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Cytochalasin B triggers a novel pertussis toxin sensitive pathway in TNF-alpha primed neutrophils Bylund, Johan; Pellmé, Sara; Fu, Huamei; Mellqvist, Ulf-Henrik; Hellstrand, Kristoffer; Karlsson, Anna; Dahlgren, Claes May 24, 2004

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ralssBioMed CentBMC Cell BiologyOpen AcceResearch articleCytochalasin B triggers a novel pertussis toxin sensitive pathway in TNF-alpha primed neutrophilsJohan Bylund1,2, Sara Pellmé1, Huamei Fu1, Ulf-Henrik Mellqvist3, Kristoffer Hellstrand4, Anna Karlsson1 and Claes Dahlgren*1Address: 1Department of Rheumatology and Inflammation Research, University of Göteborg, Göteborg, Sweden, 2Department of Paediatrics, University of British Columbia, BC Research Institute for Children's and Woman's Health, Vancouver, British Columbia, Canada V5Z 4H4, 3Department of Hematology, University of Göteborg, Göteborg, Sweden and 4Department of Virology, University of Göteborg, Göteborg, SwedenEmail: Johan Bylund - jbylund@interchange.ubc.ca; Sara Pellmé - Sara.Pellme@microbio.gu.se; Huamei Fu - Huamei.Fu@microbio.gu.se; Ulf-Henrik Mellqvist - Ulf-Hendrik.Mellqvist@medic.gu.se; Kristoffer Hellstrand - Kristoffer.Hellstrand@microbio.gu.se; Anna Karlsson - Anna.Karlsson@microbio.gu.se; Claes Dahlgren* - Claes.Dahlgren@microbio.gu.se* Corresponding author    cytokinessuperoxideprimingTNFcytoskeletonreceptor reactivationpertussis toxinG. proteinGPCRNADPH-oxidaseAbstractBackground: Cytochalasin B does not directly activate the oxygen-radical-producing NADPHoxidase activity of neutrophils but transfers desensitized G-protein coupled receptors (GPCR) intoan active signaling state by uncoupling GCPR from the cytoskeleton. The receptor uncouplingresults in respiratory burst activity when signals generated by reactivated formyl peptide receptorstrigger the NADPH-oxidase to produce superoxide anions.Results: Tumor necrosis factor alpha (TNF-alpha) primes neutrophils for subsequent activation bycytochalasin B. Pretreatment with TNF-alpha induced mobilization of receptor-storing neutrophilorganelles, suggesting that receptor up-regulation significantly contributes to the response, but thereceptor mobilization was not sufficient for induction of the cytochalasin B sensitive state. TheTNF-alpha primed state resembled that of the desensitized non-signaling state of agonist-occupiedneutrophil formyl peptide receptors. The fact that the TNF-alpha primed, cytochalasin B-triggeredactivation process was pertussis toxin sensitive suggests that the activation process involves aGPCR. Based on desensitization experiments the unidentified receptor was found to be distinctfrom the C5a receptor as well as the formyl peptide receptor family members FPR and FPRL1.Based on the fact the occupied and desensitized receptors for interleukin-8 and platelet activatingfactor could not be reactivated by cytochalasin B, also these could be excluded as receptorcandidates involved in the TNF-alpha primed state.Conclusions: The TNF-alpha-induced priming signals could possibly trigger a release of anendogenous GPCR-agonist, amplifying the response to the receptor-uncoupling effect ofcytochalasin B. However, no such substance could be found, suggesting that TNF-alpha can transferG-protein coupled receptors to a signaling state independently of agonist binding.Published: 24 May 2004BMC Cell Biology 2004, 5:21Received: 13 March 2004Accepted: 24 May 2004This article is available from: http://www.biomedcentral.com/1471-2121/5/21© 2004 Bylund et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.Page 1 of 14(page number not for citation purposes)BMC Cell Biology 2004, 5 http://www.biomedcentral.com/1471-2121/5/21BackgroundHuman neutrophil granulocytes constitute an importantpart of the innate immune defense against microbialinfections, and the bactericidal activities performed bythese cells rely on their interaction with chemoattractants,cytokines and other inflammatory mediators [1]. The che-moattractants, including C5a, platelet activating factor(PAF), interleukin-8 (IL8) and formylated peptides, bindto specific receptors [2,3], all of which belong to a familyof transmembrane G-protein coupled receptors (GPCRs).Activation of these receptors leads to directed migration,granule mobilization and activation of the neutrophilNADPH-oxidase [2]. The reactive oxygen species gener-ated by the oxidase are of importance for microbial killingand for cell-cell-signaling [4].Tumor necrosis factor-alpha (TNF-α) is one of the earliestcytokines produced at inflammatory sites by activatedmonocytes and macrophages. This cytokine affects neu-trophil function mainly through binding to type I TNFreceptor (TNFR1) [5]. The TNFR1 is a single transmem-brane glycoprotein with several intracellular motifs withknown functional significance, but it is not linked to anysignaling G-protein [5-7]. Phosphorylation of TNFR1occurs at a consensus MAPK site on its cytoplasmicdomain or through tyrosine phosphorylation [6,7],although it is not fully understood how this phosphoryla-tion control receptor signaling or processing.The biological effects of TNF-α on neutrophil functions invitro vary, as illustrated by the ability or inability of TNF-αto affect the neutrophil oxygen radical producingNADPH-oxidase. In order for TNF-α to trigger neutrophilsuperoxide production, cells need to adhere to a solid sur-face, and the magnitude of the response is determined bywhich protein that is coated on the surface [8]. TNF-α onlyweakly triggers the oxidase when the neutrophils are insuspension [8]; however, after exposure to TNF-α, thesecells are primed