Human basophil releasability. III. Genetic control of human basophil releasability

Basophil releasability implies that, in addition to the surface density of IgE molecules, biochemical events determine the capacity to release chemical mediators in response to activating stimuli. We studied the IgE (anti-IgE)-mediated and non-IgE-mediated (f-met peptide and the Ca2+ ionophore A23187) releasability of human basophils obtained from 14 monozygotic (MZ) (ages 25.7 +/- 13.3 yr; mean +/- SDM) and 13 dizygotic (DZ) twin pairs (ages 20.4 +/- 9.9 yr). A significant intrapair correlation coefficient of the maximal percent of anti-IgE-induced histamine release was found in the MZ, whereas no significant correlation was found in the DZ. The mean intrapair variance of anti-IgE-induced histamine release in MZ (VMZ) and in DZ (VDZ) gave an F value equal to 3.84 (p less than 0.01) and a heritability (H) index of 0.74. Similar findings were obtained with respect to the sensitivity to a standard concentration (10(-1) micrograms/ml) of anti-IgE. No correlation between serum IgE level and anti-IgE-induced histamine release was found in either MZ or DZ. A significant intrapair correlation coefficient of f-met peptide-induced histamine release was found in both the MZ and the DZ. The difference between MZ and DZ was not significant. The VMZ and the VDZ of the f-met peptide-induced histamine release gave an F value of 1.52 (NS) and an H value of 0.34. The intrapair correlation coefficient of A23187-induced release was significant in MZ and not significant in DZ. The mean intrapair variance of A23187-induced histamine release gave an F value of 2.33 (NS) and an H index of 0.57. Similar findings were obtained by using suboptimal (3 X 10(-1) micrograms/ml) concentrations of A23187. There was no correlation between the sensitivity of basophils to release in response to anti-IgE and their response to f-met peptide or A23187, in either the MZ or the DZ. We conclude that the ability of basophils to respond to anti-IgE and A23187 is influenced by genetic factors.     

Ca2+ and protein kinase C signaling for histamine and sulfidoleukotrienes released from human cultured mast cells

Human cultured mast cells (HCMC) release histamine and sulfidoleukotrienes (LTs) upon IgE-FcepsilonRI-mediated mast cell activation. We analyzed the Ca2+ and PKC signaling in HCMC and compared it to that in rodent mast cells. In HCMC, after IgE-mediated stimulation, an elevation of [Ca2+]i and PKC translocation to the membrane fraction was observed. As concerns Ca2+ signaling, 1) IgE-mediated histamine and LTs release was abolished after Ca2+ depletion, and the reconstitution of Ca2+ recovered the release of histamine and LTs. As regards PKC signaling, 1) staurosporine inhibited IgE-mediated mediator release. 2) PKC-downregulated mast cells did not release histamine and LTs. A23187 and PMA synergistically potentiated the activation of extracellular-regulated kinase and synergistically induced histamine and LTs release. These results demonstrated that HCMC might be useful for analysis of the signal transduction pathway for mediator release, such as histamine and LTs.     

FK-506, a potent novel inhibitor of the release of proinflammatory mediators from human Fc epsilon RI+ cells.

