Calcium ionophore potentiates chemotactic peptide and platelet activating factor in stimulating thromboxane B2 and leukotriene B4 biosynthesis in human neutrophils

Formyl-Met-Leu-Phe (FMLP) and platelet activating factor (PAF) stimulated the synthesis of thromboxane B2 (TXB2) and leukotriene B4 (LTB4) to a small degree in human neutrophils. Calcium ionophore A-23187 enhanced synergistically both FMLP and PAF induced eicosanoid synthesis, whereas phorbol ester PMA attenuated PAF but not FMLP stimulated arachidonate metabolism. These results suggest that calcium mobilization may be a rate limiting step in FMLP and PAF induced synthesis of TXB2 and LTB4 and that protein kinase C activation may play a negative regulatory role in PAF stimulated eicosanoid synthesis.     

Polymorphonuclear leukocytes cap a derivative of wheat germ agglutinin upon stimulation with formyl peptide and C5a but not leukotriene B4

Previously, we reported that a derivative of wheat germ agglutinin (termed WGA-D) specifically inhibits human polymorphonuclear leukocyte (PMN) chemotaxis to FMLP by blocking reexpression (or recycling) of formyl peptide receptors. WGA-D (? formyl peptide receptor probe) binds to a protein on the PMN membrane that exhibits the same m.w. as the formyl peptide receptor. Since clustering (i.e., capping) of ligand-receptor complexes most likely precedes their internalization, we examined the ability of normal and stimulated PMN to cap fluoresceinated WGA-D. We found that, in contrast to capping of fluoresceinated Con A, PMN cap WGA-D in a chemotactic factor-specific fashion. Fluoresceinated WGA-D (5.0 to 20 micrograms/ml) alone did not induce either PMN shape changes (i.e., activation) or capping. Both FMLP (1 to 1000 nM) and human C5a (0.1 to 1.0 nM) induced PMN to polarize and to cap bound WGA-D, in a concentration-dependent fashion. Interestingly, leukotriene B4 (LTB4) (5.0 nM), while inducing the same degree of PMN polarization as FMLP (100 nM) and C5a (0.5 nM), failed to induce PMN to cap bound WGA-D. In contrast, FMLP (100 nM), C5a (0.5 nM), and LTB4 (5.0 nM) induced PMN to cap bound fluoresceinated Con A (10 micrograms/ml) to the same extent. The effect of suboptimal concentrations of FMLP and C5a on capping of WGA-D by PMN was additive. LTB4 did not enhance either FMLP or C5a-induced capping of WGA-D by PMN. Also, FMLP and C5a (but not LTB4) were capable of inducing both desensitization and cross-desensitization of WGA-D capping by PMN. Studies using rhodamine-labeled WGA-D and a fluoresceinated analog of FMLP revealed that both capped to the same place on the PMN membrane. Thus, the data suggest that WGA-D binds to a site on the PMN membrane that is either the FMLP receptor or very closely associated with it.     

Role of a pertussis toxin substrate in the control of lectin-induced cap formation in human neutrophils.

We have examined the role of GTP-binding proteins and the associated cyclic AMP- and calcium-related transduction mechanisms in the regulation of capping in human neutrophils. Pertussis toxin (PT), a probe for the GTP-binding protein Ni, abolished capping induced by fluorescein isothiocyanate-conjugated concanavalin A (Con-A), whereas cholera toxin, a probe for the GTP-binding protein Ns, was without effect. Consistent with the latter finding, ligands acting at receptors associated with the Ns protein, namely the prostaglandin E1 and beta-adrenergic agonists, were without effect on the capping reaction. The possible role of mobilization of internal calcium was evaluated by using Quin2-loaded cells. Calcium mobilization was observed at concentrations of Con-A which yielded optimal capping (10 micrograms/ml). Treatment with PT, phorbol myristrate acetate or 8-(NN-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) abolished both calcium mobilization and capping. Colchicine, which substantially enhanced capping, had no effect on calcium mobilization. At concentrations of the lectin above those required for capping, superoxide generation and enzyme release were noted. These reactions were less susceptible to inhibition by PT, effects being observed only on the Kact. for Con-A-mediated superoxide generation with little effect on the Vmax. The degree of PT-mediated inhibition for enzyme release with Con-A was much lower than that observed with fMet-Leu-Phe. Our results imply that a step involving Ni-mediated calcium mobilization, sensitive to phorbol myristate acetate, is essential to the regulation of capping; a distinct mechanism may be involved in colchicine-mediated enhancement of capping; and Ni may play a relative minor role in the regulation of lectin-mediated exocytosis.     

