Ca2+-independent synergistic augmentation of O2- production by FMLP and PMA in HL-60 cells

N-Formyl-Met-Leu-Phe (FMLP) and phorbol 12-myristate 13-acetate (PMA) caused a synergistic augmentation of superoxide anion (O2-) production in neutrophil-like HL-60 cells differentiated with dibutyryl cAMP. The present study was undertaken to investigate the mechanism of the synergistic augmentation of O2- production. FMLP increased intracellular free Ca2+ concentration ([Ca2+]i), which was slightly suppressed by PMA and completely inhibited by an intracellular Ca2+ chelating agent, O,O'-bis(2-aminophenyl)ethyleneglycol-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM). Although FMLP-induced O2- production was inhibited by BAPTA-AM, a major part of the synergistic augmentation of O2- production by FMLP and PMA remained after BAPTA-AM treatment, suggesting that a Ca2+-independent mechanism might be involved in the augmentation. FMLP and PMA caused an activation of phospholipase D (PLD) almost additively in a Ca2+-sensitive manner. The synergistic activation of mitogen-activated protein kinase (MAPK) was evoked by combined addition of PMA and FMLP in a BAPTA-AM resistant manner. However, PD98059, a MAPK kinase inhibitor, did not affect the synergistic augmentation of O2- production, although it potently inhibited the synergistic augmentation of tyrosine phosphorylation of MAPK. Wortmannin, a phosphatidylinositol 3-kinase inhibitor, inhibited FMLP-induced O2- production, but it did not inhibit the synergistic augmentation of O2- production by PMA and FMLP. In contrast, staurosporine and GF109203X, protein kinase C inhibitors, reduced the synergistic augmentation induced by PMA and FMLP. In addition, pertussis toxin (PT) abolished the synergistic augmentation of O2- production. It is concluded that the synergistic augmentation of O2- production induced by PMA and FMLP is mediated through a PT-sensitive G protein and a protein kinase C in a Ca2+-independent manner.     

Cyclosporin A rapidly inhibits mediator release from human basophils presumably by interacting with cyclophilin.

We have examined the effects of cyclosporin A (CsA) and a series of CsA analogs that bind with decreasing affinity to cyclophilin, to evaluate the involvement of this protein in the release of preformed (histamine) and de novo synthesized (peptide leukotriene C4; LTC4) mediators of inflammatory reactions from human basophils. CsA (8 to 800 nM) concentration-dependently inhibited (5 to 60%) histamine release from peripheral blood basophils challenged with anti-IgE. CsA was more potent (92.6 +/- 1.8 vs 59.1 +/- 4.5%; p less than 0.001) and, at low concentrations, more effective when the channel-operated influx of Ca2+ was bypassed by the ionophore A23187 (IC40 = 24.1 +/- 3.9 vs 105.5 +/- 22.2 nM; p less than 0.05). CsA had no effect on the release of histamine caused by phorbol myristate and bryostatin 1 that activate different isoforms of protein kinase C. Inhibition of histamine release from basophils challenged with anti-IgE was not abolished by washing (three times) the cells before anti-IgE challenge. CsA also inhibited the de novo synthesis of LTC4 from basophils challenged with anti-IgE. The inhibitory effect of CsA was very rapid, and the drug, added from 1 to 10 min during the reaction, inhibited the ongoing release of histamine caused by anti-IgE and by A23187. The experiments with CsA analogs (CsG, CsC, CsD, and CsH) showed that CsH, which has an extremely low affinity for cyclophilin, has no effect on basophil mediator release. In addition, there is a significant correlation between the concentrations of CsA, G, C, and D that inhibited by 30% the histamine release induced by anti-IgE (r = 0.99; p less than 0.001) and by A23187 (r = 0.87; p less than 0.001) and their affinity for cyclophilin.     

Characterization of eosinophil cell activation by peptides. Differential effects of substance P, melittin, and FMET-Leu-Phe.

