Cytochalasin B triggers a novel pertussis toxin sensitive pathway in TNF-alpha primed neutrophils
© Bylund et al; licensee BioMed Central Ltd. 2004
Received: 13 March 2004
Accepted: 24 May 2004
Published: 24 May 2004
Cytochalasin B does not directly activate the oxygen-radical-producing NADPH oxidase activity of neutrophils but transfers desensitized G-protein coupled receptors (GPCR) into an active signaling state by uncoupling GCPR from the cytoskeleton. The receptor uncoupling results in respiratory burst activity when signals generated by reactivated formyl peptide receptors trigger the NADPH-oxidase to produce superoxide anions.
Tumor necrosis factor alpha (TNF-alpha) primes neutrophils for subsequent activation by cytochalasin B. Pretreatment with TNF-alpha induced mobilization of receptor-storing neutrophil organelles, suggesting that receptor up-regulation significantly contributes to the response, but the receptor mobilization was not sufficient for induction of the cytochalasin B sensitive state. The TNF-alpha primed state resembled that of the desensitized non-signaling state of agonist-occupied neutrophil formyl peptide receptors. The fact that the TNF-alpha primed, cytochalasin B-triggered activation process was pertussis toxin sensitive suggests that the activation process involves a GPCR. Based on desensitization experiments the unidentified receptor was found to be distinct from 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 activating factor could not be reactivated by cytochalasin B, also these could be excluded as receptor candidates involved in the TNF-alpha primed state.
The TNF-alpha-induced priming signals could possibly trigger a release of an endogenous GPCR-agonist, amplifying the response to the receptor-uncoupling effect of cytochalasin B. However, no such substance could be found, suggesting that TNF-alpha can transfer G-protein coupled receptors to a signaling state independently of agonist binding.
Keywordscytokines superoxide priming TNF cytoskeleton receptor reactivation pertussis toxin G. protein GPCR NADPH-oxidase
Human neutrophil granulocytes constitute an important part of the innate immune defense against microbial infections, and the bactericidal activities performed by these cells rely on their interaction with chemoattractants, cytokines and other inflammatory mediators . The chemoattractants, including C5a, platelet activating factor (PAF), interleukin-8 (IL8) and formylated peptides, bind to specific receptors [2, 3], all of which belong to a family of transmembrane G-protein coupled receptors (GPCRs). Activation of these receptors leads to directed migration, granule mobilization and activation of the neutrophil NADPH-oxidase . The reactive oxygen species generated by the oxidase are of importance for microbial killing and for cell-cell-signaling .
Tumor necrosis factor-alpha (TNF-α) is one of the earliest cytokines produced at inflammatory sites by activated monocytes and macrophages. This cytokine affects neutrophil function mainly through binding to type I TNF receptor (TNFR1) . The TNFR1 is a single transmembrane glycoprotein with several intracellular motifs with known functional significance, but it is not linked to any signaling G-protein [5–7]. Phosphorylation of TNFR1 occurs at a consensus MAPK site on its cytoplasmic domain or through tyrosine phosphorylation [6, 7], although it is not fully understood how this phosphorylation control receptor signaling or processing.
The biological effects of TNF-α on neutrophil functions in vitro vary, as illustrated by the ability or inability of TNF-α to affect the neutrophil oxygen radical producing NADPH-oxidase. In order for TNF-α to trigger neutrophil superoxide production, cells need to adhere to a solid surface, and the magnitude of the response is determined by which protein that is coated on the surface . TNF-α only weakly triggers the oxidase when the neutrophils are in suspension ; however, after exposure to TNF-α, these cells are primed with respect to NADPH-oxidase activation in response to other stimuli . Thus, while TNF-α per se does not activate the NADPH-oxidase to any significant extent in nonadherent neutrophils, it induces a state of hyper-responsiveness to other stimuli.