with respect to NADPH-oxidase activa-tion in response to other stimuli [9]. Thus, while TNF-αper se does not activate the NADPH-oxidase to any signif-icant extent in nonadherent neutrophils, it induces a stateof hyper-responsiveness to other stimuli.Several mechanisms have been proposed to account forneutrophil priming [10-14], including receptor mobiliza-tion from intracellular granule stores [15-17]. The aim ofthis study was to characterize the primed state induced inhuman neutrophils by TNF-α, using an earlier describedreceptor uncoupling system [18]. We found exposure ofnew receptors to be a part of the priming process, butmore importantly we found that neutrophils interactingwith TNF-α were transferred into a novel state, in whichsimilarities with that of neutrophils that have their formylpeptide GPCRs desensitized by a specific receptor agonist[18]. Isomerization of GPCRs, from an inactive to anactive state, occurs normally as a result of ligand bindingbut can also occur independently of agonist [19] and ourfindings are suggestive of a TNF-α induced novel activa-tion mechanism that is receptor agonist-independent.ResultsTNF-α primes the neutrophil NADPH-oxidase response to a subsequent stimulation/triggering with cytochalasin BCytochalasin B, a cytoskeleton disrupting compound,does not induce a neutrophil response by itself [18] but isknown to augment the neutrophil response to many stim-uli. We investigated whether this was true also for theminimal neutrophil response induced by a direct stimula-tion with TNF-α. We found that cytochalasin B had noeffect on the NADPH-oxidase response when added toneutrophils prior to TNF-α treatment (data not shown).However, when the cells were first treated with TNF-α andsubsequently challenged with cytochalasin B, a pro-nounced respiratory burst activity was noted (Fig 1). Thetime course of the induced response was similar to thatseen with chemoattractants such as the formylated pep-tide fMLF, an agonist that activates cells through the G-protein coupled formyl peptide receptor, FPR. The peak ofactivity was reached 1–2 minutes after the addition ofcytochalasin B and the response then rapidly declined toreach a base-line level after 3–4 min that remained con-stant throughout the observation period.In agreement with the results reported by others [8,9] wefound that TNF-α alone only poorly activated the neu-trophil NADPH-oxidase, determined as the release ofsuperoxide anions (3.2 ± 0.3 × 106 cpm; mean peak value± SD, n = 3; corresponding to a maximum value that wasless than 5% of that induced by fMLF). The oxidase activ-ity induced by cytochalasin B in TNF-α-treated cells was ofthe same magnitude as that triggered by fMLF (Figs 1 and2). In agreement with previous reports [20-22], TNF-α wasfound to prime neutrophils to a subsequent stimulationwith fMLF (Fig 2).The level of superoxide production induced by cytochala-sin B was dependent on the concentration of TNF-α. (Fig1) as well as on the duration of TNF-preactivation. Whenthe time between the addition of TNF-α and cytochalasinB was less than 5 minutes, no respiratory burst activity wasobserved upon cytochalasin B challenge. The response tocytochalasin B gradually increased with the time allowedfor TNF-α to interact with the neutrophils, reaching a pla-teau after approximately 20 min (Fig 3). The protein syn-thesis inhibitor cycloheximide did not inhibit thePage 2 of 14(page number not for citation purposes)the cytoskeleton disrupting compound cytochalasin Btriggered activation. The TNF-α primed state shows manycytochalasin B-induced NADPH-oxidase activity in TNF-αprimed cells (data not shown), suggesting that de novoBMC Cell Biology 2004, 5 http://www.biomedcentral.com/1471-2121/5/21protein synthesis was not required for the observed neu-trophil activation.Reactivation of deactivated GPCR has implications for TNF-α primingAll neutrophil chemoattractant receptors including C5aR(for the complement component C5aR), PAFR (for plate-let activating factor), CXCR1 and 2 (for IL-8), FPR (forfMLF) and FPRL1 (for WKYMVM) belong to the GPCRfamily [2]. Interaction between these receptors and theirrespective ligands results in activation of the neutrophilNADPH-oxidase (Table 1). Neutrophils that were allowedto interact with either of these agonists at 15°C and thentransferred to 37°C became deactivated, i.e., there was noburst in oxidase activity, and the cells did not respond tofurther stimulation with the same chemoattractant. Fur-thermore, cytochalasin B induced a robust burst of oxi-and C5aR) were reactivated. In contrast, no such reactiva-tion was induced by cytochalasin B in IL-8 or in PAF deac-tivated cells (Table 1).The cytochalasin B induced superoxide production inTNF-α-primed neutrophils is similar to that induced byuncoupling and reactivation of occupied and deactivatedFPR, FPRL1 or C5aR from the cytoskeleton, induced bythe same drug. The most direct interpretation of theseresults is therefore that the TNF-α primed cells express aGPCR, of hitherto unknown identity, which graduallybecomes occupied and then deactivated. Whenuncoupled from the cytoskeleton by cytochalasin B, thereceptor is reactivated and the signals generated induce anactivation of the oxidase. To test this hypothesis, we pre-treated the cells with pertussis toxin, which inactivates theheterotrimeric G-proteins coupled to the GPCRs. Since noNADPH-oxidase activity could be induced by cytochala-sin B in TNF-α primed cells that were first treated with per-tussis toxin (Fig 4), our hypothesis is valid, i.e., a GPCR isCytochalasin B stimulation of TNF-α primed neutrophilsFigure 1Cytochalasin B stimulation of TNF-α primed