Pertussis toxin as a probe of neutrophil activation

In reviewing our own and other work, it is clear that pertussis toxin treatment of neutrophils causes a time- and concentration-dependent inhibition of granule enzyme secretion induced by formylmethionylleucylphenylalanine (fMet-Leu-Phe), C5a, leukotriene (LT) B4 and platelet-activating factor (PAF). Chemotaxis, O2- generation, aggregation, and arachidonic acid production induced by fMet-Leu-Phe are also inhibited by pertussis toxin. Granule enzyme release caused by A23187 or phorbol 12-myristate 13-acetate is not inhibited. The inhibition of neutrophil function correlates closely with the NAD-ribosylation of a 41,000-dalton protein in the neutrophil plasma membrane, presumably the GTP-binding regulatory protein Ni. Pertussis toxin treatment prevents or obtunds the increased influx of Ca2+ induced by fMet-Leu-phe and LTB4, but not that caused by stimulation of neutrophils with PAF. Pertussis toxin prevents the receptor-induced breakdown of polyphosphoinositides in intact neutrophils and isolated membrane and prevents or decreases the production of inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol. The hypothesis advanced by us and others is that pertussis toxin interacts with a GTP-binding regulatory protein identical or similar to Ni, which couples receptor-chemotactic factor interaction to phospholipase C activation. Inhibition of the activation prevents the production of IP3 and the resulting release of Ca2+ from intracellular stores and of 1,2-diacylglycerol and thus, the activation of protein kinase C. The lack of these two mediators is the immediate cause of the depression of neutrophil activation resulting from pertussis toxin. Some of the limitations and uncertainties of our present knowledge with respect to this hypothesis are discussed.     

Mechanism involved in the mobilization of neutrophil calcium by 5-hydroxyeicosatetraenoate

5-Hydroxyeicosatetraenoate (5-HETE), like leukotriene B4 and platelet-activating factor, stimulated human polymorphonuclear neutrophils to mobilize intracellular calcium. The three compounds acted through mechanisms that were inhibited by pertussis toxin, cholera toxin, and PMA. Each agonist, furthermore, desensitized (or down-regulated) the neutrophil's calcium mobilization response to a second challenge with the same agonist. However, 5-HETE and leukotriene B4 had little or no activity in cross-desensitizing neutrophil responses to each other or to platelet-activating factor. Furthermore, 5-HETE interfered minimally or not at all with the binding of radiolabeled leukotriene B4 and platelet-activating factor to their respective receptors on neutrophils. Thus, 5-HETE mobilizes neutrophil calcium by a mechanism different from those used by leukotriene B4 and platelet-activating factor. This mechanism appears to involve specific 5-HETE receptors that couple to pertussis toxin-inhibitable, GTP-binding proteins.     

The anti-infective peptide, innate defense-regulator peptide, stimulates neutrophil chemotaxis via a formyl peptide receptor

The anti-infective peptide, innate defense-regulator peptide (IDR-1), has been selectively reported to modulate the innate immune response. We found that IDR-1 stimulates the chemotactic migration in human neutrophils. Moreover, IDR-1-induced neutrophil chemotaxis was completely blocked by pertussis toxin, suggesting the importance of the G_i protein in this process. The mechanism governing the IDR-1-induced neutrophil chemotaxis was found to be completely inhibited by the formyl peptide receptor (FPR) antagonist; cyclosporin H. IDR-1 was also found to induce chemotactic migration in FPR but not in vector-expressing HCT116 cells. Meanwhile, IDR-1 failed to stimulate superoxide anion generation and intracellular calcium increase in human neutrophils. Furthermore, IDR-1 was found to inhibit fMLF (an FPR agonist)-induced superoxide generation and calcium signaling in human neutrophils and FPR-expressing HCT116 cells. Taken together, the results demonstrate that IDR-1 is a partial agonist for FPR and further, stimulates neutrophil chemotaxis without inducing calcium signaling and superoxide generation.     

