Release of histamine from human leukocytes stimulated with the tumor-promoting phorbol diesters. II. Interaction with other stimuli

The tumor promoter and irritant, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), previously shown to be a potent histamine releaser, has been used to further probe the mechanism of histamine release from human basophils. TPA and the calcium ionophore, A23187, produced a synergistic response in which subeffective concentrations of each stimulus (which alone produced less than 3% release) together produced over 70% histamine release. TPA also synergized with the IgE cross-linking stimulus anti-IgE. Desensitization of cells by incubation with anti-IgE in the absence of calcium rendered the cells unresponsive to anti-IgE and super-responsive to TPA. This marked increase in the TPA response was the result of an increase in the rate of TPA-induced histamine release, and occurred in the absence of extracellular calcium. The ability of various concentrations of anti-IgE to "sensitize" cells to TPA paralleled their ability to produce histamine release in untreated cells rather than their ability to desensitize the cells. These results suggest that in the absence of calcium, anti-IgE induces desensitization of some activation of other elements of the histamine-release process. The anti-IgE dose-response pattern of this activation event further suggests that it is an integral part of the anti-IgE-induced release process itself.     

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)     

Altered surface expression of CD11 and Leu 8 during human basophil degranulation.

Immunofluorescence and flow cytometric techniques have been used to study changes in surface Ag expression and viability that occur during human basophil degranulation. Treatment with polyclonal anti-IgE, FMLP, or the calcium ionophore A23187 induced histamine release, along with rapid and sustained unimodal increases in basophil CD11b mean fluorescence intensity. In contrast, treatment with anti-IgE or FMLP resulted in a decrease in Leu 8 expression. Degranulation did not significantly affect basophil viability (as determined by exclusion of propidium iodide), scatter characteristics, or percentage of identifiable IgE-bearing cells, and an inconsistent association was seen between percent histamine release and reduction in the percent of cells identified by light microscopy after staining with alcian blue. For anti-IgE, dose-dependent changes in CD11b, CD11c, and Leu 8 expression were seen (optimal at 0.1, 0.1, and 1 microgram/ml, respectively), although CD11a expression remained unchanged. Histamine release was optimal at 0.3 microgram/ml anti-IgE, and at superoptimal concentrations, reduced CD11b expression was observed which paralleled decreases in histamine release; reduction of the expression of Leu 8, however, occurred equally at optimal and superoptimal concentrations of anti-IgE. Kinetic analyses of these responses revealed that CD11b up-regulation proceeded more rapidly than histamine release, whereas Leu 8 down-regulation was much slower and did not plateau until 120 min of stimulation. Although changes in CD11b mean fluorescence intensity correlated with the magnitude of histamine release, exposure to stimuli in the absence of calcium (which blocked degranulation) resulted in similar alterations in CD11b and Leu 8, suggesting that degranulation was not required for changes in the surface expression of these adhesion molecules. Interestingly, pretreatment of basophils with drugs that either inhibited or enhanced histamine release (isobutylmethylxanthine and cyclosporin A vs cytochalasin B, respectively) significantly decreased the magnitude of anti-IgE-induced CD11b up-regulation; down-regulation of Leu 8 expression was also partially inhibited by treatment with isobutylmethylaxanthine. These studies demonstrate that activation of human basophils by secretagogues in vitro results in a variety of phenotypic changes including alterations in surface expression of adhesion molecules, and suggest that degranulation in vivo may be accompanied or preceded by changes in adhesion-related functions.     

Characterization of the effects of phorbol esters on rat mast cell secretion

When applied to the skin, phorbol esters (PEs) elicit signs of acute inflammation, suggesting they may induce the release of mediators from mast cells. Therefore, we have studied the effects of PEs on purified rat peritoneal and thoracic mast cells both alone and in conjunction with the calcium ionophore, A23187, and various other secretagogues that interact with immunoglobulin E (e.g., anti-IgE and Con A) or other cell surface receptors, e.g., somatostatin and compd 48/80. PEs alone caused little or no release of histamine. However, the PE 12-O-tetradecanoylphorbol-13-acetate (TPA, 10 ng/ml) tremendously potentiated release induced by the calcium ionophore A23187, reducing the EC50 for A23187 from 832 ng/ml to 56 ng/ml. In the presence of suboptimal A23187 (50 ng/ml), only active tumor promoting PEs elicited histamine release. The EC50 values of the various active PEs were: TPA 5 ng/ml; 4 beta-PDD, 83 ng/ml; and 4-O-methyl-TPA, 807 ng/ml, with maximal histamine release ranging from 54 to 80%. TPA synergistically enhanced stimulation of histamine release by anti-IgE and Con A over the entire concentration-response range. In contrast, this synergism was absent when cells were stimulated with somatostatin and compd 48/80. Phorbol esters may act by increasing the activity of a calcium/phospholipid-dependent protein kinase (Ca/PL-PK). Mast cells do have Ca/PL-PK activity, and TPA in the presence of suboptimal A23187 induces protein phosphorylation comparable with other secretagogues. These results suggest that in the purified mast cell, PE-induced mediator release increases the sensitivity of release mechanisms for calcium, acts syngergistically with secretagogues interacting with IgE, and as suggested from structure-activity relationships, occurs via a specific mechanism of action perhaps involving the Ca/PL-PK.     