neu-trophils. Neutrophils were pre-incubated with TNF-α (25 ng/ml, 20 min, 37°C) after which they were stimulated with cytochalasin B The extracellular release of superoxide anions was measured by isoluminol-amplifled chemiluminescence (CL) given as Mcpm (106 counts per minutes). The figure shows the kinetics of a representative experiment. The indi-cated value shows the mean peak value ± SD, n = 3. The abil-ity of cytochalasin B to induce NADPH-oxidase activity was dependent on the priming concentration of TNF-α (inset; comparing the peak values of the responses at varying con-centrations to the value obtained with 25 ng/ml of TNF-α).CL(Mcpm)2 4 6Time (min)004080120136 ± 26160TNF-a (pg/ml)CL (% of max)0408012015050025000fMLF stimulation of TNF-α primed neutrophilsFigure 2fMLF stimulation of TNF-α primed neutrophils. Neu-trophils were pre-incubated in the absence (dashed line) or presence (solid line) of TNF-α (25 ng/ml) for 20 min at 37°C after which they were stimulated with fMLF. The extracellu-lar production of superoxide anion after addition of the pep-tide (10-7M) was measured by isoluminol-amplified CL. Responses of CL are given as Mcpm (106 counts per min-utes). The figure shows the kinetics of representative experiments.Time (min)CL (Mcpm)01002003000 2 4 6 8Page 3 of 14(page number not for citation purposes)dase activity in C5a- as well as in fMLF- and WKYMVM-deactivated cells i.e., these cells (or rather the FPR, FPRL1involved in the TNF-α-primed cytochalasin B response. Itshould be noted that pertussis toxin had no effect on theBMC Cell Biology 2004, 5 http://www.biomedcentral.com/1471-2121/5/21ability of TNF-α to mobilize CR3 to the neutrophil surface(Fig 4). On the basis of these results, we hypothesized thata preformed endogenous agonist may be released fromneutrophils during interaction with TNF-α, and we there-fore attempted to verify the existence of such an agonist.Cytochalasin B induced reactivation of FPR is inhibited by the receptor antagonist cyclosporine HTo test the hypothesis of an endogenous GPCR-agonist,we first determined the basic reactivation characteristics ofoccupied and desensitized receptors, using a modelsystem in which desensitized neutrophils were pre-incu-bated with fMLF at 15°C. When pre-warmed neutrophils(37°C for 20 min) were added to a measuring system con-taining desensitized cells, the oxidase of the non-desensi-tized cells was activated, suggesting that free ligands arepresent in the measuring system (data not shown). Inorder to determine if these free ligands are of importancefor the FPR reactivation induced by cytochalasin B, weadded the receptor specific antagonist cyclosporine H tofMLF-desensitized cells a few seconds before cytochalasinsuch inhibition by cyclosporine H was seen upon reactiva-tion, when using cells that were incubated with the FPRL1specific agonist WKYMVM or TNF-α (Fig 5).Moreover, when the fMLF specific desensitization proce-dure was performed in a dense cell population (107 cells/ml desensitized with 10-7M fMLF) and the cells werediluted to 105 cells/ml in pre-warmed measuring vialscontaining either a high concentration of peptide (finalconcentration of fMLF being 10-7M) or no peptide (finalconcentration of fMLF being 10-9M), the addition of cyto-chalasin B induced a burst in NADPH-oxidase activityonly in samples with a high concentration of fMLF (Fig 6).Taken together these results suggest that in order for cyto-chalasin B to function as an inducer of respiratory burst,the fMLF desensitized neutrophils have to be continu-Time dependency of the TNF-α effectF gure 3Time dependency of the TNF-α effect Responses to cytochalasin B after incubation with TNF-α for different peri-ods of time. Neutrophils were pre-incubated with TNF-α (25 ng/ml, 37°C) under various time-periods (5–40 min), before they were stimulated with cytochalasin B. The production of superoxide anions was measured by isoluminol-amplified CL as described above.CL(Mcpm)Time (min)0204060801001201400 10 20 30 40Pertussis toxin (PtX) sensitivity of cytochalasin B induced superoxide anion product on and CR3 upregulatio  in TNF-α rimed neutrophilsFigu e 4Pertussis toxin (PtX) sensitivity of cytochalasin B induced superoxide anion production and CR3 upreg-ulation in TNF-α primed neutrophils. Neutrophils were preincubated with TNF-α (25 ng/ml, 20 min, 37°C) before they were incubated with PtX (500 ng/ml) for various time-periods (20–140 min). The cells were either paraformalde-hyde-fixed and analyzed by flow cytometry for CR3 upregula-tion (closed squares), or stimulated with cytochalasin B, and the superoxide anion production were measured by CL (open squares). All analyzed populations of PtX incubated cells were compared to TNF-α primed cells not exposed to PtX.0204060801001200 20 40 60 80 100 120 140 160Preincubation time with PtX(min)%ofcontrolPage 4 of 14(page number not for citation purposes)B. In agreement with the specificity of the antagonist forFPR, a reduced NADPH-oxidase activity was obtained. Noously exposed to a high concentration of free ligand.BMC Cell Biology 2004, 5 http://www.biomedcentral.com/1471-2121/5/21No endogenous neutrophil activator can be identified following incubation with TNF-αNeutrophils contain several preformed agonists/chemoat-tractants [23,24] that could participate in an autocrineamplification loop. As described above, binding of neu-trophil chemoattractants to their respective neutrophilreceptors induces a rapid desensitization of the receptor,leading to an inability of the cells to respond to a secondchallenge by the same agonist. Accordingly, if TNF-αwould induce secretion of an autocrine activator, thisshould