The ability of three different peptides, substance P (SP), FMLP and melittin, to activate eosinophils purified from the peritoneal cavity of human serum-treated guinea pigs was investigated. Degranulation (eosinophil peroxidase, EPO), oxidative burst (O2-), [Ca2+]i mobilization, and arachidonic acid metabolism (thromboxane B2, TXB2) were used as indices of eosinophil activation. SP (100 nM to 100 microM), FMLP (1 to 100 microM) and melittin (10 nM to 100 microM) induced EPO release but only FMLP (1 to 100 microM) led to an elevation of [Ca2+]i. The melittin- and SP-induced EPO secretion occurred at both cytotoxic and noncytotoxic concentrations as assessed by lactate dehydrogenase release. In addition, the effect of SP was not inhibited by the SP analogue (D-Pro4, D-Trp7,9,10)SP(4-11) and SP failed to promote the generation and subsequent release of TXA2. In contrast, FMLP (10 to 100 microM) stimulated the release of TXB2 from guinea pig eosinophils that was selectively inhibited by pretreatment of the cells with BOC-FMLP. On an equimolar basis (1 microM), melittin was approximately fivefold more active at promoting TXB2 release than FMLP. The results indicate that eosinophils respond to the three peptides, SP, melittin, and FMLP in differential fashion. We conclude that activation of guinea pig eosinophils by FMLP is likely to be receptor-mediated whereas the actions of SP and melittin may act through nonspecific peptide-membrane phospholipid interactions.     

Effect of tumor necrosis factor-alpha on the stimulus-coupled responses of neutrophils and their modulation by various inhibitors.

Preincubation of human peripheral blood polymorphonuclear leukocytes (HPPMN) with recombinant human tumor necrosis factor-alpha (rHuTNF-alpha) enhanced the formylmethionyl-leucylphenylalanine (FMLP)-induced superoxide (O2-.) generation in a concentration- and preincubation time-dependent manner. The enhancement was very high for the FMLP- or opsonized zymosan (OZ)-induced O2-. generation, but was low for arachidonic acid (AA)- and phorbol myristate acetate (PMA)-induced O.2- generation. The rHuTNF-alpha has no effect on the steady state of intracellular calcium ion concentration ([Ca2+]i) nor on the membrane potential of neutrophils. The rHuTNF-alpha-primed FMLP-induced O2-. generation was inhibited by nicotineamide (NA), pertussis toxin (PT), and by the tyrosine kinase (TK) inhibitor, genistein, but was enhanced by the protein kinase C (PKC) inhibitor, H-7 (1-(5-isoquinolinesulfonyl)-3-methyl-piperazine). The inhibitory actions of NA and PT were also observed in in vivo primed guinea pig peritoneal neutrophils (GPtPMN). However, FMLP-induced O2-. generation of GPtPMN was enhanced by genistein, but was inhibited by H-7. These data indicate that TNF-alpha does not induce changes in [Ca2+]i nor in membrane potential of HPPMN, and that TNF-alpha-primed FMLP-induced O.2- generation of HPPMN is coupled with ADP-ribosylation and activation of G-proteins, and that protein kinases, especially TK, seem to exert an important role in the priming action of TNF.     

Formyl peptide stimulates and ATP gamma S potentiates [3H]cytidine 5'-diphosphate diglyceride accumulation in human neutrophils.

Although it is evident that the chemotactic peptide FMLP activates O2-formation in neutrophils via the phosphoinositidase-mediated second messenger system, it is less clear how endogenous priming agents such as ATP and platelet activating factor potentiate FMLP action. In our study, we have examined the possible effects of the stable ATP analog adenosine 5'-O-[3-thiotriphosphate] (ATP gamma S) on cellular levels of inositol 1,4,5-trisphosphate, [Ca2+]i and diglyceride (DG), in resting and in FMLP-stimulated neutrophils. Although all three measures were increased in the presence of FMLP, only the increase in DG was enhanced by pretreatment (priming) with ATP gamma S. We also measured the accumulation of the phosphoinositide cycle intermediate cytidine 5'-diphosphate (CDP)-DG to assess possible effects of priming on phosphoinositide resynthesis. The addition of FMLP to [3H]cytidine-prelabeled neutrophils elicited an increase in the accumulation of [3H]CDP-DG that was maximally enhanced in cells that were pretreated with cytochalasin B. The stimulated accumulation of [3H]CDP-DG was completely reversed by the addition of myo-inositol. Treatment of [3H]cytidine-prelabeled neutrophils with ATP gamma S (10-100 microM) resulted in a dose-dependent synergistic increase in FMLP-stimulated [3H]CDP-DG accumulation, whereas ATP gamma S alone had no effect. The observed increases in DG and in [3H]CDP-DG, in contrast to inositol 1,4,5-trisphosphate and [Ca2+]i responses, correlates well with the ATP gamma S-priming of FMLP-induced O2-formation. A similar potentiation of FMLP-induced stimulation of CDP-DG formation was also observed with platelet-activating factor. It is proposed that the priming of FMLP responses in neutrophils proceeds via a mechanism that selectively enhances DG production through a mechanism that is independent of FMLP-induced breakdown of phosphatidylinositol bisphosphate.     