Several mechanisms have been proposed to account for neutrophil priming [10–14], including receptor mobilization from intracellular granule stores [15–17]. The aim of this study was to characterize the primed state induced in human neutrophils by TNF-α, using an earlier described receptor uncoupling system . We found exposure of new receptors to be a part of the priming process, but more importantly we found that neutrophils interacting with TNF-α were transferred into a novel state, in which the cytoskeleton disrupting compound cytochalasin B triggered activation. The TNF-α primed state shows many similarities with that of neutrophils that have their formyl peptide GPCRs desensitized by a specific receptor agonist . Isomerization of GPCRs, from an inactive to an active state, occurs normally as a result of ligand binding but can also occur independently of agonist  and our findings are suggestive of a TNF-α induced novel activation mechanism that is receptor agonist-independent.
TNF-α primes the neutrophil NADPH-oxidase response to a subsequent stimulation/triggering with cytochalasin B
Reactivation of deactivated GPCR has implications for TNF-α priming
Deactivation/reactivation properties of classical chemoattractants and their receptors.
Effects on the neutrophil NADPH-oxidase*
C5a (100 ng/ml)
IL8 (100 ng/ml)
Cytochalasin B induced reactivation of FPR is inhibited by the receptor antagonist cyclosporine H
No endogenous neutrophil activator can be identified following incubation with TNF-α
Neutrophils contain several preformed agonists/chemoattractants [23, 24] that could participate in an autocrine amplification loop. As described above, binding of neutrophil chemoattractants to their respective neutrophil receptors induces a rapid desensitization of the receptor, leading to an inability of the cells to respond to a second challenge by the same agonist. Accordingly, if TNF-α would induce secretion of an autocrine activator, this should be expected to induce a desensitized state of the specific receptor population involved. The finding that TNF-α triggered cells were primed rather than desensitized to fMLF, WKYMVM/m and C5a, suggests that the potential endogenous utilizes neither FPR, FPRL1 nor C5aR.
TNF-α induces mobilization of receptor-storing granules
Effects of the MAPK inhibitor SB 203580
A prominent feature of TNF-α is its capacity to prime neutrophils to other stimuli. In this study, we show that TNF-α-treated neutrophils are primed for activation by the cytoskeleton-disrupting drug cytochalasin B. The molecular mechanisms underlying priming of the NADPH oxidase response have been extensively studied. In previous reports, we have forwarded the hypothesis that mobilization of receptors, stored within granules/vesicles, is a major mechanism involved in priming of the neutrophil response [15–17]. Other proposed mechanisms include alterations of intracellular signaling pathways (increased protein phosphorylation , phospholipase activity , intracellular Ca2+ changes ), cross-talk between Ca2+ increase and tyrosine phosphorylation , altered assembly of the oxidase , and proteolytic processing of cell surface proteins . Although we can not exclude that multiple mechanisms may be involved in TNF-α-induced priming, it is important to point out that a prerequisite for priming through the mechanisms described above is that a second agonist is required, in addition to the priming agent, in order to disclose the increased potential for neutrophils to respond.
The cytoskeleton disrupting molecule cytochalasin B has generally been regarded as a substance which lacks the ability to activate neutrophils by itself, and the results described here could possibly be explained by an ability of cytochalasin B to induce a state of receptor reactivation . This is defined as the transfer of a deactivated/desensitized receptor into an actively signaling state, achieved by uncoupling of the receptor from the cytoskeleton. Using the ligand-receptor pair fMLF-FPR as a model, it has been shown that the processing of neutrophil chemoattractant receptors includes highly regulated events that occur in a given chronological order. The binding of the agonist to its inactive cell surface receptor (FPR) generates an actively signaling receptor-ligand complex (FPR*). Shortly after binding of the chemoattractant to its receptor, the receptor-ligand complex associates with actin or other cytoskeletal proteins [34–36], inducing a physical segregation of the signaling G-protein and the receptor into different plasma membrane domains . This leads to termination of the response by a direct cessation of the transmembrane signals, and the receptor-ligand complex is thus deactivated/desensitized (FPR*des). The addition of cytochalasin B to cells with such deactivated/desensitized receptors leads to uncoupling of the receptor-ligand complex from the cytoskeleton, resulting in regained signaling capacity of the receptor (FPRre*), and the signals generated activate the oxidase .