FK-506, a macrolide that binds with high affinity to a specific binding protein, and the structurally related macrolide rapamycin (RAP) were compared to cyclosporin A (CsA) for their effects on the release of preformed (histamine) and de novo synthesized (peptide leukotriene C4) inflammatory mediators from human basophils. FK-506 (1 to 300 nM) concentration dependently inhibited histamine release from basophils activated by Der p I Ag, anti-IgE, or compound A23187. FK-506 was more potent than CsA when basophils were challenged with Ag (IC50 = 25.5 +/- 9.5 vs 834.3 +/- 79.8 nM; p less than 0.001), anti-IgE (IC50 = 9.4 +/- 1.7 vs 441.3 +/- 106.7 nM; p less than 0.001), and A23187 (IC50 = 4.1 +/- 0.9 vs 36.7 +/- 3.8 nM; p less than 0.001). The maximal inhibitory effect of FK-506 was higher than that caused by CsA when basophils were activated by Der p I (80.0 +/- 3.6 vs 49.5 +/- 4.7%; p less than 0.001) and anti-IgE (90.4 +/- 1.8 vs 62.3 +/- 2.9%; p less than 0.001). FK-506 had little or no effect on the release of histamine caused by f-met peptide, phorbol myristate (12-tetradecanoyloxy-13-acetoxy-phorbol), and bryostatin 1. RAP (30 to 1000 nM) selectively inhibited only IgE-mediated histamine release from basophils, although it had no effect on mediator release caused by f-met peptide, A23187, 12-tetradecanoyloxy-13-acetoxy-phorbol, and bryostatin 1. FK-506 also inhibited the de novo synthesis of sulfidopeptide leukotriene C4 from basophils challenged with anti-IgE. Low concentrations of FK-506 and CsA synergistically inhibited the release of mediators from basophils induced by anti-IgE or compound A23187. IL-3 (3 and 10 ng/ml), but not IL-1 beta (10 and 100 ng/ml), reversed the inhibitory effect of both FK-506 and CsA on basophils challenged with anti-IgE or A23187. RAP was a competitive antagonist of the inhibitory effect of FK-506 on A23187-induced histamine release from basophils with a dissociation constant of about 30 nM. In contrast, RAP did not modify the inhibitory effect of CsA on A23187-induced histamine release. These data indicate that FK-506 is a potent antiinflammatory agent that acts on human basophils presumably by binding to a receptor site (i.e., FK-506 binding protein).     

Protein kinase C (PKC) changes in human basophils. IgE-mediated activation is accompanied by an increase in total PKC activity

We have examined the changes in protein kinase C (PKC) which follow IgE-mediated activation of basophils. Exposure to 0.1 microgram/ml anti-IgE resulted in an increase in total cellular PKC (169 +/- 23% of control, histamine release (HR) = 33 +/- 7%, n = 12) which could be accounted for solely by the increase in membrane-associated PKC. These changes reached a maximum (280 +/- 48%) 1.0 min after challenge and declined to 190 +/- 38% after 5.0 min though histamine release was not complete until 5 to 10 min later. We found a good correlation between the increase in membrane-associated PKC and the eventual release of histamine (rs = 0.902). Donors whose basophils released less than 5% total histamine (n = 3, HR = 3 +/- 1%) showed a partial activation of PKC (173 +/- 18%) though much less than the remaining donors (increase in PKC = 346 +/- 59%, n = 9, HR = 43 +/- 7%). We observed no redistribution of cytosolic PKC at any time following exposure to anti-IgE. In contrast, 0.1 microgram/ml 2-O-tetradecanoyl-phorbol-13-acetate (HR = 36 +/- 3%, n = 3) promoted an increase in total cellular PKC, the loss of 31 +/- 4% of the cytosolic PKC and an 816 +/- 183% increase in membrane-associated PKC. Activation of PKC by anti-IgE was only partially dependent on extracellular calcium. In the absence of calcium, the increase in PKC was approximately 65% (n = 4) of that noted in the presence of 1mM calcium but these levels were sustained over much longer periods, failing to return to base line after 30 min. Higher than normal concentrations of calcium (5 to 10 mM) promoted rapid increases in PKC activity and accelerated the return to base line (back to prechallenge levels by 5 min). Suboptimal concentrations of anti-IgE (0.01 microgram/ml) attenuated the changes in membrane associated PKC and altered the kinetics of the response. The time required to reach maximum activity increased from 1.0 to 5.0 min with a corresponding decrease in the rate at which histamine was released. Higher concentrations of anti-IgE (1.0 microgram/ml) promoted a rapid increase in PKC (maximum increase in PKC = 501 +/- 59%, time = 0.5 min, HR = 28 +/- 2%) followed by an equally rapid return to base line levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Modulatory effect of HCO3- on rat mast cell exocytosis: cross-talks between bicarbonate and calcium.