Differential modulation by N4, 2'-O-dibutyryl cytidine 3':5'-cyclic monophosphate of neutrophil activation.

The cyclic pyrimidine nucleotide, cCMP, is an endogenous substance in mammalian cells but little is known on its functional role. We studied the effects of cCMP, its cell-permeant analogue, N4,2'-O-dibutyryl cytidine 3':5'-cyclic monophosphate (Bt2cCMP), and of butyrate on superoxide (O2-) formation and cytosolic Ca2+ [( Ca2+]i) in human neutrophils. Bt2cCMP inhibited O2- formation and the rise in [Ca2+]i induced by a chemotactic peptide at submaximally effective concentrations. O2- formation induced by platelet-activating factor was potentiated by Bt2cCMP, whereas the cyclic nucleotide had no effect on the rise in [Ca2+]i induced by this agonist. Bt2cCMP enhanced O2- formation induced by tau-hexachlorocyclohexane at a submaximally effective concentration. O2- formation stimulated by complement C5a, concanavalin A, NaF, A 23187, phorbol myristate acetate and arachidonic acid was not affected by Bt2cCMP. cCMP was less effective than Bt2cCMP to inhibit fMet-Leu-Phe-induced O2- formation, and butyrate was without effect on any of the functional parameters studied. Our data show that a cell-permeant analogue of cCMP differentially inhibits and potentiates activation of human neutrophils.     

Stimulus-response coupling in monocytes infected with Leishmania. Attenuation of calcium transients is related to defective agonist-induced accumulation of inositol phosphates.

Mononuclear phagocytes infected with Leishmania have been shown to have defective responses to extracellular stimuli. To investigate the potential relationship of these findings to alterations in calcium-dependent signaling pathways, the regulation of [Ca2+]i concentrations was examined in human peripheral blood monocytes infected with amastigotes of Leishmania donovani. Measurements of [Ca2+]i in fura-2-loaded monocytes were made at the single cell level by microfluorimetry. In normal monocytes, resting [Ca2+]i was 56 +/- 2 nM (mean +/- SEM). In contrast, in monocytes infected with Leishmania there was an approximately twofold increase in basal [Ca2+]i (122 +/- 5 nM, p less than 0.01 vs control). Treatment of cells with pertussis toxin before infection did not abrogate infection-induced increases in basal [Ca2+]i, suggesting that this effect was not mediated via the activation of a G protein coupled to phospholipase C. However, elevated resting [Ca2+]i did correlate with increased rates of 45Ca2+ uptake by infected monocytes. As expected, in response to treatment with 10(-7) M FMLP, control monocytes showed rapid net increases in [Ca2+]i of 303 +/- 19 nM. In contrast, net transients of [Ca2+]i in infected monocytes in response to FMLP were attenuated to only 137 +/- 9 nM (p less than 0.01 vs control). This result was not related to excess buffering of [Ca2+]i in infected cells as both control and infected monocytes showed equivalent transients of [Ca2+]i in response to the calcium ionophore A23187. Rather, inhibition of agonist-induced calcium release in infected cells appeared related to defective generation of second messenger because compared to control cells labeled with myo-[2-3H]inositol, little accumulation of inositol 1,4,5-trisphosphate was detected in infected monocytes. Attenuation of inositol phosphate accumulation and calcium release in response to chemotactic peptide correlated with decreased FMLP-induced superoxide and hydrogen peroxide production by infected monocytes. These results provide direct evidence for defective regulation of [Ca2+]i and calcium-dependent signaling in Leishmania-infected monocytes and provide a basis for understanding abnormalities in activation-related responses that involve signaling through Ca(2+)-regulated pathways.     

Human neutrophil Fc gamma RIIIB and formyl peptide receptors are functionally linked during formyl-methionyl-leucyl-phenylalanine-induced chemotaxis.