The cytochalasin B induced activation of the NADPH-oxidase in TNF-α primed neutrophils is very similar to that achieved by an uncoupling of FPR*des from the cytoskeleton and a transfer of the receptor into an FPRre* state. The assumption that GPCRs are involved also in TNF-α/cytochalasin B induced oxidase activity was validated by experiments using pertussis toxin, a specific inhibitor of the heterotrimeric G-proteins linked to all the neutrophil chemoattractant receptors yet characterized . The cytochalasin B-induced oxidase activity in TNF-α primed cells was clearly pertussis toxin sensitive; in contrast, the toxin did not affect TNF-α induced mobilization of CR3 to the cell surface, which is in agreement with the fact that the TNF-α receptor itself is not a member of the GPCR family . It is interesting to note that cytoskeleton-dependent receptor reactivation is not achieved with all neutrophil GPCRs. FPR*des, FPRLl*des and C5aR*des were all reactivatable by cytochalasin B. No such reactivation was however obtained with the occupied receptors for IL-8 and PAF. Neither the IL-8-R*des nor the PAF-R*des could thus be reactivated with cytochalasin B (Table 1). These data suggest that these two receptor-ligand pairs should be excluded from being responsible for the TNF-α induced change in sensitivity for cytochalasin B, but more importantly the data also suggest that there are fundamental differences with respect to signaling between different groups of chemoattractant receptors. This is in agreement with recently presented data describing distinct signaling pathways for GPCRs that mediate directional migration by chemoattractants, guiding the neutrophils out of the vasculature (i.e., interleukins and lipid mediators) and those that guide the cells through the interstitium to a site of infection (i.e. bacterial chemoattractants and activated complement factors) . Our results suggest that TNF-α induces a neutrophil state that shares basic the signaling properties with the FPR*des, FPRLl*des and C5aR*des, i.e., a state that is reactivated when the cytoskeleton is disrupted by cytochalasin B.
Neutrophils contain known, and probably also unknown, secretable chemoattractants [24, 40], as well as known and possibly not yet identified receptors for such agonists (i.e., CXCR1 and 2 for IL-8 and FPRL1 for LL37). The combined TNF-α/cytochalasin B-dependent activation process was pertussis toxin sensitive suggesting that the activation process involves a heterotrimeric G-protein, possibly (but not known for certain) linked to a receptor. An attractive hypothesis that directly explains our results would be that TNF-α induces secretion of an endogenous agonist which binds back to a neutrophil receptor sharing its basic signaling properties with the cytoskeleton-regulated group of GPCRs. This endogenous agonist would occupy its surface receptors and being a ligand-receptor pair of the cytoskeleton-binding type, the receptors would then become desensitized upon agonist binding and the cells would be transferred into a cytochalasin B-activated state. Although we can not exclude this possibility, the experimental evidence presented is inconsistent with this scenario. On the one hand pretreatment with secretagogues induces mobilization of neutrophil storage organelles, but on the other hand, such a mobilization is insufficient for induction of the cytochalasin B sensitive state as illustrated by the finding that IL8, a potent secretagogue (Fig 9), failed to prime neutrophils for subsequent activation by cytochalasin B (Table 1). In addition, we assumed that provided that a hypothetical agonist secreted in response to TNF-α has a binding affinity for its receptor, that is of the same magnitude as the hitherto identified/characterized neutrophil activators, it should be present in the extracellular environment, but despite the use of several experimental approaches we were unable to find any evidence for the existence of such an agonist.