Mechanism and regulation of neutrophil priming by platelet-activating factor

Although a weak direct stimulus of superoxide anion (O₂^?) production, platelet-activating factor (PAF) markedly enhances responses to chemotactic peptides (such as n-formyl-met-leu-phe, FMLP) and phorbol esters (such as phorbol myristate acetate, PMA) in human neutrophils. The mechanism of priming was explored first through inhibition of steps in the signal transduction pathway at and following PAF receptor occupation. Priming was not altered by pertussis toxin or intracellular calcium chelation, but the PAF receptor antagonist WEB 2086 and the protein kinase C (PKC) inhibitors sphinganine and staurosporine significantly inhibited the primed response. In order to study the regulation of PAF priming, the effect of PAF alone was desensitized by exposure to escalating doses of PAF prior to exposure to the secondary stimuli. The priming effect of PAF was not desensitized under these conditions. The role of PKC in desensitization was also studied. Prior exposure to PAF also desensitized the increase in membrane PKC activity evoked by a single concentration of PAF. However, when the PAF response was desensitized, PKC priming of the response to FMLP or PMA still occurred, suggesting that PKC activity may play a role in the maintenance of the primed state despite PAF desensitization. These data suggest that: (1) PAF priming is receptor- and PKC-mediated but is independent of pertussis toxin-inhibitable G-proteins or intracellular calcium, (2) during migration in vivo, neutrophils may be desensitized to the direct effects of PAF but maintain the capacity for enhanced responses to other stimuli, (3) desensitization of PAF-induced particulate PKC activity also occurs, but PAF primes PKC activity despite PAF desensitization, and (4) distinct mechanisms govern the direct and priming effects of PAF on oxidative metabolism. © 1993 Wiley-Liss, Inc.     

Production by K 562 cells of an inhibitor of adherence-related functions of human neutrophils

Certain tumor cells generate factors that inhibit neutrophil chemotaxis. Our study was designed to explore whether such factors are produced by K 562 malignant cells and whether these have a broader effect in altering neutrophil functions. After 48 h of in vitro culture of K 562 cells, the culture medium and the cells were separated, lyophilized, and extracted with ethanol. These K 562 products, i.e., either the cell or supernatant extract, inhibited both nonstimulated locomotion and locomotion induced either by FMLP or activated serum. Furthermore, K 562 products inhibited neutrophil adherence and oxidative burst induced by opsonized zymosan, whereas oxidative burst induced by PMA or FMLP was not altered. K 562 products had an inhibitory effect on the PMN binding to iC3b-coated particles. They did not modify Mo1 expression of resting cells, did not alter the up-regulation of the receptor induced by FMLP but inhibited the FMLP-induced capping of Mo1 Ag. Con A capping was also inhibited. Actin polymerization in FMLP-stimulated PMN, as measured by flow cytometry and phalloidin binding to F-actin, was inhibited by K 562 products. The inhibitory factor present in K 562 products (cell and culture supernatant) was purified in three steps including gel filtration, ion-exchange chromatography, and IEF. The eluted active fraction corresponded to single band of about 8 kDa on SDS-PAGE. From these experiments, it is concluded that K 562 malignant cells in culture contain and release a low molecular mass factor (congruent to 8 kDa) that inhibits all adherence-related functions of neutrophils, whereas it does not alter FMLP- or PMA-induced oxidative burst. Further studies are needed to assess whether products of other tumor cells also act on the neutrophil by inhibiting adherence-related functions, Mo1 function and capping, and actin polymerization.     