be expected to induce a desensitized state of thespecific receptor population involved. The finding thatTNF-α triggered cells were primed rather than desensi-tized to fMLF, WKYMVM/m and C5a, suggests that thepotential endogenous utilizes neither FPR, FPRL1 norC5aR.To identify the hypothetical endogenous agonist, we pre-pared cell free supernatants of TNF-α treated neutrophilsand added these to new populations of primed orunprimed cells. No oxidase activation could be inducedby compounds secreted from the TNF-α primed cells(data not shown). As stated above, neutrophils added to apopulation of fMLF desensitized cells were rapidly acti-vated to generate superoxide by the fMLF present in themedium. When repeating this experiment with TNF-primed instead of fMLF desensitized cells, no such activitywas seen (Fig 7). Furthermore, the newly added cells (nonTNF-α primed) were not triggered by the addition of cyto-chalasin B (Fig 7), as illustrated by the finding that theNADPH-oxidase activity in mixed population of cells(50% primed and 50% non-primed) was only half of thatof a cell population (with the same number of cells) inwhich all the cells were primed. Taken together these datasuggest that no free agonist is present in the TNF-α primedcell suspension that could be responsible for transfer ofTNF-α induces mobilization of receptor-storing granulesPrevious studies have shown that different receptor struc-tures as well as potential activating agonists are stored insecretory granules of peripheral blood neutrophils[17,25,26], suggesting that mobilization of theseorganelles will prime neutrophils to certain stimuli. Wemonitored the amount of the complement receptor 3(CR3) and found that TNF-α-primed cells exposed anincreased number of CR3 on their surface (Fig 8). Inaddition, TNF-α priming was accompanied by anincreased specific binding of radiolabeled fMLF (10.2 ±3.3 moles/106cells bound to TNF-α treated cells com-pared to 4.0 ± 1.6 fmoles/106 cells for corresponding con-trol cells; mean ± SEM, n = 6), reflecting an increasedamount of formyl peptide receptors (FPR) on the neu-trophil surface. Hence, FPR and integrin-storingorganelles were significantly mobilized upon treatment ofneutrophils with TNF-α. We also found it of interest todetermine whether induction of the cytochalasin B sensi-tive state is unique for TNF-α, or if it occurs also with othersecretagogues. To investigate the precise role of granulemobilization in induction of the cytochalasin B sensitivestate, we monitored the amount of the complement recep-tor 3 (CR3) on the surface of neutrophils. The finding thatneutrophil receptors for IL-8 were not reactivated by cyto-chalasin B (Table 1) suggested that IL-8 is a suitable con-trol priming agent in these experiments. No respiratoryburst was obtained in response to cytochalasin B whenTNF-α was replaced by IL-8 (Table 1), despite an induc-tion of storage organelle mobilization also by IL-8 (Fig 9).It should be pointed out that the activation potency inrelation to cytochalasin B was retained also when the con-centration of TNF-α was reduced to concentrations thatgave a level of CR3 mobilization similar to that inducedby IL-8.Table 1: Deactivation/reactivation properties of classical chemoattractants and their receptors.Effects on the neutrophil NADPH-oxidase*Agonist (conc.) Receptor(s) Activation Deactivation ReactivationfMLF (l0-7M) FPR + + +WKYMVM (10-7M) FPRL1 + + +C5a (100 ng/ml) C5aR + + +PAF (10-7M) PAFR + + -IL8 (100 ng/ml) CXCR 1/2 + + -*Indicated chemoattractants were used to stimulate human neutrophils directly (activation), or added to the cells at 15 (according to the protocol described in Methods) after which these cells were transferred to 37 and stimulated with the same agonist (deactivation) or reactivated with cytochalasin B (reactivation). The superoxide anion production was measured by isolummol-enhanced CL. The + and - indicate whether or not the process occurred. The experiments were repeated between 2–10 times.Page 5 of 14(page number not for citation purposes)the cells into a cytochalasin B-sensitive state.BMC Cell Biology 2004, 5 http://www.biomedcentral.com/1471-2121/5/21Cyclosporin H sensitivity of cytochalasin B induced superoxide anion production in fMLF desensitized, WKYMVM desensitized or TNF-α primed neutrophilsFigure 5Cyclosporin H sensitivity of cytochalasin B induced superoxide anion production in fMLF desensitized, WKYMVM desensitized or TNF-α primed neutrophils. Neutrophils were preincubated with fMLF (10-7M; 10 min A), WKYMVM (10-7M; 10 min B) or TNF-α (25 ng/ml; 20 min C). The cells were challenged with cytochalasin B in samples without (solid lines) and with cyclosporine H (final concentration in A, 10-7M and in B and C, 10-6M; dashed lines) that was added 1 min before cytochalasin B. The production of superoxide anions was measured by isoluminol-amplified CL as described above. The figure shows the kinetics of representative experiments and the ratios between the peak response in the absence and presence 0204060800 2 4 6AfMLFRatio:≈5CL(Mcpm)0204060800 2 4 6BWKYMVMRatio: 1.06±0.09(mean ± SD; n=3)CL(Mcpm)0204060800 2 4 6TNF-a CRatio: 0.96±0.28(mean ± SD; n=6)Time (min)CL(Mcpm)Page 6 of 14(page number not for citation purposes)of cyclosporine H are given.BMC Cell Biology 2004, 5 http://www.biomedcentral.com/1471-2121/5/21Effects of the MAPK inhibitor SB 203580There is a well described link between TNF-α-inducedactivation of the p38 mitogen-activated protein kinase(MAPK) and superoxide anion formation in adherentneutrophils (reviewed by Berton [27]). We investigatedthe effect of the specific p38 MAPK inhibitor SB203580 inour model