Over the past few years, studies on the biology of GPCR have been focused on the activation achieved through binding of a specific agonist to its receptor. It has, however, become increasingly clear that GPCRs can be transferred from a non-signaling R state to an actively signaling R* state also in the absence of any bound ligand [19, 41]. It is obvious that the R* state can not be constitutively active, and irrespectively of whether the receptor reaches the R* state following agonist binding or if this state is reached independently of any activating agonist, the deactivation mechanisms appears to be put in action. The physical separation of the R* from the G-protein occurring through a linkage of the receptor to the cytoskeleton, constitute an important termination mechanism, and R*des state can be reversed through an uncoupling from the cytoskeleton . The fact that we were unable to find any secreted components that could fulfill the role of a receptor agonist in the TNF-α/cytochalasin B dependent activation system, suggests that TNF-α may transfer neutrophil GPCR's to a cytoskeleton associated R*des state independently of agonist binding.
A transformation from R to R* can, thus, occur in the absence of a specific ligand, but very little is known about the regulatory mechanisms that determines the rate of transfer or the levels of the two forms at equilibrium. The R/R* equilibrium differs for individual wild-type isoforms of a given receptor, and the ratio between the two forms can also be changed both by defined point mutations localized to several different intracellular receptor domains, as well as by the degree of glycosylation of the receptor . It seems reasonable to hypothesize that the R/R* transformation rate and/or ratio at equilibrium can be changed not only through direct structural changes in the receptor protein, but also indirectly through signals generated by a second receptor such as the one for the TNF-α (receptor communication), or through a direct receptor-receptor interaction in the membrane.
Neutrophils triggered with TNF-α mobilize receptor storing organelles and concomitantly but independently, the cells are primed to respond to the microfilament disrupting toxin cytochalasin B. These findings are suggestive of two mechanisms involved in neutrophil priming by TNF-α. While the enhanced response to fMLF is explained by recruitment of new formyl peptide receptors to the cell surface, the cytochalasin B-sensitive state is achieved through a novel mechanism that transfers G-protein coupled receptors to a primed state which is fully activated when cytochalasin B uncouples the receptors from the cytoskeleton. Such a reaction could possibly be induced by an endogenous secreted receptor agonist. We were, however, unable to find any components that could fulfill the role of an agonist in the TNF-α/cytochalasin B dependent activation system. The presented data support the recently introduced concept that receptor activation can occur independently of a specific receptor ligand . Accordingly, we suggest that TNF-α transfers neutrophil receptors to a desensitized and cytoskeleton associated state independently of agonist binding.
Isolation of neutrophils
Leukocytes were isolated from freshly prepared leukopacks (The Blood Center, Sahlgrenska University Hospital, Göteborg) obtained from healthy blood donors. After removal of erythrocytes through dextran sedimentation, the leukocyte-rich supernatant was carefully layered onto Ficoll-Paque (Lymphoprep, Nyegaard, Norway). After centrifugation at 380 × g for 30 minutes, the pellet was resuspended in Krebs-Ringer phosphate buffer (KRG, pH 7.3; 120 mM NaCl, 5 mM KC1, 1.7 mM KH2PO4, 8.3 mM NaHPO4 and 10 mM glucose) supplemented with Ca2+ (1 mM) and Mg2+ (1.5 mM), and kept on melting ice until used in experiments .
The hexapeptide Trp-Lys-Tyr-Met-Val-Met-NH2 (WKYMVM) was synthesized and HPLC-purified by Alta Bioscience (University of Birmingham, Birmingham, United Kingdom). The formylated peptide N-formylmethionyl-leucyl-phenylalanine (fMLF), the chemotactic fragment of complement factor 5 (C5a), platelet activating factor (PAF), and TNF-α were from Sigma Chemical Co., St. Louis. Human recombinant IL-8 was provided by R&D Systems, Oxon, UK. The peptide agonists were dissolved in dimethyl sulfoxide to 10-2M and stored at -70°C until use. Further dilutions were made in KRG.