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)     

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.     

IgE-mediated release of histamine from human cutaneous mast cells

We investigated the ability of antigen-IgE interactions to stimulate histamine release from human infant cutaneous mast cells. Skin obtained at circumcision contained numerous perivascular mast cells, as assessed by light and electron microscopy. The histamine content of this tissue averaged 17.7 ng (+/- 1.5 SEM)/mg wet weight. Challenge of 200-microns thick sections of unsensitized skin with varying concentrations of monoclonal murine antibodies to human IgE caused no net release of histamine. After skin sections were incubated in the presence of 5 micrograms/ml of human myeloma IgE (S) for 120 min at 37 degrees C, monoclonal anti-IgE challenge resulted in 40.1% (+/- 6.0 SEM) histamine release. Similar passive sensitization with 1/20 dilutions of serum from humans expressing IgE to purified Juniperus sabinoides (JS) antigen rendered the tissue responsive to specific antigen challenge. Dose-related histamine release occurred over 30 min with optimal release of 12.6% (+/- 2.4 SEM) after stimulation with 100 ng/ml of JS antigen. This reaction required sensitization with serum containing IgE to JS and was antigen-specific. Optimal reactions to antigen occurred at 3 mM added Ca++, 34 degrees C to 37 degrees C, pH 7.2. Antigen-induced release was markedly influenced by the added Ca++ concentration; no release occurred in the absence of Ca++, 54% of the optimal response was observed at 2 mM Ca++, and 28% of the optimal response occurred at 4 mM Ca++. The addition of Mg++ did not influence antigen-induced release. The results of this study provide functional evidence that 1) human infant cutaneous mast cells express Fc-epsilon receptors; 2) these receptors are largely unoccupied in vivo; and 3) stimulation of passively sensitized infant mast cells with anti-IgE or specific antigen leads to immediate histamine release. This new system should permit detailed in vitro studies of immediate hypersensitivity reactions in human skin.     

Histamine-containing cells obtained from the nose hours after antigen challenge have functional and phenotypic characteristics of basophils.

The pattern of mediators and appearance of cells that stain with alcian blue during human experimental early and late phase allergic reactions suggest that basophils accumulate in nasal secretions within hours of local Ag stimulation. To further explore whether the histamine containing cells that enter the nose after Ag challenge are mast cells or basophils, we studied their functional and phenotypic characteristics. Approximately 24 h after intranasal Ag provocation of subjects with allergic rhinitis, nasal lavage was performed, and the cells were isolated for degranulation studies, analysis of surface Ag, and viability. The average histamine content per alcian blue staining cell was 0.78 +/- 0.2 pg (n = 7), similar to that reported for peripheral blood basophils. Nasal cells were challenged in vitro with anti-IgE, ragweed Amb a I, and FMLP and their responses were compared to those of peripheral blood basophils isolated simultaneously from the same donors. Nasal leukocytes released histamine maximally at 0.1 micrograms/ml of anti-IgE (35.8 +/- 7.8%, n = 7) and responded to FMLP (25.4 +/- 9.9%, n = 7). The response of the cells to ragweed Amb a I and anti-IgE was attenuated compared to peripheral blood basophils. Anti-IgE-induced histamine release was calcium and temperature dependent. Dual color immunofluorescence and flow cytometric analysis of the recovered nasal cells coexpressed CD18, a leukocyte marker not expressed by mast cells. The nasal cells consistently had high levels of spontaneous histamine release (19.5 +/- 2.0%, n = 22). The viability of all cells, assessed by erythrosin B dye exclusion, was 70 +/- 2% (n = 15). However, the viability of IgE-bearing cells was only 28.3 +/- 5.7% (n = 4). The characteristics of histamine release and the nature of the cellular surface markers provide functional proof that the histamine-containing cells accumulating after nasal Ag challenge are basophils and not mast cells.     

Mechanisms of mouse mast cell activation and inactivation for IgE-mediated histamine release.

The IgE-mediated histamine release from mouse mast cells requires Ca++, is optimal at 37 degrees C, and is enhanced by phosphatidylserine. The rate of release is relatively slow. The mast cells can be activated to release histamine by either anti-IgE or anti-Fab antibodies and, in the case of cells from sensitized mice, by the immunizing antigen. The incubation of mast cells with antigen in the absence of Ca++ or phosphatidylserine fails to release histamine. Such cells are desensitized to the further addition under optimal conditions of the same antigen. Desensitization is antigen specific, requires optimal levels of antigen, and occurs at both 30 degrees and 37 degrees C. In contrast, anti-IgE desensitizes all IgE-mediated histamine release reactions.     