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.     

The involvement of calcium and protein kinase C in modulating agonist-stimulated chemotaxis of human neutrophils

Chemotaxis of human neutrophils in response to a gradient of the chemotactic peptide, fmet-leu-phe (FMLP), was measured by the under-agarose technique. The dose-response curve for FMLP was biphasic; low concentrations were stimulatory, and the response was reduced at higher concentrations. The response to FMLP was partially inhibited (about 50%) in the absence of extracellular Ca2 (EGTA added). NiCl2 dose-dependently inhibited FMLP-stimulated chemotaxis in the presence of extracellular Ca2+; the maximum inhibition obtainable with NiCl2 was similar to that with the absence of extracellular Ca2+. These results suggest that FMLP-stimulated chemotaxis is, at least partially, dependent on stimulation of Ca2+ influx. The phorbol ester, PMA, dose-dependently inhibited chemotaxis; the response was almost completely inhibited by 10 nM PMA. This result indicates that activation of protein kinase C inhibits chemotaxis. These results are discussed in relation to the physiological responses of neutrophils.     

The effect of the NADPH oxidase inhibitor diphenyleneiodonium on aerobic and anaerobic microbicidal activities of human neutrophils.

NADPH-dependent superoxide production by intact human neutrophils is inhibited by DPI (diphenyleneiodonium), when stimulated by either FMLP (N-formylmethionyl-leucyl-phenylalanine) or PMA (phorbol 12-myristate 13-acetate). Addition of 10 microM-DPI abolished the reduction of both the FAD and the cytochrome b components of the NADPH oxidase. DPI inhibition of the oxidase was associated with defective aerobic killing of staphylococci by human neutrophils. Anaerobic killing, phagocytosis, chemotaxis and motility were relatively unaffected by 10 microM-DPI. Degranulation of the azurophil and specific granules, induced by the soluble stimuli FMLP or PMA, and by particulate stimuli was decreased by the presence of DPI. The above effects of DPI on human neutrophils are similar to those found in chronic granulomatous disease.     

Neutrophil thrombospondin receptors are linked to GTP-binding proteins

The extracellular matrix (ECM) protein thrombospondin (TSP) binds to specific receptors on polymorphonuclear leukocytes (PMNs) and stimulates motility. TSP can also enhance the response of PMNs to the formylated peptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP). Our initial evidence suggesting that PMN TSP receptors were linked to GTP-binding proteins (G-proteins) came from studies using pertussis toxin (PT) and cholera toxin (CT) to inhibit TSP-mediated motility. Both PT and CT inhibited TSP-mediated chemotaxis and substrate-associated random migration. Inhibition was not indirectly caused by a rise in cAMP since neither dibutyryl cAMP (300 μM) nor 8-bromo-cAMP (300 μM) significantly affected TSP-mediated motility. In fact, TSP itself caused a significant rise in intracellular cAMP levels (from 7.2 ± 0.3 to 14.2 ± 0.1 pmol/10⁶ cells). Although we could not test the PT sensitivity of TSP priming for FMLP-mediated chemotaxis (as PT inhibits FMLP-mediated chemotaxis itself), we evaluated the effect of CT on this response. CT completely abolished TSP-dependent priming of FMLP-mediated chemotaxis. Direct evidence for an interaction between TSP receptors and G-proteins was obtained by examining the effect of TSP on α-subunit ADP-ribosylation, GTPase activity, and GTPγS binding. We observed a decrease in the ability of FMLP to stimulate GTPase activity on membranes isolated from PMNs incubated with TSP. Furthermore, the PT-dependent ribosylation of G_iα2,3 stimulated by FMLP was eliminated by TSP treatment. These data indicated that the two receptors share a pool of G-proteins. However, TSP did not block the CT-dependent ribosylation stimulated by FMLP, suggesting that TSP receptors may also interact with a different pool of G_iα2,3. TSP itself significantly (P < 0.005) increased GTP hydrolysis in PMN membranes (to 110.6 ± 2.7% of control values). In addition, GTPγS binding to membranes increased significantly (P < 0.005) following exposure to 10 nM TSP (to 108 ± 1.4% of control values). Conversely, GTP treatment reduced the affinity of TSP for its receptor without altering total binding. These data demonstrate that TSP receptors are linked to G-proteins, a subpopulation of which also associates with FMLP receptors. © 1996 Wiley-Liss, Inc.     