Neutrophil priming and NADPH-oxidase activity
Neutrophils (1–2 × l06cells/ml) were incubated at 37°C for 5–40 minutes in the presence or absence (control) of a priming agent. The NADPH-oxidase activity of these cells was then recorded using luminol/isoluminol-enhanced chemiluminescence (CL) systems . The CL activity was measured in a six-channel Biolumat LB 9505 (Berthold Co. Wildbad, Germany), using disposable 4-ml polypropylene tubes with a 0.90-ml reaction mixture containing 1–2 × 106 neutrophils. In order to differentiate between intracellularly and extracellularly generated reactive oxygen species, two different reaction mixtures were used. Tubes used for the measurement of extracellular release of superoxide anion contained neutrophils, horseradish peroxidase (HRP; a cell impermeable peroxidase; 4 U) and isoluminol (a cell impermeable CL substrate; 2 × 10-5M). Tubes used for measurement of intracellular generation of reactive oxygen species contained neutrophils, SOD (a cell impermeable scavenger for O2-; 50 U), catalase (a cell impermeable scavenger for H2O2; 2000 U), and luminol (a cell permeable CL substrate; 2 × 10-5M). The tubes were equilibrated at 37°C, after which the stimulus (0.1 ml) was added. The light emission was recorded continuously.
Determination of receptor exposure
The amount of fMLF-receptors expressed on the cell surface in the different neutrophil populations was determined by incubating the cells with radiolabeled fMLF in the presence or absence of unlabeled fMLF. Unbound peptide was removed by centrifugation of the cells through an oil layer. The oil layer which was composed of a mixture of dibutylphtalate and dinonylphtalate (10:3, v/v; 100 μl) was layered on top of 10 μl of urea (6 M) in Eppendorf tubes. The radiolabeled peptide (50 μl [3H]fMLF; 8 × 10-8M) was then layered on top of the oil followed by 50 μl of unlabeled fMLF (4 × 10-5M) in KRG or vehicle alone. Neutrophils (2 × 106, 100 μl) were added to the fMLF-solution and the tubes were incubated on melting ice for 1 h. After centrifugation at 9000 × g for 15 seconds in a Beckman microfuge (Beckman Instruments, Fullerton, CA), the bottom of the centrifuge tubes (containing the pelleted cells) was excised and collected for determination of radioactivity.
To measure surface exposure of CR3, the cells were labeled with phycoerythrin-conjugated monoclonal antibodies specific for CD11b (DAKO M741; 10 μl to a cell pellet of 106). The cells were examined by use of flow cytometry (FACScan; Becton Dickinson, Mountain View, CA).
Deactivation and reactivation
In order to deactivate neutrophils to GPCR agonists, the cells were first equilibrated for 10 min at 15°C. The agonist, e.g., the chemoattractant fMLF (final concentration 10-7M) was added and the incubation was continued at 15°C for an additional 5 min . The cells were then transferred to 37°C for 5 minutes, and reactivation was subsequently achieved by adding cytochalasin B (5 μg/ml).
Human recombinant TNF-α, fMLF, luminol, isoluminol, cycloheximide, Pertussis toxin, SB 203580 and cytochalasin B were obtained from Sigma Chemical Co., St. Louis. Superoxide dismutase (SOD), catalase and horse radish peroxidase (HRP) were from Boehringer-Mannheim, Germany. Radiolabeled fMLF was from Du Pont NEN (Boston, Mass).
The Student's t-test (two-tailed) was performed to determine statistical significance.
List of abbreviations
cluster of differentiation number 11b
chemiluminescence, GPCR, G-protein coupled receptor, CR3, complement receptor 3, CXCR, the IL 8 (CXC cytokine) receptor, C5a, the chemotactic split product from complement factor 5
the C5a receptor, fMLF, formylmet-leu-phe
the formyl peptide receptor
the formyle peptide like receptor 1
tumor necrosis factor, TNFR, the TNF-recepor
Research grants: Supported by the Swedish Medical Research Council, King Gustaf the V's 80-year foundation and the Swedish Society for Medical Research.
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