Fluoride-mediated activation of guinea pig neutrophils

In guinea pig periotoneal neutrophils NaF at a concentration of above 5 mM elicited a dose-dependent, delayed and sustained activation of NADPH oxidase. Unlike in human neutrophils, in guinea pig cells, this response was independent of extracellular calcium. Fura2 fluorescence measurements indicated also a fluoride-mediated moderate elevation in the level of cytosolic calcium concentration. Pretreatment of neutrophils with pertussis toxin, blocked fluoride-promoted activation of NADPH oxidase, indicating that NaF stimulation was mediated by a G protein which is a pertussis toxin substrate. NaF-elicited calcium elevation was insensitive to the toxin. Upon transfer of NaF-stimulated cells to a fluoride-free medium, superoxide release declined and calcium levels diminished. The response of the deactivated, fluoride-prestimulated guinea pig neutrophils to a secondary stimulation with phorbol myristate acetate (PMA) or fMet-Leu-Phe, was either unaffected by the previous challenge with NaF (PMA) or augmented by it (the chemotactic peptide). In parallel to the activation of NADPH oxidase, NaF also induced translocation of protein kinase C to cell membranes. This effect was also abolished by a pretreatment with pertussis toxin.     

Fluoride-mediated activation of guinea pig neutrophils

In guinea pig peritoneal neutrophils NaF at a concentration of above 5 mM elicited a dose-dependent, delayed and sustained activation of NADPH oxidase. Unlike in human neutrophils, in guinea pig cells, this response was independent of extracellular calcium. Fura2 fluorescence measurements indicated also a fluoride-mediated moderate elevation in the level of cytosolic calcium concentration. Pretreatment of neutrophils with pertussis toxin, blocked fluoride-promoted activation of NADPH oxidase, indicating that NaF stimulation was mediated by a G protein which is a pertussis toxin substrate. NaF-elicited calcium elevation was insensitive to the toxin. Upon transfer of NaF-stimulated cells to a fluoride-free medium, superoxide release declined and calcium levels diminished. The response of the deactivated, fluoride-prestimulated guinea pig neutrophils to a secondary stimulation with phorbol myristate acetate (PMA) or fMet-Leu-Phe, was either unaffected by the previous challenge with NaF (PMA) or augmented by it (the chemotactic peptide). In parallel to the activation of NADPH oxidase, NaF also induced translocation of protein kinase C to cell membranes. This effect was also abolished by a pretreatment with pertussis toxin.     

Intracellular calcium rise produced by platelet-activating factor is deactivated by fMet-Leu-Phe and this requires uninterrupted activation sequence: role of protein kinase C

Stimulation of the neutrophils with fMet-Leu-Phe inhibits the rise in intracellular concentration of free calcium produced by the subsequent addition of platelet-activating factor. This deactivation is not observed in pertussis toxin treated cells. In addition, preincubation of the cells with the protein kinase C activator phorbol 12-myristate 13-acetate for three minutes abolishes completely the rise in calcium produced by platelet-activating factor. This inhibition is prevented by the addition of the protein kinase C inhibitor 1-(5-isoquinoline-sulfonyl)-2-methyl piperazine prior to the addition of the phorbol ester. Phorbol 12-myristate 13-acetate, at a concentration that does not produce significant inhibition, accelerates the rate of calcium removal from the cytoplasm, and this is abolished by the protein kinase C inhibitor. In contrast, the deactivation by fMet-Leu-Phe is not prevented by the protein kinase C inhibitor. The results presented here suggest that the protein kinase C system may regulate the opening by platelet-activating factor of possible plasma membrane associated pertussis toxin independent calcium channels and/or the binding of platelet-activating factor to the receptors. In addition, protein kinase C activation increases the rates of the calcium efflux pump and/or calcium sequestering by intracellular organelles. The most simple and straightforward explanation of the observed deactivation by fMet-Leu-Phe is that the addition of fMet-Leu-Phe to neutrophils stimulates the production of platelet-activating factor which then binds to and deactivates the receptors.     

Signalling for increased cytoskeletal actin in neutrophils

The addition of fMet-Leu-Phe, platelet-activating factor, leukotriene B4 or sodium propionate to rabbit neutrophils causes an increase in the amount of actin associated with the cytoskeletal actin. The increase is rapid, transient and inhibitable by pertussis toxin. On the other hand, the addition of phorbol 12-myristate 13-acetate or NH4Cl causes a pertussis toxin-insensitive increase in cytoskeletal actin. The effects of the phorbol ester and fMet-Leu-Phe are additive, and in the presence of the phorbol ester, the fMet-Leu-Phe induced effect declines to the level produced by the phorbol ester. These results suggest that: one of the signalling pathways for actin polymerization involves a guanine-nucleotide binding protein; actin polymerization mediated through this pathway is rapid, transient and inhibitable by pertussis toxin, and a second signalling pathway is independent of this guanine-nucleotide binding protein; actin polymerization, mediated by this second pathway, is somewhat slower, sustained and insensitive to pertussis toxin. These results are discussed in terms of a model which includes gelsolin, profilin and the pertussis toxin-sensitive guanine-nucleotide binding protein.     