A guanine nucleotide regulatory protein controls polyphosphoinositide metabolism, Ca2+ mobilization, and cellular responses to chemoattractants in human monocytes

Previous studies demonstrated that oligopeptide chemoattractant receptors on PMN and macrophages exist in high and low affinity states which are interconvertible by guanosine di- and triphosphates. These observations suggest that guanine nucleotide regulatory (N) proteins play a role in phagocyte activation by chemotactic factors. The data presented here indicate that chemotactic factor receptors on monocytes utilize an N protein to activate phospholipase C and subsequent biologic responses by the cells. This conclusion is based on the findings that inactivation of an N protein of 41,000 m.w. by Bordetella pertussis toxin (PT) treatment abolishes monocyte responsiveness to chemoattractants but not to lectins, PMA, or the Ca2+ ionophore A23187. Treatment with PT inhibited IP3 production, Ca2+ mobilization, and cellular activation as assessed by chemotaxis and changes in forward light scattering in response to the chemoattractants by at least 80%. Therefore, a PT-sensitive N protein plays an important role in the activation of monocytes by chemoattractants.     

A monoclonal antibody that detects a specific human neutrophil antigen involved in phorbol myristate acetate- and formyl-methionyl-leucyl-phenylalanine-triggered respiratory burst.

A mouse IgM mAb termed P1E3 was raised against resting human peripheral blood neutrophils and has been shown to recognize a cell-surface Ag with an apparent molecular mass of 155 kDa, as assessed by immunoprecipitation analysis. In addition to the main 155-kDa protein, an additional band of about 210 kDa was also recognized by P1E3 in Western blot analysis. Sequential immunoprecipitation assays showed that the Ag recognized by P1E3 differed from the CD29 and CD45 Ag. However, sequential immunoprecipitation assays carried out with two distinct anti-CD15 mAb and P1E3 showed that P1E3 reacted with CD15 or with a CD15-like Ag. P1E3 stained strongly resting human peripheral blood neutrophils, hardly reacted with peripheral blood monocytes and did not react with PBL and platelets, as assessed by immunofluorescence flow cytometry. P1E3 inhibited the respiratory burst induced by PMA or FMLP, but not the oxidative response induced by Con A or the calcium ionophores A23187 or ionomycin. Furthermore, P1E3 inhibited the activation of the Na+/H+ antiporter in response to PMA or FMLP and the phosphorylation of a protein of about 50 kDa in response to PMA. However, preincubation of neutrophils with P1E3 did not affect the increase in cytosolic free calcium concentration induced by FMLP. These data suggest that the Ag recognized by P1E3 may play a role in modulating the activation of the respiratory burst induced by PMA or FMLP, and that P1E3 seems to affect protein kinase C-mediated signal transduction mechanisms coupled to the induction of the respiratory burst.     