The formyl peptide receptor (FPR) and the glycosyl-phosphatidylinositol-linked type III receptor for the Fc portion of IgG (Fc gamma RIIIB; CD16) play important roles in various inflammatory responses in human neutrophils. The mechanisms of signaling by the glycosyl phosphatidylinositol-anchored Fc gamma RIIIB are not known. Therefore, we investigated the possibility that Fc gamma RIIIB and FPR may act in concert to mediate neutrophil functions. We observed that pretreatment of normal human neutrophils with Fab fragments of a mAb to the Fc gamma RIII (3G8) specifically inhibited their chemotaxis into micropore filters in response to the formylated peptides FMLP or formyl-norleucyl-leucyl-phenylalanine. Pretreatment of neutrophils with a saturating concentration of 3G8 Fab (100 nM or 5 micrograms/ml) followed by exposure to FMLP (0.5 to 500 nM) indicated that significant inhibition of chemotaxis was observed at peptide concentrations greater than 5 nM. However, 3G8 Fab had no effect on the neutrophil response to a wide range (0.05 to 500 nM) of other chemotactic factors, including C5a, leukotriene B4, IL-8 (neutrophil-activating peptide-1), and platelet-activating factor. Moreover, pretreatment of neutrophils with mAb to other cell surface molecules (decay-accelerating factor, Fc gamma RII, and HLA class I) did not affect chemotaxis to FMLP. Inhibition of movement was not due to degradation of FMLP by the cell surface endopeptidase 24.11 (CD10), because neutrophils pretreated with the CD10 inhibitor phosphoramidone and 3G8 Fab displayed the same altered response to FMLP as cells pretreated with 3G8 Fab alone. Ligation of the Fc binding site of Fc gamma RIIIB appears to be essential for altering the FMLP-induced response, since soluble aggregated IgG and other anti-Fc gamma RIII antibodies, all of which recognize the ligand binding site, mimic the inhibitory effect of the 3G8 Fab on FMLP-induced chemotaxis. In contrast, a mAb (214.1) that does not recognize the Fc binding site of Fc gamma RIIIB had no effect on FMLP-induced chemotaxis. Not only did anti-Fc gamma RIII inhibit neutrophil chemotaxis to FMLP in a filter-based migration assay, but 3G8 Fab also inhibited FMLP-induced neutrophil transendothelial migration. Scatchard plot analysis of radioligand binding experiments indicated that 3G8 Fab did not significantly alter the number of FMLP binding sites on neutrophils but significantly increased the affinity of the FPR for [3H]FMLP. Removal of greater than 80% of cell surface Fc gamma RIIIB by phospholipase C abolished the neutrophil chemotactic response to FMLP but did not affect movement toward C5a, IL-8, or leukotriene B4.(ABSTRACT TRUNCATED AT 400 WORDS)     

Chemotactic factor-induced generation of superoxide radicals by human neutrophils: evidence for the role of sodium.

The role of sodium ion in superoxide (O2-) generation by human peripheral neutrophils was investigated. Cells were activated by exposure to the synthetic tripeptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP), and O2- release was assessed by ferricytochrome c reduction after 5 min of incubation at 37 degrees C in the presence of FMLP 4 X 10(-8) M. In the absence of monovalent cations (isotonic glucose), negligible O2- generation occurred. There was a progressive increase in the magnitude of FMLP-induced O2- generation with increasing Na+ concentration up to 90 mM, where the response was noted to plateau. Varying the K+ concentration (1 to 10 mM) had no effect on the amount of O2- produced in the presence of Na+ 140 mM. FMLP also stimulated 22Na+ and 48Ca2+ uptake by the cells in a dose- and time-dependent fashion. FMLP-induced 22Na+ uptake appeared to be independent of the external Ca2+ concentration ( to 4 mM). In contrast, there was a progressive decrease in themagnitude of the FMLP-induced increase in 45Ca2+ uptake as the Na+ concentration was reduced by replacement with choline+ or glucose. These studies support a requirement for Na+ in FMLP-induced O2- generation and suggest that a Na+ influx may underlie the nature of this requirement. The data are also consistent with the hypothesis that a Na+ influx may precede the Ca2+ influx in the FMLP-induced activation sequence.     

Divergent effects of propranolol on neutrophil superoxide release: involvement of phosphatidic acid and diacylglycerol as second messengers.

Relatively high levels of propranolol (170 microM) markedly attenuated the generation of 1,2 diacylglycerol in neutrophils stimulated with either FMLP plus cytochalasin B or with 20.0 mM NaF. This effect resulted from inhibition of phosphatidic acid phosphohydrolase as it was accompanied by a corresponding increase in the recovery of phosphatidic acid in organic extracts of stimulated cells. Although propranolol enhanced phosphatidic acid levels in neutrophils treated with FMLP alone, the drug had only a slight inhibitory influence on diglyceride generation in these cells. The effect of propranolol on enhancement of PA levels in neutrophils treated with FMLP alone strongly correlated with enhancement of FMLP-induced O2- generation. However, propranolol induced a similar dose-dependent inhibition of O2- generation in neutrophils stimulated with either FMLP + cytochalasin B or with 20.0 mM NaF. These results are consistent with the hypothesis that both phosphatidic acid and diacylglycerol are required for optimal initiation of neutrophil O2- release.     