HCO-3 modulation of histamine release and its relationship with the Ca2+ signal were studied in serosal rat mast cells. Histamine release was induced by Ca2+ mobilizing stimuli, namely compound 48/80, thapsigargin, Ca2+ chelators, ionophore A23187, and PMA and ionophore A23187 in a HCO-3-buffered medium or a HCO-3-free medium. The presence of HCO-3 reduced histamine release by 48/80, Ca2+ chelators, A23187, and PMA/A23187, but increased histamine release induced by thapsigargin. Histamine release by PMA was significantly higher in a HCO-3-free medium than in a HCO-3-free medium, as it was the PMA potentiation of histamine release by A23187. [Ca2+]i changes induced by these drugs were measured in fura-2-loaded mast cells. In thapsigargin and EGTA or BAPTA preincubated mast cells [Ca2+]i increase was higher in a HCO-3-buffered medium than in a HCO-3-free medium in the presence of Ca2+. On the contrary, in compound 48/80 and PMA/A23187 activated mast cells the [Ca2+]i increase is the same both in the presence and in the absence of HCO-3. The effect of HCO-3 on histamine release in serosal rat mast cells depends on the stimulus, but it is not related to the presence of Cl-. In thapsigargin-stimulated mast cells the effect of HCO-3 on histamine release may be related to the Ca2+ signal, but in compound 48/80, EGTA, and PMA/A23187-activated mast cells there is no relationship between intracellular Ca2+ and the inhibitory effect of HCO-3 on histamine release. Additionally, the PKC pathway is implicated in the inhibitory effect of HCO-3 on histamine release, the higher the chelation of calcium rendering the higher the enhancement of the response after adding calcium in the absence of HCO-3.     

The human recombinant c-kit receptor ligand, rhSCF, induces mediator release from human cutaneous mast cells and enhances IgE-dependent mediator release from both skin mast cells and peripheral blood basophils.

The gene product of the steel locus of the mouse represents a growth factor for murine mast cells and a ligand for the c-kit proto-oncogene receptor, a member of the tyrosine kinase receptor class of oncogenes (for review, see O. N. Witte. 1990. Cell 63:5). We have studied the effect of the human recombinant c-kit receptor ligand stem cell factor (rhSCF) on the release of inflammatory mediators from human skin mast cells and peripheral blood basophils and compared its activity to that of rhIL-3, rhSCF (1 ng/ml to 1 microgram/ml) activated the release of histamine and PGD2 from mast cells isolated from human skin. Analysis by digital video microscopy indicated that purified human skin mast cells (84 +/- 5% pure) responded to rhSCF (0.1 to 1 microgram/ml) challenge with a rapid, sustained rise in intracellular Ca2+ levels that was accompanied by secretion of histamine. A brief preincubation (10 min) of mast cells with rhSCF (0.1 pg/ml to 1 ng/ml) significantly enhanced (100 +/- 35%) the release of histamine induced by anti-IgE (3 micrograms/ml), but was much less effective on IgE-mediated release of PGD2. In contrast, a short term incubation with rhSCF did not potentiate the secretion of histamine activated by substance P (5 microM). A 24-h incubation of mast cells with rhSCF did not affect the release of mediators induced by anti-IgE (3 micrograms/ml), probably due to receptor desensitization, rhSCF (1 ng/ml to 3 micrograms/ml) neither caused release of histamine or leukotriene C4 (LTC4) release from leukocytes of 14 donors, nor induced a rise in intracellular Ca2+ levels in purified (greater than 70%) basophils. Brief preincubation (10 min) of leukocytes with rhSCF (1 ng/ml to 3 micrograms/ml) caused an enhancement (69 +/- 11%) of anti-IgE-induced release of histamine that was significant at concentrations as low as 3 ng/ml (p less than 0.05), whereas it appeared less effective in potentiating IgE-mediated LTC4 release. In contrast, a prolonged incubation (24 h) with rhSCF (0.1 pg/ml to 100 ng/ml) did not enhance the release of histamine or LTC4 induced by anti-IgE (0.1 microgram/ml), whereas rhIL-3 (3 ng/ml) significantly potentiated the release of both mediators.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of antibiotics on immediate hypersensitivity reactions in vitro: suppression of IgE-mediated histamine release from peripheral blood basophils by fosfomycin