Granulocyte-macrophage colony-stimulating factor is a stimulant of platelet-activating factor and superoxide anion generation by human neutrophils.

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) was studied for its ability to stimulate the synthesis and release of the inflammatory mediator platelet-activating factor (PAF) from human neutrophils as measured by bioassay and incorporation of [3H]acetate into PAF. GM-CSF stimulated the synthesis but not the release of PAF from neutrophils. PAF synthesis took place in a time- and concentration-dependent manner, was dependent on a pertussis toxin-sensitive G protein and could be inhibited by antibodies to GM-CSF. On the other hand, pre-incubation of neutrophils with GM-CSF followed by stimulation with the bacterial tripeptide formylmethionylleucylphenylalanine caused PAF synthesis and release. The effect of GM-CSF was qualitative and not simply the result of larger amounts of PAF being synthesized since similar amounts were generated in response to the calcium ionophore A23187 but no released PAF could be detected. In functional studies GM-CSF stimulated superoxide anion generation from neutrophils with a time and dose relationship that paralleled PAF synthesis. In addition, the serine protease inhibitor L-1-tosylamide-2-phenylethyl chloromethyl ketone, which inhibits PAF synthesis, reduced PAF accumulation as well as superoxide generation, raising the possibility of a causal relationship between cell-associated PAF and cell activation. These results identify PAF as a direct product of GM-CSF stimulation in neutrophils where it may play a role in signal transduction and demonstrate that PAF is released only after subsequent neutrophil stimulation. The selective release of PAF may play a role in regulating and amplifying the inflammatory response.     

Activation of human neutrophils by a synthetic anti-microbial peptide, KLKLLLLLKLK-NH2, via cell surface calreticulin.

We previously reported that a synthetic anti-bacterial peptide, KLKLLLLLKLK-NH2 (L5), showed significant chemotherapeutic activity in methicillin-resistant Staphylococcus aureus-infected mice, and its ability to activate human neutrophils was related to its chemotherapeutic activity. In this study, we found that activation of neutrophils by L5 was inhibited by pertussis toxin, suggesting that GTP-binding protein (G-protein) participates in this process. We isolated an L5-binding protein, which turned out to be human calreticulin, with a molecular mass of 60 kDa from neutrophil membranes. From experiments using an anti-calreticulin antibody, we proposed that calreticulin is partly localized on the surface of neutrophils, and L5-bound calreticulin transmits a signal into cells via G-protein to activate neutrophils to generate superoxide anion.     

Activation of human neutrophils by Mycobacterium tuberculosis-derived sulfolipid-1.

The principal sulfatide of a group of acidic lipids from virulent Mycobacterium tuberculosis, sulfolipid-1 (SL-1), stimulates neutrophil superoxide (O2-) generation and, at lower concentrations, primes neutrophil response to several other metabolic agonists including FMLP, and PMA. These responses to SL-1 were examined in relation to diacylglycerol (DAG) generation, Ca2+ availability and activation of guanine nucleotide binding proteins to clarify the signal transduction pathways involved. Pertussis toxin inhibited the ability of SL-1 to both stimulate neutrophils directly and to prime neutrophils for subsequent responses induced by PMA, suggesting a role for one or more guanine nucleotide regulating proteins in both responses. SL-1 induced a rise in neutrophil DAG levels. DAG generation was inhibited by pretreatment of cells with pertussis toxin. Depletion of extracellular Ca2+ ablated O2- release induced by stimulatory levels of SL-1 but did not inhibit the priming effect induced by substimulatory concentrations of the lipid. Investigation of the activation of the neutrophil NADPH oxidase in a cell-free system revealed that the SL-1-priming effect was associated with translocation of the soluble cytosolic factors required for activation of the enzyme. Cytosolic factor translocation was not observed in pertussis toxin pretreated cells. Our results provide evidence for the role of a guanine nucleotide binding protein in both priming and direct activation of neutrophils by SL-1. This G protein regulates both SL-1-induced DAG generation and cytosolic cofactor translocation involved in neutrophil activation and priming. The multiplicity of effects of SL-1 on signal transduction pathways leading to phagocyte activation and priming may exert a profound influence on the pathogenicity of M. tuberculosis.     