Pertussis toxin effects on chemoattractant-induced response heterogeneity in human PMNs utilizing Fluo-3 and flow cytometry

Flow cytometric methods were utilized to determine N-formylpeptide-induced cytosolic calcium levels in human polymorphonuclear leukocytes (PMNs) detected with the calcium indicator Fluo-3. Fluo-3 was readily loaded into PMNs as the acetoxymethyl ester. At room temperature Fluo-3 extrusion was minimal (less than 10%) over a 2 h time period. Flow cytometric histograms yielded symmetric distributions indicating homogeneous labelling of the cells. Stimulation of the cells with N-formyl-met-leu-phe (FMLP) caused homogeneous activation of all cells as indicated by a shift of the fluorescence distribution to higher fluorescence levels while still maintaining a symmetrical distribution. Resting values or FMLP-induced cytosolic calcium levels were similar in cells loaded over a 20-fold range of Fluo-3-acetoxymethyl ester. The effect of graded pertussis toxin (PT) treatment on the calcium response was determined by incubating cells with different concentrations of pertussis toxin for a time period that yielded a range of ADP ribosolation levels inside the cells. When these cells were activated with FMLP, the fluorescence histograms showed that pertussis toxin treatment resulted in a conversion of cells from responders to nonresponders. The responding cells responded with maximum calcium elevations similar to controls. This behavior may reflect heterogeneous insertion of the A-protomer of PT or a very sharp threshold of coupled G-proteins required to transduce the responses.     

A comparison of the effects of soluble stimuli on free cytoplasmic and membrane bound calcium in human neutrophils

Calcium dynamics in human neutrophils have been studied using Quin 2 fluorescence as a measure of free cytoplasmic calcium and chlortetracycline fluorescence as an indicator of membrane-bound calcium. The results show that 1) FMLP-induced increased cytoplasmic calcium likely comes from at least two different pools. Calcium is released from one only after a high affinity receptor interaction and from the second also after a lower affinity interaction. The initial increment in cytosolic calcium does not appear to originate in the pool(s) reflected by CTC fluorescence. 2) Cytochalasin B strikingly alters the FMLP effect on membrane associated calcium, inducing a marked “recovery” phase which could be a reflection of fusion of granule membranes with the plasma membrane. 3) PMA, at concentrations inducing extensive specific granule release (≤ 10 ng/ml) has no measurable direct effect on membrane-bound or cytosolic calcium. However, PMA inhibits a subsequent CTC fluorescence response to FMLP and following the ionophore, A23187, it induces a clear decrease in cytosolic calcium. These indirect effects may be explained in terms of PMA's activation of protein kinase C.     

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.     

Enhancement of phorbol ester-induced protein kinase activity in human neutrophils by platelet-activating factor

We have shown that platelet-activating factor (PAF), a weak primary stimulus for neutrophil superoxide generation, synergistically enhances neutrophil oxidative responses to the tumor promoter phorbol myristate acetate (PMA). Since PMA is known to cause cytosol-to-membrane shift of calcium-activated, phospholipid-dependent protein kinase (protein kinase c, PKC) in human neutrophils, we investigated the role of PAF in modifying PMA-induced PKC activation/translocation. Protein kinase activity was measured as the incorporation of 32P from gamma-32P-ATP into histone H1 induced by enzyme in cytosolic and particulate fractions from sonicated human neutrophils. PAF did not alter the sharp decrease in cytosolic PKC activity induced by PMA. However, in the presence of PAF and PMA, total particulate protein kinase activity increased markedly over that detected in the presence of PMA alone (144 +/- 9 pmoles 32P/10(7)PMN/minute in cells treated with 20 ng/ml PMA compared to 267 +/- 24 pmoles 32P in cells exposed to PMA and 10(-6)M PAF). The increase in total particulate protein kinase activity was synergistic for the two stimuli, required the presence of cytochalasin B during stimulation, and occurred at PAF concentrations of 10(-7) M and above. Both PKC and calcium-, phospholipid-independent protein kinase activities in whole particulate fractions were augmented by PAF as were both activities in detergent-extractable particulate subfractions. PAF did not directly activate PKC obtained from control or PMA-treated neutrophils. However, the PKC-enhancing effect of PAF was inhibited in the absence of calcium during cellular stimulation. PAF also increased particulate protein kinase activity in cells simultaneously exposed to FMLP but the effect was additive for these stimuli. These results suggest that PAF enhances PMA-induced particulate PKC activity by a calcium-dependent mechanism. The enhancing effect of PAF may be directly involved in the mechanism whereby the phospholipid "primes" neutrophils for augmented oxidative responses to PMA.     

