend(32K)Tumor necrosis fActor(Tnf)Is regArded As the first cytokinetriggering inflAmmAtory response(DriScoll et Al,1995).We next exAmined tnf Activity in the joints After the injection ofEither il8or gro.As shown in Figure3A, no tnf Activity wAs detected After il8 injection.However, injection of gro induced A significAnt level of tnf Activity in the joints. Tnf Activity wAS meASurABle even At1 hour, increASed rApidly And peAked At2 hourS After the injection (Fig.3A). When AntiTnf mAB wAS coinjected with Gro, the Groinduced Il8 level At12 hourS, But not2 hourS, wAS inhiBited By 50% (Fig.3B).Thus, il8 And gro Are eAch cApABle of producing the other chemokine when they Are injected into the joints.Gro, But not il8, induces tnf Activity, which in turn contriButes to the production of il8 in A lAte phAse.There were no detectABle levels of il8, gro, And tnf in sAlinetreAted controls(DAtA Not shown). A, Il8Or gro(10 g each)WAs injected into knee joints.At indicAted intervAls After the injection, synoviAl fluids were hArvested And tnf Activity in the joints wAs meAsured.VAlues represent the meAn se of 9 estimAtions from sepArAte joints.Below detection level.B, gro(10 g)WAs injected into knee joints with AntiTnf mAB (10 g).NormAl mouse igg1 wAs used As A control. At2 And12 hourS After the injection, SynoviAl fluidS were hArveSted And the concentrAtion of Il8 wAS meASured.VAlues represent the meAn se of 8 estimAtions from sepArAte joints.Full Figure And legend(37K)Neutrophil infiltrAtion After neutrAlizAtion of the endogenous cytokinesto investigAte the involvement of endogenously produced cytokines in the neutrophil influx, Antiil8 igg, Antigro mAB, or Antitnf mAB were injected together with il8 or gro.Il8 or groinduced neutrophil infiltrAtion wAs completely Blocked By the coAdministrAtion of Antiil8 igg or Antigro mAB, respectively(Not shown).The dAtA in Figure 4A show thAt il8induced neutrophil infiltrAtion wAs not Altered By the AdministrAtion of Antigro mAB And/or Antitnf mAB At Any time point exAmined. In contrASt, Groinduced neutrophil infiltrAtion After 9 hourS wAS SignificAntly inhiBited By the treAtment with AntiIl8 IgG, Although there were no chAngeS At2 to6 hours After the injection (Fig.4B).When Antitnf mAB wAs injected with gro, the neutrophil infiltrAtion After 4 hours wAs significAntly inhiBited By 30% to 50%.CoAdministrAtion of Antiil8 igg And Antitnf mAB further enhAnced the inhiBition(Fig.4B).These dAtA indicAte thAt il8 Acts directly on neutrophils, whereAs gro AttrActs neutrophils, in pArt, viA the production of tnf And il8.Il8 gro does not AppeAr to Be effective for AttrActing neutrophils.Either il8(A)Or gro(B) (10 g each)WAs injected into knee joints with10 g of AntiTnf mAB, AntiGro mAB, or AntiIl8 IgG, Alone or in comBinAtion. At indicAted intervAlS After the injection, the numBers of infiltrAting neutrophilswere counted.VAlues represent the meAn se of 8 estimAtions from sepArAte joints.P mab alone.Full Figure And legend(56K)Mrna expression for tnf by il8 and groto further examine whether il8 is incapable of inducing tnf, a cellular source of tnf was next examined in vivo and the tnfproducing capability by the cells was investigated in vitro.As shown in Figure 5a, tnf activity in the joints after gro injection was similar in normal and in neutrophildepleted rabbits, suggesting that resident cells in the joints were responsible for tnf production.Immunohistochemistry demonstrated that synovial cells were stained positively for tnf(Fig.5B). Therefore, SynoviAl cellS hArveSted from heAlthy rABBitS were cultured And StimulAted withEither il8or Gro.A, gro(10 g)WAs injected into knee joints of normAl or neutrophildepleted rABBitS. At2 hourS After the injection, SynoviAl fluidS were hArveSted And Tnf Activity wAS meASured.VAlues represent the meAn se of 9 estimAtions from sepArAte joints. B, SynoviAl tiSSueS oBtAined At2 hourS After Gro injection (10 g)Were stained with antitnf igg.Preimmune goat serum was used as a control. OriginAl mAgnificAtion1, 000.Full Figure And legend(22K)The dAtA Shown in Figure6 indicAted thAt Il8 fAiled to induce Tnf Activity in cultured SynoviAl cellS even At A concentrAtion AS high AS1 g/ml.On the other hand, gro dosedependently induced tnf activity(Fig.6).Heatinactivated gro(1 g/ml,100 C for 5 minuteS)Did not induce the activity(Not shown).Mrna expression for tnf in synovial cells was next examined by semiquantitated rtpcr, which showed that il8 induced equivalent levels of the mrna expression in synovial cells as compared with gro(Fig.7, A and B), despite the absence of TNF activity in the culture supernatants.Thus, it is likely that tnf may be inactivated in case of il8 stimulation. A, SynoviAl cellS hArveSted from knee jointS of A heAlthy rABBit were StimulAted with1 g/ml Il8Or grofor1 hour At 37 C.Rna was extracted from the cells and mrna expression for tnf was examined by rtpcr.Expression of gapdh was used as a control.Shown are representative photographs of three individual experiments.B, ethidium bromide stained pcr products were photographed, and then the images were digitized and analyzed.Results are expressed as a ratio of each pcr product/gapdh band density.Values represent the mean se of six different estimations of pcr data.Pcr was performed in duplicate for each experiment.

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