Arachidonic acid as a second messenger. Interactions with a GTP-binding protein of human neutrophils.

Arachidonic acid (20:4) and other cis-unsaturated fatty acids exert direct effects on a variety of cells, effects that do not depend on the metabolism of fatty acids via cyclooxygenase or lipoxygenase pathways. In these studies arachidonic acid and other cis-unsaturated fatty acids (but not trans-unsaturated or saturated fatty acids) increased the specific binding of the nonhydrolyzable analog of GTP, [35S]GTP gamma S, to purified neutrophil membrane preparations and elicited superoxide anion generation from intact neutrophils. There was a positive correlation (r = 0.70) between the capacity of fatty acids to increase nucleotide binding and to elicit the respiratory burst. Scatchard plot analysis of binding at equilibrium demonstrated an increase in the number of available GTP binding sites in the presence of 50 microM arachidonic acid. Nonsteroidal antiinflammatory agents interfered with the arachidonic acid effect on [35S]GTP gamma S binding. ADP-ribosylation of the pertussis toxin substrate Gi alpha within the plasmalemma-reduced specific [35S]GTP gamma S binding and blocked arachidonate-dependent enhancement of binding. Moreover, pertussis toxin treatment of intact neutrophils inhibited arachidonic acid-induced superoxide anion generation. The data indicate that arachidonic acid directly activates a GTP binding protein in the neutrophil plasma membrane and may thereby act as a second messenger in signal transduction.     

Further evidence for the involvement of a phosphoprotein in the respiratory burst oxidase of human neutrophils.

Phosphorylation of a 47 kDa protein in human neutrophils is induced by phorbol 12-myristate 13-acetate (PMA), opsonized latex beads, fMet-Leu-Phe, calcium ionophore A23187 and fluoride. All of these stimuli activate the specialized microbicidal respiratory burst of neutrophils, and in each case the kinetics of activation correspond with the kinetics of phosphorylation of the 47 kDa protein. Trifluoperazine (50 microM) and chlorpromazine (100 microM), inhibitors of calmodulin and protein kinase C, abolish the increase in oxygen consumption and selectively prevent phosphorylation of the 47 kDa protein after PMA stimulation. Treatment of neutrophils with pertussis toxin totally inhibits both superoxide production and phosphorylation of this protein in response to fMet-Leu-Phe, but not in response to PMA, indicating that a GTP-binding protein modulates the fMet-Leu-Phe receptor signal. Phosphorylation of the 47 kDa protein, a phenomenon absent from the neutrophils of subjects with autosomal recessive chronic granulomatous disease, which lack the respiratory burst, appears to be the common trigger for activation of the burst in normal neutrophils.     

Tyrosine phosphorylation in human neutrophil

Protein tyrosine phosphorylation in human neutrophils was examined by immunoblotting with antibodies specific for phosphotyrosine. The addition of the human hormone granulocyte-macrophage colony stimulating factor to human neutrophils caused an increase in the tyrosine phosphorylation levels of several proteins. The increases in at least two of these proteins having molecular masses of 40 kDa (p40) and 54 kDa (p54) were rapid and were inhibited in pertussis toxin treated cells. The newly synthesized tyrosine kinase inhibitor ST 638 inhibited the increases in the levels of the tyrosine phosphorylation in p92, p78, p54 and p40 proteins. The epidermal growth factor receptor tyrosine kinase inhibitors were less effective. The addition of the chemotactic factor fMet-Leu-Phe to human neutrophils also caused an increase in tyrosine phosphorylation in some of these proteins. The pattern of the fMet-Leu-Phe-induced tyrosine phosphorylation was different from that produced by GM-CSF. The increases were also inhibited by ST 638. In addition, ST 638 inhibited superoxide production but not actin polymerization in control and GM-CSF-treated cells stimulated with fMet-Leu-Phe. Moreover, the active but not inactive phorbol esters increase the tyrosine phosphorylation only in the 40 kDa protein. These results suggest several points: (a) some of the responses produced by GM-CSF and fMet-Leu-Phe are mediated through tyrosine phosphorylation, (b) the GM-CSF receptor is coupled to a pertussis toxin sensitive G-protein, (c) the 40 kDa protein is probably the Gi alpha 2, and (d) the 78 or the 92 kDa protein is most likely the receptor for GM-CSF, which indicates that the receptor may have a tyrosine kinase domain.     