The effect of antibiotics on allergic reactions was studied in vitro using the release of histamine from human peripheral blood leukocytes (basophils) after incubation with anti-IgE. For the several antibiotics we tested, including beta-lactams and aminoglycosides, none had the capacity to enhance antigen-induced histamine release, but some of them (minocycline, polymyxin B, and fosfomycin) suppressed the release of histamine in a dose-dependent manner. Since fosfomycin has proved to be capable of suppressing IgE-mediated histamine release non-cytotoxically, the effect of fosfomycin on histamine release induced by other secretagogues was further studied. The suppression of histamine release was also demonstrated when the leukocytes, preincubated with fosfomycin, were challenged with either Ca ionophore A 23187 or a synthetic peptide, formyl-methionyl-leucyl-phenylalanine (FMLP). We concluded that some antibiotics, particularly fosfomycin, have the capacity to suppress histamine release mediated by various secretagogues, suggesting they may possess an anti-allergic property as well as a bactericidal activity.     

Possible role of calmodulin in the control of histamine release from human basophil leukocytes

We investigated the possible role of calmodulin (CaM) in the control of histamine release from human basophil leukocytes using several CaM antagonists. Trifluoperazine (TFP) (10(-6)-2 X 10(-5) M), pimozide (10(-6)-1.5 X 10(-5) M), chlorpromazine (CPZ) (10(-5)-10(-4) M) and promethazine (PMZ) (2 X 10(-5)-10(-4) M) inhibited in vitro histamine secretion from human basophils induced by several immunological (antigen, anti-IgE, and formyl-L-methionyl-L-leucyl-L-phenylalanine: f-met peptide) and nonimmunological (Ca2+ ionophore A23187 and the tumor promoter 12-0-tetradecanoyl-phorbol-13-acetate: TPA) stimuli. Trifluoperazine sulfoxide (TFP-S) and chlorpromazine sulfoxide (CPZ-S), which have very low affinity to CaM, had practically no inhibitory effect on histamine release from human basophils. The inhibitory effect of TFP could be made irreversible by irradiating the cells with UV light. A sulfonamide derivative, the compound N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7) (2.5 X 10(-5)-2 X 10(-4) M), which selectively binds to CaM, inhibited the release of histamine from basophils. In contrast, the chloride deficient analogue, W-5, which interacts only weakly with CaM, had practically no inhibiting effect. The IC50 for enzyme release by a series of eight CaM antagonists was closely correlated (r = 0.91; p less than 0.001) with the CaM specific binding, supporting the concept that these agents act by binding to CaM and thereby inhibiting histamine release. TFP and W-7 inhibited histamine release in the absence and in the presence of increasing concentrations of extracellular Ca2+. These results emphasize the possible role of CaM in the control of histamine secretion from human basophils.     

Monoclonal antibodies to the leukocyte common antigen (CD45) inhibit IgE-mediated histamine release from human basophils.

mAb were selected that inhibited IgE-mediated histamine release from human basophils. The two mAb, HB 9AB6 and HB 10AB2, are of the IgG1 subclass and have a 50% inhibitory concentration of 0.16 to 1.1 micrograms/ml. The mAb required several hours of incubation with the basophils at 37 degrees C to induce maximum inhibition. Neither mAb directly released histamine from human basophils nor did they inhibit release induced by formylmethionine tripeptide, calcium ionophore A23187, or PMA. There was little inhibition of IgE-mediated release when the cells were preincubated with the mAb at 4 degrees C. By FACS analysis the 2 mAb bound to all peripheral blood leukocytes and immunoprecipitated a approximately 200-kDa protein from peripheral blood leukocytes and several cell lines of human origin. In binding studies and by sequential immunoprecipitation the 2 mAb and a known anti-CD45 mAb bound to the same protein. However, the mAb recognized different epitopes. Therefore, mAb to the CD45 surface Ag, a membrane protein tyrosine phosphatase, inhibits IgE-receptor mediated histamine release from human basophils. The data suggest a link between protein tyrosine phosphorylation and high affinity IgE receptor-mediated signal transduction in human basophils.     