system. As shown in Fig 8, SB203580 signifi-cantly attenuated the TNF-α priming, whereas no reduc-tion was observed when neutrophils were first incubatedwith SB203580 and then stimulated with PMA, a phorbolthrough protein kinase C (Fig 10A). Furthermore, the p38MAPK inhibitor attenuated the effect of TNF-α inducedgranule mobilization, as determined by a reduced expo-sure of CR3 on the surface of cells challenged with TNF-αin the presence of SB 203580 (Fig 10B). Hence, p38MAPKis involved not only in the TNF-α-induced NADPH-oxi-dase activation in adherent cells, but also in the primingeffects (degranulation and cytochalasin B-sensitivity)induced by the cytokine.Dilution effects on cytochalasin B induced activationFigure 6Dilution effects on cytochalasin B induced activation Neutrophils were incubated in the presence of fMLF (10-7M; A) or TNF-α (25 ng/ml; B) for 20 min at 37°C and then diluted in measuring vials containing the same concentration (high; solid lines) or no agonist (low; dashed lines), respectively. The cells were then immediately challenged with cytochalasin B. The production of superoxide anion was measured by isoluminol-amplified CL as described above. The figure shows the kinetics of represent-ative experiments and the ratios, determined from the peak response in the presence of high and low concentrations of agonist.040801201602000 2 4 6fMLF ARatio: ≈ 50CL(Mcpm)Time (min)02040600 2 4 6BTNF-aRatio: 1.08±0.17(mean ± SD; n=3)CL(Mcpm)Time (min)Page 7 of 14(page number not for citation purposes)ester known to activate the NADPH oxidase directlyBMC Cell Biology 2004, 5 http://www.biomedcentral.com/1471-2121/5/21DiscussionA prominent feature of TNF-α is its capacity to prime neu-trophils to other stimuli. In this study, we show that TNF-α-treated neutrophils are primed for activation by thecytoskeleton-disrupting drug cytochalasin B. Themolecular mechanisms underlying priming of theNADPH oxidase response have been extensively studied.icles, is a major mechanism involved in priming of theneutrophil response [15-17]. Other proposed mecha-nisms include alterations of intracellular signaling path-ways (increased protein phosphorylation [28],phospholipase activity [29], intracellular Ca2+ changes[30]), cross-talk between Ca2+ increase and tyrosine phos-phorylation [31], altered assembly of the oxidase [32],Addition of non-primed cells to TNF-α primed cells and activation by cytochalasin BFigure 7Addition of non-primed cells to TNF-α primed cells and activation by cytochalasin B. Neutrophils incubated for 20 min at 37°C for 20 min were added to a population of TNF-α primed cells. The amount of superoxide release by the newly added cells was determined (A; dashed line). For comparision the NADPH-oxidase activity induced by cytochalasin B when added to the TNF-primed cells (A; solid line) is also shown. The NADPH-oxidase activity induced by cytochalasin B in a cell population where all the cells were primed with TNF-α for 20 min (B; solid line) was compared to that of a cell population where 50% of the cells were primed with TNF-α for 20 min and the other 50% for 1 min (B; dashed line). The production of superoxide anions was measured by isoluminol-amplified CL as described above. The figure shows the kinetics of representa-tive experiments and the ratios between the peak responses in the populations are also given.0204060801000 2 4 6BRatio: 1.97±0.17(mean ± SD; n=4)Time (min)CL(Mcpm)02040600 2 4 6ARatio: ≈ 50Time (min)CL(Mcpm)Page 8 of 14(page number not for citation purposes)In previous reports, we have forwarded the hypothesisthat mobilization of receptors, stored within granules/ves-and proteolytic processing of cell surface proteins [33].Although we can not exclude that multiple mechanismsBMC Cell Biology 2004, 5 http://www.biomedcentral.com/1471-2121/5/21may be involved in TNF-α-induced priming, it is impor-tant to point out that a prerequisite for priming throughthe mechanisms described above is that a second agonistis required, in addition to the priming agent, in order todisclose the increased potential for neutrophils torespond.The cytoskeleton disrupting molecule cytochalasin B hasgenerally been regarded as a substance which lacks theability to activate neutrophils by itself, and the resultsdescribed here could possibly be explained by an ability ofcytochalasin B to induce a state of receptor reactivation[18]. This is defined as the transfer of a deactivated/desen-sitized receptor into an actively signaling state, achievedby uncoupling of the receptor from the cytoskeleton.Using the ligand-receptor pair fMLF-FPR as a model, it hasbeen shown that the processing of neutrophil chemoat-tractant receptors includes highly regulated events thatoccur in a given chronological order. The binding of theagonist to its inactive cell surface receptor (FPR) generatesreceptor, the receptor-ligand complex associates withactin or other cytoskeletal proteins [34-36], inducing aphysical segregation of the signaling G-protein and thereceptor into different plasma membrane domains [37].This leads to termination of the response by a direct cessa-tion of the transmembrane signals, and the receptor-lig-and complex is thus deactivated/desensitized (FPR*des).The addition of cytochalasin B to cells with such deacti-vated/desensitized receptors leads to uncoupling of thereceptor-ligand complex from the cytoskeleton, resultingin regained signaling capacity of the receptor (FPRre*),and the signals generated activate the oxidase [18].The cytochalasin B induced activation of the NADPH-oxi-dase in TNF-α primed neutrophils is very similar to thatachieved by an uncoupling of FPR*des from the