Activation of superoxide formation and lysozyme release in human neutrophils by the synthetic lipopeptide Pam3Cys-Ser-(Lys)4. Involvement of guanine-nucleotide-binding proteins and synergism with chemotactic peptides.

Upon exposure to the bacterial chemotactic peptide fMet-Leu-Phe, human neutrophils release lysozyme and generate superoxide anions (O2.-). The synthetic lipoamino acid N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteine (Pam3Cys), which is derived from the N-terminus of bacterial lipoprotein, when attached to Ser-(Lys)4 [giving Pam3Cys-Ser-(Lys)4], activated O2.- formation and lysozyme release in human neutrophils with an effectiveness amounting to about 15% of that of fMet-Leu-Phe. Palmitic acid, muramyl dipeptide, lipopolysaccharide and the lipopeptides Pam3Cys-Ala-Gly, Pam3Cys-Ser-Gly, Pam3Cys-Ser, Pam3Cys-OMe and Pam3Cys-OH did not activate O2.- formation. Pertussis toxin, which ADP-ribosylates guanine-nucleotide-binding proteins (G-proteins) and functionally uncouples formyl peptide receptors from G-proteins, prevented activation of O2.- formation by fMet-Leu-Phe and inhibited Pam3Cys-Ser-(Lys)4-induced O2.- formation by 85%. Lipopeptide-induced exocytosis was pertussis-toxin-insensitive. O2.- formation induced by Pam3Cys-Ser-(Lys)4 and fMet-Leu-Phe was enhanced by cytochalasin B, by a phorbol ester and by a diacylglycerol kinase inhibitor. Addition of activators of adenylate cyclase and removal of extracellular Ca2+ inhibited O2.- formation by fMet-Leu-Phe and Pam3Cys-Ser-(Lys)4 to different extents. Pam3Cys-Ser-(Lys)4 synergistically enhanced fMet-Leu-Phe-induced O2.- formation and primed neutrophils to respond to the chemotactic peptide at non-stimulatory concentrations. Our data suggest the following. (1) Pam3Cys-Ser-(Lys)4 activates neutrophils through G-proteins, involving pertussis-toxin-sensitive and -insensitive processes. (2) The signal transduction pathways activated by fMet-Leu-Phe and Pam3Cys-Ser-(Lys)4 are similar but not identical. (3) In inflammatory processes, bacterial lipoproteins and chemotactic peptides may interact synergistically to activate O2.- formation, leading to enhanced bactericidal activity.     

Superoxide responses to formyl-methionyl-leucyl-phenylalanine in primed neutrophils. Role of intracellular and extracellular calcium.

For superoxide (O2-) responses of human neutrophils stimulated by FMLP, experiments were designed to assess the requirement of extracellular calcium [( Ca2+]o) for priming of O2- responses by platelet-activating factor (PAF), PMA, or ionomycin. Although priming by PMA did not require [Ca2+]o, there was, as expected, a requirement for [Ca2+]o for the optimal priming effects of PAF and ionomycin. The ED50 value for [Ca2+]o in the priming function of PAF was 105 microM. The [Ca2+]o-dependent priming with ionomycin was bimodal with two ED50 values for [Ca2+]o of 90 microM and 3.2 mM. Optimal priming by PAF required at least 4-min exposure of cells to [Ca2+]o. Cells primed by PAF exhibited faster initial rates of O2-production after addition of FMLP, but the duration of O2- production was not prolonged. Whereas PAF-primed responses to FMLP are usually associated with increases in intracellular calcium [( Ca2+]i) after addition of FMLP, two conditions were found in which O2- responses to FMLP in PAF-primed cells occurred in the absence of any detectable increase in [Ca2+]i. When cells were loaded with the calcium chelator, bis-(O-aminophenoxy)-ethane-H,N,N',N'-tetraacetic acid, and then primed with PAF, normal amounts of inositol 1,4,5-trisphosphate were formed, but no increase in [Ca2+]i occurred after addition of FMLP even though the cells exhibited a fully primed O2- response; in Ca2(+)-depleted and ionomycin-permeabilized cells that were primed with PAF and then stimulated with FMLP, O2- was generated in amounts comparable to reference control (primed) cells, but there was suppressed production of inositol 1,4,5-trisphosphate and no increase in [Ca2+]i after addition of FMLP to PAF-primed cells. These data confirm the requirement of [Ca2+]o for optimal priming of neutrophils by PAF and ionomycin (but not cells primed by PMA) and indicate that, under certain conditions, generation of O2- in response to FMLP in PAF-primed neutrophils can occur independent of any increase in [Ca2+]i.     