Ouabain-induced enhancement of rat mast cells response. Modulation by protein phosphorylation and intracellular pH

The digitalic glicoside ouabain induces potentiation of rat mast cell histamine release in response to several stimuli, which is mediated by Na+/Ca2+ exchanger. In this work, we studied the effect of ouabain on cytosolic calcium, intracellular pH and histamine release with Ca2+ ionophore A23187 in conditions designed to maximize ouabain-induced potentiation of rat mast cells response. The effect of protein kinase C (PKC), cAMP and phosphatase inhibition was also tested. Ouabain induced an enhancement in histamine release, cytosolic calcium and intracellular pH. The adenylate cyclase activator forskolin reduced the effect of ouabain on histamine release and intracellular pH, but enhanced the effect on cytosolic calcium. PKC activator PMA enhanced the effect of ouabain on histamine release and cytosolic calcium, without affecting intracellular pH. A PKC inhibitor, GF-109203X, reduced ouabain-induced enhancement of histamine release and intracellular pH, but increased the enhancement on cytosolic calcium. Finally, inhibition of protein phosphatases 1 and 2A with okadaic acid, increased the effect of ouabain on histamine release and intracellular pH, but reduced cytosolic calcium in presence of ouabain. This result suggest that ouabain-induced potentiation of rat mast cell histamine release with A23187 is modulated by kinases, and this modulation may be carried out by changes in intracellular alkalinization. However, the mechanism underlying cellular alkalinization remains to be elucidated.     

Intracellular Ca2+ mobilization and not calcium influx promotes phorbol ester-stimulated thromboxane A2 synthesis in human platelets.

Phorbol esters, potent activators of protein kinase C (PKC), greatly enhance the release of arachidonic acid and its metabolites (TXA2, HETES, HHT) by Ca2+ ionophores in human platelets. In this paper, we report the relationship between intracellular Ca2+ mobilization and external calcium influx into platelets and the ability of PMA plus A23187 to promote thromboxane A2 (TXA2) synthesis. The enhanced levels of TXA2 due to the synergistic stimulation of the platelets with A23187 and phorbol esters are not affected significantly by the presence of external Ca2+ or the calcium-chelator EGTA. PKC inhibitors, staurosporine and sphingosine, abolished phorbol myristate acetate (PMA) potentiation of TXA2 production which strongly supports the role of PKC in the synergism. Platelet aggregation is more sensitive to PMA and external calcium than TXA2 formation. PMA increased TXA2 production as much as 4-fold at low ionophore concentrations. The A23187-induced rise in [Ca2+]i was reduced by pretreatment of human platelets with phorbol esters, both in the presence and absence of EGTA, and staurosporine reversed this inhibitory effect. These results indicate that the synergistic stimulation of TXA2 production by A23187 and phorbol esters is promoted by intracellular Ca2+ mobilization and not by external calcium influx. Our data also suggest that PKC is involved in the regulation of Ca2+ mobilization from some specific intracellular stores and that PKC may also stimulate the Ca(2+)-dependent phospholipase A2 at suboptimal Ca2+i concentrations.     

Protein L. A bacterial Ig-binding protein that activates human basophils and mast cells.

Peptostreptococcus magnus strain 312 (10(6) to 10(8)/ml), which synthesizes a protein capable of binding to kappa L chains of human Ig (protein L), stimulated the release of histamine from human basophils in vitro. P. magnus strain 644, which does not synthesize protein L, did not induce histamine secretion. Soluble protein L (3 x 10(-2) to 3 micrograms/ml) induced histamine release from human basophils. The characteristics of the release reaction were similar to those of rabbit IgG anti-Fc fragment of human IgE (anti-IgE): it was Ca2(+)- and temperature-dependent, optimal release occurring at 37 degrees C in the presence of 1.0 mM extracellular Ca2+. There was an excellent correlation (r = 0.82; p less than 0.001) between the maximal percent histamine release induced by protein L and that induced by anti-IgE, as well as between protein L and protein A from Staphylococcus aureus (r = 0.52; p less than 0.01). Preincubation of basophils with either protein L or anti-IgE resulted in complete cross-desensitization to a subsequent challenge with the heterologous stimulus. IgE purified from myeloma patients PS and PP (lambda-chains) blocked anti-IgE-induced histamine release but failed to block the histamine releasing activity of protein L. In contrast, IgE purified from myeloma patient ADZ (kappa-chains) blocked both anti-IgE- and protein L-induced releases, whereas human polyclonal IgG selectively blocked protein L-induced secretion. Protein L acted as a complete secretagogue, i.e., it activated basophils to release sulfidopeptide leukotriene C4 as well as histamine. Protein L (10(-1) to 3 micrograms/ml) also induced the release of preformed (histamine) and de novo synthesized mediators (leukotriene C4 and/or PGD2) from mast cells isolated from lung parenchyma and skin tissues. Intradermal injections of protein L (0.01 to 10 micrograms/ml) in nonallergic subjects caused a dose-dependent wheal-and-flare reaction. Protein L activates human basophils and mast cells in vitro and in vivo presumably by interacting with kappa L chains of the IgE isotype.     