cytoskele-ton and a transfer of the receptor into an FPRre* state. Theassumption that GPCRs are involved also in TNF-α/cyto-chalasin B induced oxidase activity was validated byexperiments using pertussis toxin, a specific inhibitor ofthe heterotrimeric G-proteins linked to all the neutrophilchemoattractant receptors yet characterized [2]. The cyto-chalasin B-induced oxidase activity in TNF-α primed cellswas clearly pertussis toxin sensitive; in contrast, the toxindid not affect TNF-α induced mobilization of CR3 to thecell surface, which is in agreement with the fact that theTNF-α receptor itself is not a member of the GPCR family[38]. It is interesting to note that cytoskeleton-dependentreceptor reactivation is not achieved with all neutrophilGPCRs. FPR*des, FPRLl*des and C5aR*des were all reactivat-able by cytochalasin B. No such reactivation was howeverobtained with the occupied receptors for IL-8 and PAF.Neither the IL-8-R*des nor the PAF-R*des could thus bereactivated with cytochalasin B (Table 1). These datasuggest that these two receptor-ligand pairs should beexcluded from being responsible for the TNF-α inducedchange in sensitivity for cytochalasin B, but more impor-tantly the data also suggest that there are fundamentaldifferences with respect to signaling between differentgroups of chemoattractant receptors. This is in agreementwith recently presented data describing distinct signalingpathways for GPCRs that mediate directional migrationby chemoattractants, guiding the neutrophils out of thevasculature (i.e., interleukins and lipid mediators) andthose that guide the cells through the interstitium to a siteof infection (i.e. bacterial chemoattractants and activatedcomplement factors) [39]. Our results suggest that TNF-αinduces a neutrophil state that shares basic the signalingproperties with the FPR*des, FPRLl*des and C5aR*des, i.e., astate that is reactivated when the cytoskeleton is disruptedby cytochalasin B.Neutrophils contain known, and probably also unknown,CR3-upregulation in TNF-α primed neutrophilsFigure 8CR3-upregulation in TNF-α primed neutrophils. Neu-trophils were incubated with TNF-α (25 ng/ml, 20 min, 37°C, solid line) and were paraformaldehyde-fixed, incubated with phycoerythrin-conjugated anti-CR3 antibodies (CD18/CD11b) and analysed by flow cytometry and compared with control cells (dashed line). Representative histograms of binding are shown, and the inset shows the exposure of CR3 on fMLF-primed neutrophils. The numbers indicate the mean fluorescence intensity as percent of control ± SD, n = 3.control+TNF 204 ± 46% of control 020015010050100 101 102 103 10fluorescence intensitynumberofcells+fMLF 185 ± 35 % of control015010050100 101 102 103numberof cellsPage 9 of 14(page number not for citation purposes)an actively signaling receptor-ligand complex (FPR*).Shortly after binding of the chemoattractant to itssecretable chemoattractants [24,40], as well as known andpossibly not yet identified receptors for such agonists (i.e.,BMC Cell Biology 2004, 5 http://www.biomedcentral.com/1471-2121/5/21CXCR1 and 2 for IL-8 and FPRL1 for LL37). The combinedTNF-α/cytochalasin B-dependent activation process waspertussis toxin sensitive suggesting that the activationprocess involves a heterotrimeric G-protein, possibly (butnot known for certain) linked to a receptor. An attractivehypothesis that directly explains our results would be thatTNF-α induces secretion of an endogenous agonist whichbinds back to a neutrophil receptor sharing its basic sign-aling properties with the cytoskeleton-regulated group ofGPCRs. This endogenous agonist would occupy its surfacereceptors and being a ligand-receptor pair of the cytoskel-eton-binding type, the receptors would then becomedesensitized upon agonist binding and the cells would betransferred into a cytochalasin B-activated state. Althoughwe can not exclude this possibility, the experimental evi-dence presented is inconsistent with this scenario. On theone hand pretreatment with secretagogues induces mobi-lization of neutrophil storage organelles, but on the otherhand, such a mobilization is insufficient for induction ofthe cytochalasin B sensitive state as illustrated by thefinding that IL8, a potent secretagogue (Fig 9), failed toprime neutrophils for subsequent activation by cytochala-sin B (Table 1). In addition, we assumed that providedthat a hypothetical agonist secreted in response to TNF-αtrophil activators, it should be present in the extracellularenvironment, but despite the use of several experimentalapproaches we were unable to find any evidence for theexistence of such an agonist.Over the past few years, studies on the biology of GPCRhave been focused on the activation achieved throughbinding of a specific agonist to its receptor. It has, how-ever, become increasingly clear that GPCRs can be trans-ferred from a non-signaling R state to an actively signalingR* state also in the absence of any bound ligand [19,41].It is obvious that the R* state can not be constitutivelyactive, and irrespectively of whether the receptor reachesthe R* state following agonist binding or if this state isreached independently of any activating agonist, the deac-tivation mechanisms appears to be put in action. Thephysical separation of the R* from the G-protein occur-ring through a linkage of the receptor to the cytoskeleton,constitute an important termination mechanism, andR*des state can be reversed through an uncoupling fromthe cytoskeleton [18]. The fact that we were unable to findany secreted components that could fulfill the role of areceptor agonist in the TNF-α/cytochalasin B dependentactivation system, suggests that TNF-α may