Phospholipase A2 activation in chemotactic peptide-stimulated HL60 granulocytes: synergism between diacylglycerol and Ca2+ in a protein kinase C-independent mechanism

In dimethylsulfoxide-differentiated HL60 granulocytes, the chemotactic peptide N-formyl-Met-Leu-Phe (FMLP) augments arachidonic acid (AA) release via phospholipase A2 activity induced by the Ca2+-ionophore, A23187. Evidence indicates that this augmentation is mediated by diacylglycerols formed endogenously during FMLP receptor activation: The augmentation is mimicked by the synthetic diglyceride 1-oleoyl-2-acetyl-glycerol (OAG) and the tumor promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate; Pertussis toxin inhibits FMLP-induced augmentation but not OAG-induced augmentation: At suboptimal concentrations FMLP and OAG act cooperatively to augment ionophore A23187-induced AA release but not at optimal concentrations. These data indicate that phospholipase A2 activation in FMLP-stimulated HL60 granulocytes involves cooperative interactions between diacylglycerol formed endogenously and Ca2+. Interestingly, this effect of diacylglycerol appears not to be mediated by protein kinase C, since a specific protein kinase C inhibitor, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) does not inhibit receptor-mediated release of AA by stimulated HL60 granulocytes.     

Analysis of calcium homeostasis in activated human polymorphonuclear leukocytes. Evidence for two distinct mechanisms for lowering cytosolic calcium

The stimulation of polymorphonuclear leukocytes (PMNs) by chemoattractants triggers a rapid rise in cytosolic free calcium concentration(s) ([Ca2+]i), which quickly returns to base line, suggesting a role for calcium removal in the homeostasis of activated PMNs. To investigate cytosolic calcium homeostasis, PMNs were treated with a fluoroprobe and ionomycin to induce a sustained elevation of [Ca2+]i. The cells were then stimulated, and attenuation of the fluorescence signal was measured as an indication of calcium loss from the cytosol. The formyl peptide chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP), phorbol myristate acetate (PMA), and 1,2-dioctanoyl-sn-glycerol, but not the inactive phorbol ester 4 alpha-phorbol didecanoate, induced a dose-dependent decrease in [Ca2+]i in ionomycin-pretreated cells. However, the decline in [Ca2+]i caused by PMA was sustained and occurred following a lag time, whereas the response to fMLP was immediate, lasted approximately 2 min, and then was followed by a return of [Ca2+]i to its initial level. The restoration of [Ca2+]i required extracellular calcium. Varying the ionomycin concentration allowed studies at different initial [Ca2+]i, which in untreated PMNs was approximately 135 nM. In contrast to fMLP, PMA did not lower calcium at concentrations below 200 nM. The decline in [Ca2+]i induced by fMLP, but not PMA, was blocked by pertussis toxin. In contrast, the decrease in [Ca2+]i caused by PMA and 1,2-dioctanoyl-sn-glycerol, but not fMLP, was inhibited by the protein kinase C antagonists staurosporine, H-7, and sphingosine. These results suggest that formyl peptide chemoattractants transiently stimulate an activity which lowers [Ca2+]i to normal intracellular levels. Activation of this process appears to be independent of protein kinase C. An additional cytosolic calcium lowering activity, dependent on protein kinase C, operates at [Ca2+]i above 200 nM. Thus, activated PMNs can use at least two processes for attentuation of elevated cytosolic calcium levels.     

Signal transduction events and Fc gamma R engagement in human neutrophils stimulated with immune complexes.