Platelet-activating factor activates two distinct effector pathways in human eosinophils

In granulocytes, platelet-activating factor (PAF) shares many of its biological effects with other chemotactic factors, such as FMLP, complement fragments, and lipid mediators. Two unique effects are that PAF is relatively resistant to pertussis toxin (PTX) and that PAF activates the inflammatory functions of eosinophils more strongly than it activates those of neutrophils. To investigate the molecular mechanisms of the responses of eosinophils to PAF, we analyzed superoxide anion production by a chemiluminescence method that provides real-time kinetic data for the cellular responses. We found that PAF induced bimodal superoxide anion production in human eosinophils, consisting of an intense, but transient, first phase and a larger and sustained second phase. In contrast, PAF induced essentially a transient unimodal response in human neutrophils. The two phases of eosinophil response were mediated by distinct cellular mechanisms: the second phase was highly dependent on cellular adhesion and beta(2) integrins, but the first phase was independent of both adhesion and beta(2) integrins. The upstream signaling mechanisms were also different: the second phase was mediated by PTX-resistant G-protein(s) and through activation of phosphatidylinositol 3-kinase, while the first phase was mediated by PTX-sensitive G-protein(s). Furthermore, the second-phase response was approximately 100-fold more resistant to inhibition by a competitive PAF receptor antagonist than the first phase. Thus, eosinophils and neutrophils react differently to PAF, and PAF activates two separate and distinct effector pathways in human eosinophils. These two activation pathways may explain the eosinophils' strong and diverse biological responses to PAF.     

Neutrophil activation by surface bound IgG: pertussis toxin insensitive activation

Surface bound IgG induces neutrophil degranulation and production of superoxide radicals by a mechanism that is not inhibited by either pertussis toxin or cholera toxin, whereas these functions induced by soluble mediators such as FMLP and soluble aggregates of IgG are profoundly inhibited by pertussis toxin. Interaction of neutrophils with surface bound IgG triggers the loss of 32P labeled PIP2 and PIP and the influx of extracellular calcium. Neither of these cellular events when induced by surface bound IgG is inhibited by pertussis toxin. These observations suggest that neutrophil activation induced by surface bound IgG proceeds along a pathway which is not regulated by proteins which are inhibited by either pertussis or cholera toxins.     

Propionic acid-induced calcium mobilization in human neutrophils

The ability of propionic acid to elicit an increase in the level of cytoplasmic free calcium in human neutrophils was examined in detail. Propionic acid induced a rapid and dose-dependent mobilization of calcium that relied on both internal and external sources of calcium. The effects of propionic acid on the mobilization of calcium were inhibited by pertussis toxin, but not cholera toxin, implicating a guanine nucleotide binding protein. Furthermore, preincubation of the neutrophils with phorbol 12-myristate 13-acetate resulted in a decreased mobilization of calcium. This inhibitory activity of phorbol myristate acetate was antagonized by the protein kinase C inhibitor H-7. Preincubation of the cells with the synthetic chemotactic factor fMet-Leu-Phe caused a reduction in the magnitude of the calcium transient elicited by propionic acid. However, the calcium response to propionic acid was not affected by antagonists of fMet-Leu-Phe and platelet-activating factor binding or by an inhibitor of leukotriene synthesis. Propionic acid did not elicit a mobilization of calcium in monocytes, platelets, lymphocytes, or undifferentiated HL-60 cells. However, the treatment of the HL-60 cells with dimethylsulfoxide resulted in the appearance of a calcium response to propionic acid. The potential physiological significance of these findings are discussed.     