transferCR3-upregulation in IL-8 and TNF-α primed neutrophilsFigure 9CR3-upregulation in IL-8 and TNF-α primed neutrophils. Neutrophils were incubated with or without IL-8 (100 ng/ml) or TNF-α (25 ng/ml) for 20 min at 37°C. The cells were then paraformaldehyde-fixed, incubated with phycoeryterin-conju-gated anti-CR3 antibodies (CD18/CD11b), analysed by flow cytometry and compared with control cells. The data is calculated from the mean fluorescence intensity of each cell population and expressed as percentage of the value obtained in control cells, n = 6.0100200300control IL-8 TNF-a400**%ofexposureoncontrolcellsPage 10 of 14(page number not for citation purposes)has a binding affinity for its receptor, that is of the samemagnitude as the hitherto identified/characterized neu-neutrophil GPCR's to a cytoskeleton associated R*des stateindependently of agonist binding.BMC Cell Biology 2004, 5 http://www.biomedcentral.com/1471-2121/5/21Effects of SB203580 on TNF-α primingFigure 10Effects of SB203580 on TNF-α priming. Neutrophils were primed by TNF-α (25 ng/ml, 20 min, 37°C) in the absence or presence of the p38 MAPK inhibitor SB203580 (1 µM). (A) The cells were stimulated with cytochalasin B (5 µg/ml) and the extracellular production of superoxide anion was measured by CL. The effect of SB203580 on PMA-induced superoxide anion production was used as control. (B) Neutrophils were incubated with TNF-α (25 ng/ml) in the absence or presence of SB203580 (1 µM)), or with SB203580 alone (20 min, 37°C) and were paraformaldehyde-fixed, incubated with phycoerythrin-conjugated anti-CR3 antibodies (CD18/CD11b), analysed by flow cytometry and compared with control cells. Calculation were made from the mean fluorescence intensity of each cell population and expressed as percentage of the value obtained in CL(Mcpm)PMA +SB2035800100200300400TNF-a TNF-a +SB203580PMAA*CR3 exposure(% of control)control TNF-a + SB203580TNF-aSB203580B100200150502500Page 11 of 14(page number not for citation purposes)control cells. The results are given as mean ± SE of three independent experiments.BMC Cell Biology 2004, 5 http://www.biomedcentral.com/1471-2121/5/21A transformation from R to R* can, thus, occur in theabsence of a specific ligand, but very little is known aboutthe regulatory mechanisms that determines the rate oftransfer or the levels of the two forms at equilibrium. TheR/R* equilibrium differs for individual wild-type isoformsof a given receptor, and the ratio between the two formscan also be changed both by defined point mutationslocalized to several different intracellular receptordomains, as well as by the degree of glycosylation of thereceptor [19]. It seems reasonable to hypothesize that theR/R* transformation rate and/or ratio at equilibrium canbe changed not only through direct structural changes inthe receptor protein, but also indirectly through signalsgenerated by a second receptor such as the one for theTNF-α (receptor communication), or through a directreceptor-receptor interaction in the membrane.ConclusionsNeutrophils triggered with TNF-α mobilize receptor stor-ing organelles and concomitantly but independently, thecells are primed to respond to the microfilament disrupt-ing toxin cytochalasin B. These findings are suggestive oftwo mechanisms involved in neutrophil priming by TNF-α. While the enhanced response to fMLF is explained byrecruitment of new formyl peptide receptors to the cellsurface, the cytochalasin B-sensitive state is achievedthrough a novel mechanism that transfers G-protein cou-pled receptors to a primed state which is fully activatedwhen cytochalasin B uncouples the receptors from thecytoskeleton. Such a reaction could possibly be inducedby an endogenous secreted receptor agonist. We were,however, unable to find any components that could fulfillthe role of an agonist in the TNF-α/cytochalasin Bdependent activation system. The presented data supportthe recently introduced concept that receptor activationcan occur independently of a specific receptor ligand [19].Accordingly, we suggest that TNF-α transfers neutrophilreceptors to a desensitized and cytoskeleton associatedstate independently of agonist binding.MethodsIsolation of neutrophilsLeukocytes were isolated from freshly prepared leuko-packs (The Blood Center, Sahlgrenska University Hospi-tal, Göteborg) obtained from healthy blood donors. Afterremoval of erythrocytes through dextran sedimentation,the leukocyte-rich supernatant was carefully layered ontoFicoll-Paque (Lymphoprep, Nyegaard, Norway). Aftercentrifugation at 380 × g for 30 minutes, the pellet wasresuspended in Krebs-Ringer phosphate buffer (KRG, pH7.3; 120 mM NaCl, 5 mM KC1, 1.7 mM KH2PO4, 8.3 mMNaHPO4 and 10 mM glucose) supplemented with Ca2+ (1mM) and Mg2+ (1.5 mM), and kept on melting ice untilNeutrophil activatorsThe hexapeptide Trp-Lys-Tyr-Met-Val-Met-NH2(WKYMVM) was synthesized and HPLC-purified by AltaBioscience (University of Birmingham, Birmingham,United Kingdom). The formylated peptide N-formylme-thionyl-leucyl-phenylalanine (fMLF), the chemotacticfragment of complement factor 5 (C5a), platelet activat-ing factor (PAF), and TNF-α were from Sigma ChemicalCo., St. Louis. Human recombinant IL-8 was provided byR&D Systems, Oxon, UK. The peptide agonists were dis-solved in dimethyl sulfoxide to 10-2M and stored at -70°Cuntil use. Further dilutions were made in KRG.Neutrophil priming and NADPH-oxidase activityNeutrophils (1–2 × l06cells/ml) were incubated at 37°Cfor 5–40 minutes in the presence or absence (control) ofa priming agent. The NADPH-oxidase activity of thesecells was then recorded using luminol/isoluminol-enhanced chemiluminescence (CL) systems [43]. The CLactivity was measured in a six-channel Biolumat LB 9505(Berthold Co. Wildbad, Germany), using disposable 4-mlpolypropylene tubes with a 0.90-ml reaction mixturecontaining 1–2 × 106 neutrophils. In order to differentiatebetween intracellularly and extracellularly generated reac-tive oxygen species, two different reaction mixtures wereused. Tubes used for the measurement of extracellularrelease of superoxide anion contained neutrophils, horse-radish peroxidase (HRP; a cell impermeable peroxidase; 4U) and isoluminol (a cell impermeable CL substrate; 2 ×10-5M). Tubes used for measurement of intracellulargeneration of reactive oxygen species contained neu-trophils, SOD (a cell impermeable scavenger for O2-; 50U), catalase (a cell impermeable scavenger for H2O2; 2000U), and luminol (a cell permeable CL substrate; 2 × 10-5M). The tubes were equilibrated at 37°C, after which thestimulus (0.1 ml) was added. The light emission wasrecorded continuously.Determination of receptor exposureThe amount of fMLF-receptors expressed on the cell sur-face in the different neutrophil populations was deter-mined by incubating the cells with radiolabeled fMLF inthe presence or absence of unlabeled fMLF. Unboundpeptide was removed by centrifugation of the cellsthrough an oil layer. The oil layer which was composed ofa mixture of dibutylphtalate and dinonylphtalate (10:3, v/v; 100 µl) was layered on top of 10 µl of urea (6 M) inEppendorf tubes. The radiolabeled peptide (50 µl[3H]fMLF; 8 × 10-8M) was then layered on top of the oilfollowed by 50 µl of unlabeled fMLF (4 × 10-5M) in KRGor vehicle alone. Neutrophils (2 × 106, 100 µl) were addedto the fMLF-solution and the tubes were incubated onmelting ice for 1 h. After centrifugation at 9000 × g for 15Page 12 of 14(page number not for citation purposes)used in experiments [42]. seconds in a Beckman microfuge (Beckman Instruments,Fullerton, CA), the bottom of the centrifuge tubes (con-BMC Cell Biology 2004, 5 http://www.biomedcentral.com/1471-2121/5/21taining the pelleted cells) was excised and collected fordetermination of radioactivity.To measure surface exposure of CR3, the cells were labeledwith phycoerythrin-conjugated monoclonal antibodiesspecific for CD11b (DAKO M741; 10 µl to a cell pellet of106). The cells were examined by use of flow cytometry(FACScan; Becton Dickinson, Mountain View, CA).Deactivation and reactivationIn order to deactivate neutrophils to GPCR agonists, thecells were first equilibrated for 10 min at 15°C. The ago-nist, e.g., the chemoattractant fMLF (final concentration10-7M) was added and the incubation was continued at15°C for an additional 5 min [44]. The cells were thentransferred to 37°C for 5 minutes, and reactivation wassubsequently achieved by adding cytochalasin B (5 µg/ml).ReagentsHuman recombinant TNF-α, fMLF, luminol, isoluminol,cycloheximide, Pertussis toxin, SB 203580 and cytochala-sin B were obtained from Sigma Chemical Co., St. Louis.Superoxide dismutase (SOD), catalase and horse radishperoxidase (HRP) were from Boehringer-Mannheim, Ger-many. Radiolabeled fMLF was from Du Pont NEN (Bos-ton, Mass).Statistical analysisThe Student's t-test (two-tailed) was performed to deter-mine statistical significance.List of abbreviationsCD11b, cluster of differentiation number 11b; CL, chemi-luminescence, GPCR, G-protein coupled receptor, CR3,complement receptor 3, CXCR, the IL 8 (CXC cytokine)receptor, C5a, the chemotactic split product from comple-ment factor 5; C5aR, the C5a receptor, fMLF, formylmet-leu-phe; FPR, the formyl peptide receptor; FPRL1, theformyle peptide like receptor 1; IL-8, interleukin 8; SOD,superoxide dismutase; TNF, tumor necrosis factor, TNFR,the TNF-recepor; WKYMVM, Trp-Lys-Tyr-Met-Val-Met.Authors contributionsThe scientific question raised in the paper was formulatedduring discussions between all the authors, about themechanisms behind the priming phenomenon in relationto receptor resensitization (see [18]). JB, SP, HF and UMHperformed all the experiments using techniques devel-oped by CD, JB and AK. All authors participated in theplanning of the work and in analyzing the results. UMHand CD wrote the first version of the paper, but contribu-tions from all authors were important for the final out-AcknowledgementsResearch grants: Supported by the Swedish Medical Research Council, King Gustaf the V's 80-year foundation and the Swedish Society for Medical Research.References1. Matsukawa A, Hogaboam CM, Lukacs NW, Kunkel Sl: Chemokinesand innate immunity. Rev Immunogenet 2000, 2:339-358.2. Ye RD, Boulay F: Structure and function of leukocyte chemoat-tractant receptors. Adv Pharmacol 1997, 39:221-289.3. Dewitt S, Laffafian I, Hallett MB: Phagosomal oxidative activityduring beta2 integrin [CR3)-mediated phagocytosis by neu-trophils is triggered by a non-restricted Ca2+ signal: Ca2+controls time not space. J Cell Sci 2003, 116:2857-2865.4. Werner E: GTPases and reactive oxygen species: switches forkilling and signaling. J Cell Sci 2004, 117:143-153.5. 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Lundqvist H, Gustafsson M, Johansson A, Sarndahl E, Dahlgren C:Neutrophil control of formylmethionyl-leucyl-phenylalanineinduced mobilization of secretory vesicles and NADPH-oxi-dase activation: effect of an association of the ligand-recep-tor complex to the cytoskeleton. Biochim Biophys Acta 1994,1224:43-50.yours — you keep the copyrightSubmit your manuscript here:http://www.biomedcentral.com/info/publishing_adv.aspBioMedcentralPage 14 of 14(page number not for citation purposes)chemotactic gradients. J Cell Biol 2002, 159:91-102.


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