Signal transduction in monocytes and granulocytes measured by multiparameter flow cytometry.

The novel calcium indicator fura red and the oxidative burst indicator dihydrorhodamine (both excited at 488 nm) were used in combination with multiparameter flow cytometry to allow simultaneous kinetic measurements of calcium fluxes and oxidative bursts in monocytes and granulocytes. Using this method it was possible to obtain direct evidence for the following cell type- and stimulus-specific differences in signal transduction pathways: 1) n-formyl-methionyl-leucyl-phenylalanine (FMLP)/cytochalasin B-induced oxidative burst is several-fold higher in granulocytes than in monocytes although the calcium fluxes have similar amplitudes in the two cell types; 2) stimulus-induced calcium fluxes in granulocytes are mainly due to release from intracellular stores, whereas monocytes mobilize calcium mainly by influx from the medium; 3) the FMLP/cytochalasin B-induced calcium flux in monocytes is less sensitive to the G-protein inhibitor pertussis toxin than the flux in granulocytes; 4) in contrast to FMLP/cytochalasin B, the protein kinase C activator phorbol myristate acetate (PMA) induces an oxidative burst that is not preceded by a cytoplasmic calcium flux; 5) the PMA-induced oxidative burst can be triggered in monocytes and granulocytes that are depleted of intracellular calcium ions, whereas that induced by FMLP/cytochalasin B can not; 6) the G-protein inhibitor pertussis toxin blocks an early event in the signal transduction pathway of FMLP/cytochalasin B, as shown by inhibition of both calcium fluxes and oxidative burst; and 7) 100 nM of the protein kinase inhibitor staurosporine blocks the FMLP/cytochalasin B-induced respiratory burst by interfering with a step downstream to cytoplasmic calcium fluxes, whereas only 10-20 nM is necessary to block PMA-induced oxidative burst.     

Stimulation of human polymorphonuclear leukocyte phosphatidylinositol-4-phosphate kinase by concanavalin A and formyl-methionyl-leucyl-phenylalanine is calcium-independent. Correlation with maintenance of actin assembly.

Chemoattractants directly stimulate the enzyme activity that synthesizes phosphatidylinositol-4,5-bisphosphate (PIP2), phosphoinositol-4-monophosphate (PIP) kinase. The present study determined whether stimulation of this enzyme correlates with actin assembly by assessing the calcium dependence of this reaction. Incubation of neutrophils with 5 to 100 micrograms/ml Con A caused a concentration-dependent increase in PIP kinase activity ranging from 1.38- to 3.4-fold. The effective concentration which stimulated PIP kinase by 50% (17 micrograms/ml, EC50) corresponded with the EC50 for Con A-induced superoxide production (32 micrograms/ml). Like chemoattractants, the increase in PIP kinase by Con A was characterized by a 2.6-fold increase in the maximum velocity (Vmax) of the enzyme, and no change in the Km for ATP. The kinetics of FMLP- and Con A-induced filamentous actin formation preceded stimulation of PIP kinase and was sustained over the same time period that this increased enzyme activity was noted. Although transmembrane signaling by FMLP and Con A requires an increase in intracellular calcium for some polymorphonuclear leukocyte (PMN) functional responses, calcium depletion of PMN by incubation with 100 microM Quin 2 A/M and 5 mM EGTA did not prevent the stimulation of PIP kinase by FMLP or Con A. In addition, calcium depletion did not prevent the increase in filamentous actin formation by FMLP and Con A in PMN. These findings demonstrate that Con A increases PIP kinase activity in human PMN and that PIP kinase stimulation and maintenance of actin assembly are independent of calcium fluxes in these cells. Because PIP2 controls the function of the actin-regulatory proteins, profilin and gelsolin, changes in the synthetic rate of PIP2 through regulation of PIP kinase may provide a molecular basis for the prolonged stimulation of actin assembly in human PMN by agonists such as Con A and FMLP.     