Metabolic comparison between basophils and other leukocytes from human blood

Basophilic granulocytes were purified from the blood of normal individuals by successive isopyknic centrifugation and elutriation centrifugation. Starting with the leukocyte-rich fraction of 500 ml of blood, we recovered 31 to 80% (mean 51%, n = 20) of the basophils in 45 to 87% purity (mean 69%, n = 23). The contaminating cells were mainly lymphocytes. The basophils were greater than 98% vital (exclusion of ethidium bromide and hydrolysis of fluorescein diacetate). The histamine content of the basophils was 1.1 to 2 pg/cell (mean 1.6 pg/cell, n = 22). With anti-IgE, 30 to 50% of the histamine was released; with phorbol myristic acetate (PMA) or the calcium ionophore A23187, 70 to 100% of the histamine was released. Serum-opsonized zymosan (STZ) did not induce histamine release. Reactions with monoclonal antibodies revealed that the basophils expressed the C3bi receptor (CR3) and the leukocyte function-associated antigen 1 (LFA1), but not the gp 150,95 antigen, the C3b receptor (CR1), or the low avidity Fc gamma receptor. Basophils carry class I but not class II HLA antigens. During incubation of the basophils with serum-opsonized Staphylococcus aureus or Escherichia coli, these bacteria were neither phagocytized nor killed. STZ, PMA, A23187, or anti-IgE did not initiate an "oxidative burst" in the basophils. This was tested with oxygen consumption, cytochrome c reduction, NBT reduction, chemiluminescence, and release of hydrogen peroxide. Moreover, we did not detect cytochrome b558, superoxide dismutase, catalase, or peroxidase in the basophils. Of the typical granule-associated enzymes lysozyme, Vitamin B12-binding protein, and beta-glucuronidase, only beta-glucuronidase was present in the basophils in detectable amounts. This enzyme was released, together with histamine, on incubation of the cells with PMA, A23187, or anti-IgE, but not with STZ. We conclude that basophils from normal human blood are not phagocytes and are probably not involved in the oxidative defense of the host against foreign antigens.     

Complement-induced histamine release from human basophils. II. Mechanism of the histamine release reaction.

The activation of human serum complement by incubation with zymosan generates C5a which releases histamine from autologous basophils. The characteristics of the C5a-induced histamine release were investigated. It is similar to IgE-mediated reactions in requiring Ca++ and in being inhibited by EDTA. However, it has marked differences from IgE-mediated reactions. C5a, at all concentrations, released histamine completely in less than 2 min. The C5a reaction has a narrow pH optimum that antigen-induced release and occurs well at 17 degrees to 37 degreesC but not at 0 degreesC. The optimal reaction temperature is 25 degrees to 30 degrees C. Unlike the antigen-induced release, no two-stage activation with C5a for the release of histamine could be demonstrated. There was additive release between C5a- IgE-mediated reactions. Leukocytes could be desensitized to the C5a-mediated reaction by 1) incubating the cells at 37 degrees C for 45 min, 2) pretreating the leukocytes with activated serum in the presence of EDTA, and 3) adding the activated serum to the leukocytes at 0 degrees C before transferring to the optimal reaction temperatures. Cells desensitized to the complement-induced release have normal reactions to IgE-mediated histamine release. In parallel experiments, cells from allergic donors desensitized for IgE-mediated reactions by incubation with antigen under sub-optimal conditions release histamine normally upon the addition of C5a. The results indicate that histamine release by C5a involves a mechanism of basophil activation that is different from the pathway involved in the IgE-induced reaction.     