Signal transduction events have been evaluated in human neutrophils stimulated with immune complexes consisting of polyclonal rabbit antibody complexed with BSA. Immune complexes induced dose-related O2- responses, but very small increases in intracellular calcium ([Ca2+]i) levels were observed, in contrast to FMLP-stimulated cells. Measurements employing [45Ca2+] demonstrated that calcium influx and efflux in cells stimulated with immune complexes was substantially less than fluxes found in FMLP-stimulated cells. With respect to inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) formation under conditions in which the O2- responses to immune complexes or FMLP were similar, the Ins(1,4,5)P3 response to immune complexes was much smaller (by 65%) as compared to that induced by FMLP. Although pertussis toxin-treated cells showed a greatly diminished O2- response (by 89%) to FMLP, the response to immune complexes was largely resistant (only 26% reduction) to the inhibitory effects of this toxin. Antibodies to Fc gamma R indicated that engagement of Fc gamma RII and Fc gamma RIII, but not Fc gamma RI, receptors was related to the O2- response of neutrophils to immune complexes. O2- formation occurred in neutrophils incubated with Staphylococcus aureus cell walls bearing antibodies to Fc gamma RII or Fc gamma RIII. These data indicate that, in human neutrophils stimulated with immune complexes, signal transduction events involve engagement of Fc gamma RII and Fc gamma RIII. The O2- response is largely pertussis-toxin insensitive, is not associated with a significant increase in levels of [Ca2+]i, and is associated with relatively little formation of Ins(1,4,5)P3. This is in contrast to cells stimulated with FMLP in which O2- responses are largely pertussis toxin-sensitive and associated with large increases in [Ca2+]i as well as formation of Ins(1,4,5)P3. Signal transduction events involving Fc gamma R appear to be quite different from those events related to engagement of FMLP receptors.     

The immunosuppressant cyclosporin A antagonizes human formyl peptide receptor through inhibition of cognate ligand binding

Cyclosporin A (CsA) is a fungus-derived cyclic undecapeptide with potent immunosuppressive activity. Its analog, cyclosporin H (CsH), lacks immunosuppressive function but can act as an antagonist for the human formyl peptide receptor (FPR). More recent studies have shown that CsA also inhibits fMLF-induced degranulation in differentiated HL-60 promyelocytic leukemia cells. However, it is unclear whether CsA interferes with ligand-receptor interaction, G protein activation, or other downstream signaling events. In this study we used human neutrophils, differentiated HL-60 cells, and rat basophilic leukemia (RBL)-2H3 cells expressing human FPR (RBL-FPR) to identify the action site of CsA. In functional assays, CsA inhibited fMLF-stimulated degranulation, chemotaxis, calcium mobilization, and phosphorylation of the MAPKs ERK 1/2 and the serine/threonine protein kinase Akt. CsA also blocked Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm)-induced functions in RBL-FPR cells. Concentrations for half-maximal inhibition with CsA are generally 6- to 50-fold higher than that of CsH. CsA was compared with another immunosuppressant, ascomycin, relative to the inhibitory effects on FPR-mediated chemotaxis, calcium mobilization, and degranulation. In these experiments, ascomycin produced no inhibitory effects at low micromolar concentrations (1-4 microM), whereas the inhibitory effects of CsA were prominent at comparable concentrations. Finally, CsA dose-dependently inhibited the uptake of fNle-Leu-Phe-Nle-Tyr-Lys-fluoresceine and [3H]fMLF or [125I]WKYMVm binding to FPR. However, CsA and CsH did not show any obvious inhibitory effect on FPR-like 1-mediated cellular functions. These results demonstrate that CsA is a selective antagonist of FPR and that its inhibition of fMLF-stimulated leukocyte activation is at the level of cognate ligand binding.     

Inhibition of macrophage secretion by tetanus toxin is not directly linked to cytosolic calcium homeostasis

Tetanus toxin (TT) inhibits secretion of neurotransmitters from neurons and lysozyme from human macrophages (Mphi). Because these secretory events are associated with changes in cytosolic free calcium [Ca2+]i, we examined the effect of TT on Mphi calcium homeostasis and secretion in response to ionomycin and phorbol myristate acetate (PMA). Using Quin 2 to report [Ca2+]i, basal [Ca2]i was similar for control cells (133 nM) and Mphi treated with TT (127 nM). In response to ionomycin (50 nM) [Ca2+]i increased to 548 +/- 74 nM in control cells and to 357 +/- 36 nM in TT-treated Mphi (p less than 0.02, N = 12). Despite this rise in [Ca2+]i, neither control Mphi nor TT-treated Mphis secreted the lysosomal enzyme lysozyme in response to this concentration of ionomycin (50 nM). In both control and TT-treated Mphi, stimulation with a higher concentration of ionomycin (1000 nM) caused saturation of the quin 2 fluorescence signal. However, lysozyme secretion from TT-Mphi was inhibited. In response to the phorbol ester, PMA (3 uM), [Ca2+]i did not increase in either control Mphi or TT-treated Mphi. However, secretion of lysozyme from TT-treated Mphi was also inhibited in response to this stimulus (70.8% of control, p less than 0.02, N = 3). These data indicate that the ability of TT to inhibit secretion from Mphi is not directly linked to alterations of cytosolic calcium homeostasis.     