Extracellular ATP itself elicits superoxide generation in guinea pig peritoneal macrophages.

Extracellular ATP itself elicited the generation of superoxide (O2-) in guinea pig peritoneal macrophages associated with an increase in cytosolic calcium ([Ca2+]i). The ATP-induced O2- generation was completely inhibited by pretreatment with pertussis toxin (PT) accompanied by the suppression of [Ca2+]i mobilization. Pre-exposure to a small amount of phorbol myristate acetate (PMA) primed the ATP-induced generation of O2- without a change of [Ca2+]i. The results suggest that ATP-induced O2- generation is mediated by [Ca2+]i mobilization and by PT-sensitive G protein.     

Inhibition of the oxidative burst in human neutrophils by sphingoid long-chain bases. Role of protein kinase C in activation of the burst

The neutrophil oxidative burst is characterized by increased cellular O2 consumption due to the activation of a membrane-associated superoxide-generating NADPH-oxidase. The response is triggered by a variety of stimuli, including opsonized zymosan, formylmethionylleucinephenylalanine (FMLP), arachidonate, short-chain diacylglycerols, and phorbol myristate acetate (PMA). We herein demonstrate that incubation of cells with sphinganine or sphingosine blocks or reverses activation by these agonists. The inhibition is reversible, does not affect cell viability, and does not affect another complex cell function, phagocytosis. Inhibitory concentrations of sphinganine did not significantly affect cytoplasmic calcium levels or FMLP-generated calcium transients. Structural requirements for inhibition of the oxidative burst include a long aliphatic chain and an amino-containing head-group, and there is modest specificity for the native (erythro) isomer of sphinganine. Inhibition involves stimulus-induced activation mechanisms rather than a direct effect on the NADPH oxidase, since sphinganine did not inhibit NADPH-dependent superoxide generation in isolated membranes containing the active enzyme. Activation by FMLP, diacylglycerol, PMA, opsonized zymosan, and arachidonate was blocked by the same concentrations of sphinganine, indicating that these agonists share a common inhibited step. Three lines of evidence indicate that this step involves protein kinase C. First, in a micelle system and in platelets, long-chain bases are inhibitors of this enzyme (Hannun, Y., Loomis, C., Merrill, A., and Bell, R. M. (1986) J. Biol. Chem. 261, 12604-12609). Second, sphinganine blocks PMA-stimulated incorporation of 32PO4 into neutrophil proteins. Third, sphinganine inhibits the binding of [3H]phorbol dibutyrate to its cellular receptor, known to be protein kinase C. We suggest that long-chain bases function as physiologic modulators of cellular regulatory pathways involving protein kinase C.     

Effects of glutathione-oxidizing agents on microtubule assembly and microtubule-dependent surface properties of human neutrophils

In human peripheral blood polymorphonuclear leukocytes and lymphocytes, GSH-oxidizing agents promote the movement of surface-bound concanavalin A (Con A) into caps and inhibit the assembly of microtubules (MT) that is normally induced by Con A binding. Con A capping and inhibition of MT assembly occur when GSH levels in cell suspensions are decreased by 30-70%, and return to GSH to control levels is accompanied by the appearance of cytoplasmic MT and by inhibition of the capping response with Con A. Oxidation of GSH markedly stimulates the hexose monophosphate shunt, and regeneration of GSH occurs rapidly. The data indicate that MT cannot be assembled or maintained in the face of decreased GSH levels. Thus, GSH homeostasis becomes critical during physiological events such as phagocytosis which simultaneously induce the assembly of MT and the production of agents like H2O2 that can oxidize GSH.