Different protein kinase C isozymes could modulate bradykinin-induced extracellular calcium-dependent and -independent increases in osteoblast-like MC3T3-E1 cells.

Effects of protein kinase C (PKC) on bradykinin (BK)-induced intracellular calcium mobilization, consisting of rapid Ca2+ release from internal stores and a subsequent sustained Ca2+ inflow, were examined in Fura-2-loaded osteoblast-like MC3T3-E1 cells. The sustained Ca2+ inflow as inferred with Mn2+ quench method was blocked by Ni2+ and a receptor-operated Ca2+ channel blocker SK&F 96365, but not by nifedipine. The short-term pretreatment with phorbol 12-myristate 13-acetate (PMA), inhibited BK-stimulated Ca2+ inflow, and the prior treatment with PKC inhibitors, H-7 or staurosporine, enhanced the initial internal release and reversed the PMA effect. Moreover, 6 h pretreatment with PMA caused similar effect on the BK-induced inflow to that obtained with PKC inhibitors, whereas 24 h pretreatment was necessary to affect the internal release. On the other hand, the translocation and down-regulation of PKC isozymes were examined after PMA treatment of MC3T3-E1 cells by immunoblot analyses of PKCs with the isozyme-specific antibodies. 6 h treatment with PMA induced down-regulation of PKC beta, whereas longer treatment was needed for down-regulation of PKC alpha. Taken together, it was suggested that the BK-induced initial Ca2+ peak and the sustained Ca2+ inflow through the activation of a receptor-operated Ca2+ channel, are differentially regulated by PKC isozymes alpha and beta, respectively, in osteoblast-like MC3T3-E1 cells.     

Extracellular signal-regulated kinases regulate leukotriene C4 generation, but not histamine release or IL-4 production from human basophils

Human basophils secrete histamine and leukotriene C4 (LTC4) in response to various stimuli, such as Ag and the bacterial product, FMLP. IgE-mediated stimulation also results in IL-4 secretion. However, the mechanisms of these three classes of secretion are unknown in human basophils. The activation of extracellular signal-regulated kinases (ERKs; ERK-1 and ERK-2) during IgE- and FMLP-mediated stimulation of human basophils was examined. Following FMLP stimulation, histamine release preceded phosphorylation of ERKs, whereas phosphorylation of cytosolic phospholipase A2 (cPLA2), and arachidonic acid (AA) and LTC4 release followed phosphorylation of ERKs. The phosphorylation of ERKs was transient, decreasing to baseline levels after 15 min. PD98059 (MEK inhibitor) inhibited the phosphorylation of ERKs and cPLA2 without inhibition of several other tyrosine phosphorylation events, including phosphorylation of p38 MAPK. PD98059 also inhibited LTC4 generation (IC50 = approximately 2 microM), but not histamine release. Stimulation with anti-IgE Ab resulted in the phosphorylation of ERKs, which was kinetically similar to both histamine and LTC4 release and decreased toward resting levels by 30 min. Similar to FMLP, PD98059 inhibited anti-IgE-mediated LTC4 release (IC50, approximately 2 microM), with only a modest effect on histamine release and IL-4 production at higher concentrations. Taken together, these results suggest that ERKs might selectively regulate the pathway leading to LTC4 generation by phosphorylating cPLA2, but not histamine release or IL-4 production, in human basophils.     