The chymotrypsin inhibitor carbobenzyloxy-leucine-tyrosine-chloromethylketone interferes with the neutrophil respiratory burst mediated by a signaling pathway independent of PtdInsP2 breakdown and cytosolic free calcium.

The effects of carbobenzyloxy-leucine-tyrosine-chloromethylketone (zLYCK), an inhibitor of chymotrypsin, were investigated on the activation pathways of the human neutrophil respiratory burst. At 10 microM zLYCK, a parallel inhibition was observed of superoxide production stimulated with the chemo-attractant FMLP and of chymotrypsin-like activity of human neutrophils. By contrast, superoxide production induced by PMA was minimally affected by zLYCK. The known transduction pathways triggered by FMLP were analyzed. zLYCK did not affect either the FMLP-induced cytosolic free calcium transient, inositol 1,4,5 trisphosphate formation, nor the PMA-induced phosphorylation of the 47-kDa substrate of protein kinase C. zLYCK did not affect the activity of protein kinase C extracted from neutrophils. In Ca(2+)-depleted cells, in which phosphatidylinositol 4,5-biphosphate breakdown does not occur, zLYCK inhibited the FMLP-induced respiratory burst in cells primed by low doses of PMA. The activity of the NADPH oxidase tested with active membranes from stimulated neutrophils or in a cell-free system was not inhibited by zLYCK. We conclude that: 1) zLYCK inhibits superoxide production through the inhibition of a chymotrypsin-like protease of the neutrophil, 2) zLYCK inhibits FMLP-induced activation of NADPH oxidase through a pathway independent of PtdInsP2 breakdown and cytosolic free calcium, and 3) zLYCK may prove a useful probe for the characterization of its target protease in neutrophil activation.     

Phorbol 12, myristate 13, acetate potentiates the respiratory burst while inhibits phosphoinositide hydrolysis and calcium mobilization by formyl-methionyl-leucyl-phenylalanine in human neutrophils

It is widely believed that the transduction pathway in the activation of the NADPH oxidase by formyl-methionyl-leucyl-phenylalanine (FMLP) in neutrophils involves the stimulation of phosphoinositide hydrolysis, the increase in [Ca2+]i and the activity of the Ca2+ and phospholipid dependent protein kinase C. The results presented here show that the activation of the respiratory burst by FMLP can be dissociated by the stimulation of the hydrolysis of phosphatidylinositol 4,5-bisphosphate and Ca2+ changes. In fact, in neutrophils pretreated (primed) with non stimulatory doses of phorbol myristate acetate the respiratory burst by chemotactic peptide is greatly potentiated while the increase in [3H] inositol phosphates formation and in [Ca2+]i are depressed due to the inhibition of phospholipase C. This finding indicates that FMLP can trigger also a sequence of transduction reactions for the activation of the NADPH oxidase different from that involving the formation of the second messengers diacylglycerol and inositol phosphates and the increase in free Ca2+ concentration.     

An immunosuppressive retrovirus-derived hexapeptide interferes with intracellular signaling in monocytes and granulocytes through N-formylpeptide receptors.

We have previously found that the retroviral p15E-derived hexapeptide LDLLFL is a potent inhibitor of the FMLP-induced polarization response that is an early event in chemotaxis of monocytes and granulocytes. We investigated the mechanism of action of LDLLFL. LDLLFL inhibited the changes in [Ca2+]i in response to FMLP, but not to C5a or leukotriene B4. The reverse peptide LFLLDL was not inhibitory. In the presence of LDLLFL, the FMLP dose-response curve shifted to higher concentrations, indicating that LDLLFL interfered with binding of FMLP to its receptor. Indeed, binding of [3H]FMLP to neutrophilic granulocytes was inhibited in the presence of LDLLFL. Furthermore, immunosuppressive LDLLFL homologs also inhibited binding of FMLP to granulocytes, whereas noninhibitory LDLLFL homologs did not. Our results suggest that retroviral p15E and p15E-like factors, which can be found in serum of patients with cancer or chronic upper airway infections, may interfere with the interaction of N-formylpeptides derived from (opportunistic) bacteria, with monocytes and granulocytes. This receptor interference may impair monocyte and granulocyte reactivity toward these agents.