A step sensitive to pertussis toxin and phorbol ester in human neutrophils regulates chemotaxis and capping but not phagocytosis

Treatment of human neutrophils with pertussis toxin (PT) abolishes chemotaxis in response to either platelet-activating factor (PAF) or f-Met-Leu-Phe (FMLP), and capping induced via the concanavalin A (Con A) receptor. These functional effects are accompanied by the inhibition of calcium mobilization by PAF, FMLP and Con A. The agent phorbol 12-myristate-13-acetate (PMA) also inhibits chemotaxis and capping as well as calcium mobilization by these receptors. In sharp contrast, neither PT, cholera toxin (CT), nor PMA, inhibits the phagocytosis of non-opsonized and opsonized Candida albicans, sheep erythrocytes or fluorescent latex beads. Our results suggest that receptor-initiated chemotaxis and capping involve a step that is sensitive to PT and PMA, and that phagocytosis is not regulated in a similar fashion.     

The priming of neutrophils by lipopolysaccharide for production of intracellular platelet-activating factor. Potential role in mediation of enhanced superoxide secretion

LPS priming of the neutrophil results in enhanced release of superoxide upon subsequent stimulation, but the mechanism of this effect remains obscure. The recent recognition that neutrophils synthesize and retain platelet-activating factor within the cell led us to hypothesize that enhanced synthesis of platelet-activating factor in the LPS-primed cell might account for the observed effects of lipopolysaccharide. Using human neutrophils isolated on plasma-Percoll gradients, we found that incubation with 100 ng/ml LPS for 60 min resulted in a small but significant increase in intracellular platelet-activating factor assessed after lipid extraction, TLC, and bioassay. The further stimulation of primed neutrophils with FMLP resulted in a marked increase in neutrophil platelet-activating factor compared with non-LPS-treated controls. The priming effect of LPS was time dependent (30 to 60 min), dose dependent, and inhibited at 0 degree C and did not require protein synthesis. Platelet-activating factor so generated was not released but rather retained within the neutrophil, and the molecular species of platelet-activating factor produced was predominantly 1-O-hexadecyl-2-acetyl-sn-3-phosphorylcholine. Platelet-activating factor production in LPS-treated neutrophils was also enhanced by PMA, suggesting that receptor-mediated events could not account exclusively for the enhancement. Considering the ability of nanomolar concentrations of exogenously added platelet-activating factor to prime the neutrophil for enhanced release of superoxide, the rapid intracellular accumulation of platelet-activating factor that accompanies stimulation of an LPS-primed cell by FMLP may modulate the secretory events that accompany such stimulation.     

Granulocyte-macrophage colony-stimulating factor primes neutrophils by activating a pertussis toxin-sensitive G protein not associated with phosphatidylinositol turnover

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic cytokine which produces diverse biological effects in target cells of myeloid origin. GM-CSF enhances the production of superoxide anion (O2-) by mature neutrophils in response to chemotactic peptides such as formyl-methionyl-leucyl-phenylalanine (fMLP), but alone it has no effect on this system. This process has been termed "priming." fMLP activates neutrophils via a pertussis toxin-sensitive GTP-binding protein, leading to the rapid production of the second messengers diacylglycerol (DAG) and inositol trisphosphate, via phosphatidylinositol turnover, and arachidonic acid (AA) by a presumptive phospholipase A2-mediated mechanism. All three second messengers may lead to the generation of O2-. We investigated the effect of priming of GM-CSF on these systems. GM-CSF had no effect on fMLP-stimulated DAG and inositol trisphosphate levels, nor did it amplify the response to exogenously added phorbol ester (to mimic the action of DAG) or calcium ionophore. Neutrophils primed with the cytokine showed a small, but significant, enhancement of fMLP-stimulated AA release. Compared with unprimed controls, primed neutrophils also showed a significant increase in O2- production when stimulated with either AA or the nonhydrolyzable GTP analogue, GTP-gamma-S. The magnitude of enhanced O2- production was similar to that observed after fMLP treatment of primed cells. All of these effects, including the increased sensitivity to AA treatment, were inhibited by pertussis toxin. These data show that GM-CSF primes neutrophils by modulating the activity of at least one pertussis toxin-sensitive G protein coupled to a metabolic pathway that mobilizes and utilizes arachidonic acid.     

Reactivation of formyl peptide receptors triggers the neutrophil NADPH-oxidase but not a transient rise in intracellular calcium

In neutrophils, coupling of chemoattractants to their cell surface receptor at low temperature (相似文献