Effects of rhinovirus infection on histamine and cytokine production by cell lines from human mast cells and basophils

To understand the biochemical events that occur in the airways after rhinovirus (RV) infection, we developed for the first time a model in which the cell lines from human mast cells (HMC-1) and basophils (KU812) can be infected with RV14, a major group RV. Viral infection was confirmed by demonstrating that viral titers in culture supernatants, and RV RNA increased with time. RV14 infection alone and a combination of PMA plus calcium ionophore A23187, did not increase histamine production by these cells, although IgE plus anti-IgE increased the histamine production. However, histamine content in the supernatants increased in response to PMA plus A23187, or IgE plus anti-IgE after RV14 infection. PMA plus A23187 or IgE plus anti-IgE induced the production of IL-8 and GM-CSF in supernatants of HMC-1 cells and IL-4 and IL-6 in supernatants of KU812 cells. RV14 infection further increased the production of the cytokines, whereas RV14 infection alone did not alter the production of the cytokines by these cells. An Ab to ICAM-1 inhibited RV14 infection of the cells and decreased the production of cytokines and histamine after RV14 infection. RV14 infection enhanced the increases in intracellular calcium concentration and activation of NF-kappaB by PMA plus A23187 in the cells. These findings suggest that RV14 infection may prime the cytokine and histamine production from mast cells and basophils and may cause airway inflammation in asthma.     

Histamine release from human basophils by synthetic block co-polymers composed of polyoxyethylene and polyoxypropylene and synergy with immunologic and non-immunologic stimuli

Co-polymers composed of polyoxyethylene and polyoxypropylene have been shown previously to trigger histamine release from mouse peritoneal mast cells; this property quantitatively is directly related to the ionophorous ability of these compounds to cause a functional exchange of intracellular K+ for extracellular Na+ across the cell membrane. We investigated the effect of an inflammatory copolymer, T130R2, on human basophils. The data demonstrate that T130R2 can cause calcium-dependent histamine release from human basophils in vitro. Further, at concentrations that do not cause histamine release, this co-polymer markedly augments release by suboptimal concentrations of the lectin Con A or anti-IgE antibody and the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate but not the calcium ionophore A23187. Thus, these co-polymers induce mediator release from cells of both rodents and humans. In both instances it is likely that calcium-dependent cell triggering is the result of an influx of sodium ions with concomitant depolarization of the transmembrane potential. In common with the calcium ionophore A23187, the co-polymer T130R2 has the ability to synergize with stimuli which trigger the IgE receptor as well as those which directly activate the cellular calcium- and phospholipid-dependent protein kinase.     

Modulation of histamine-induced Ca2+ release by protein kinase C. Effects on cytosolic and mitochondrial [Ca2+] peaks

In HeLa cells, histamine induces production of inositol 1,4,5-trisphosphate (InsP3) and release of Ca2+ from the endoplasmic reticulum (ER). Ca2+ release is typically biphasic, with a fast and brief initial phase, followed by a much slower and prolonged one. In the presence of inhibitors of protein kinase C (PKC), including staurosporine and the specific inhibitors GF109203X and Ro-31-8220, the fast phase continued until the ER became fully empty. On the contrary, treatment with phorbol 12,13-dibutyrate inhibited Ca2+ release. Staurosporine had no effect on InsP3-induced Ca2+ release in permeabilized cells and did not modify either histamine-induced InsP3 production. These data suggest that histamine induces Ca2+ release and with a short lag activates PKC to down-regulate it. Consistently, Ca2+ oscillations induced by histamine were increased in amplitude and decreased in frequency in the presence of PKC inhibitors. We show also that mitochondrial [Ca2+] was much more sensitive to changes in ER-Ca2+ release induced by PKC modulation than cytosolic [Ca2+]. PKC inhibitors increased the histamine-induced mitochondrial [Ca2+] peak by 4-fold but increased the cytosolic [Ca2+] peak only by 20%. On the contrary, PKC activation inhibited the mitochondrial [Ca2+] peak by 90% and the cytosolic one by only 50%. Similarly, the combination of PKC inhibitors with the mitochondrial Ca2+ uniporter activator SB202190 led to dramatic increases in mitochondrial [Ca2+] peaks, with little effect on cytosolic ones. This suggests that activation of ER-Ca2+ release by PKC inhibitors could be involved in apoptosis induced by staurosporine. In addition, these mechanisms allow flexible and independent regulation of cytosolic and mitochondrial [Ca2+] during cell stimulation.