Concanavalin A-induced histamine release from normal rat mast cells.

Concanavalin A- (con A) induced release of histamine from normal rat mast cells was studied. In the presence of phosphatidylserine (PS) con A induced a concentration and temperature-dependent, noncytotoxic histamine release at con A concentrations ranging from 0.1 to 100 mug/ml. The optimal con A concentration, 100 mug/ml, caused a 27.3% (+/- 2.7 S.E.M.) net histamine release. Release began approximately 30 sec after addition of con A and was complete within 45 min. In the absence of PS, no net con A-induced release occurred. The effect of PS was concentration dependent from 1 to 100 mgg/ml. PS alone, however, did not cause histamine release. Binding studies indicated that mast cells bound up to 16 X 10(6) con A molecules per cell without histamine release. Upon removal of unbound con A and the addition of PS, normal histamine release occurred. Alpha-Methyl-D-mannose (50 mM) prevented both con A binding and histamine release and if added after Con A, caused a rapid cessation of histamine release and a reversal of con A binding. This study indicates several important advantages of the con A-induced histamine release system. Binding of con A to mast cells can be dissociated from histamine release by omitting PS from the medium. Release can then be induced by the addition of PS. Alpha-Methyl-D-mannose can be used to terminate rapidly the ongoing release reaction at any phase of the interaction. This system is potentially very useful for investigation of metabolic events during histamine release.     

The mechanism of mediator release from human basophils induced by platelet-activating factor.

Platelet-activating factor (PAF) is a lipid mediator able to induce a variety of inflammatory processes in human peripheral blood cells. We have investigated the effect of PAF on the release of chemical mediators from human basophils of allergic and normal donors. PAF (10 nM to 1 microM) caused a concentration-dependent, noncytotoxic histamine release (greater than or equal to 10% of total) in 27 of 44 subjects tested (24 atopic and 20 nonatopic donors). The release process was either very rapid (t1/2 approximately equal to 10 s) or quite slow (t 1/2 approximately equal to 10 min), temperature- and Ca2(+)-dependent (optimal at 37 degrees C and 5 mM Ca2+). Coincubation of PAF with cytochalasin B (5 micrograms/ml) enhanced the release of histamine induced by PAF and activated the release process in most donors (42 of 44). Atopics did not release significantly more histamine than normal subjects, and the percentage of PAF responders (greater than or equal to 10% of total) was nearly the same in the two groups. Histamine release was accompanied by the synthesis and release of leukotriene C4, although this lagged 1 to 2 min behind histamine secretion. Lyso-PAF (100 nM to 10 microM), alone or together with cytochalasin B, did not release significant amounts of histamine. The release of histamine activated by PAF was inhibited by the specific PAF receptor antagonist, L-652,731, with an IC50 of 0.4 microM. There was a partial desensitization to PAF when the cells were preincubated with PAF (100 nM to 1 microM) for 2 min in the absence of Ca2+, whereas the cells remained responsive to anti-IgE (0.1 micrograms/ml). If neutrophils were removed from the basophil preparation by a Percoll gradient or a countercurrent elutriation technique, there was a significant decrease in PAF-induced histamine release. PAF (1 microM) was able to induce a very rapid, transient rise (peak less than 10 s) in [Ca2+]i in purified basophils analyzed by digital video microscopy. Finally, among human histamine-containing cells, the basophils are unique in degranulating following a PAF challenge. Mast cells from human lung, skin, or uterus failed to respond to PAF (10 nM to 1 microM) regardless of the presence or absence of cytochalasin B (5 micrograms/ml). Our results demonstrate that PAF is able to induce the release of inflammatory mediators from human basophils, and that neutrophils can influence this response. It is suggested that PAF-induced basophil activation can play a role in the pathogenesis of allergic disorders.     

Signal transduction events in human basophils. A comparative study of the role of protein kinase C in basophils activated by anti-IgE antibody and formyl-methionyl-leucyl-phenylalanine.

We have compared the transmembrane signals generated in human basophils by two distinct stimuli, anti-IgE antibody and FMLP (f-met peptide). Although both stimuli resulted in the activation of protein kinase C (PKC) and an increase in intracellular free calcium, there were substantial differences between the two which suggested that distinct signal transduction mechanisms were operating. We have confirmed an earlier observation that the cross-linking of IgE led to an increase in membrane PKC activity with no apparent concomitant loss of cytosolic PKC and established that in contrast, the univalent stimulus, f-met peptide, resulted in the canonical translocation of cytosolic PKC to the membrane. Furthermore, unlike anti-IgE-stimulated basophils, there was no clear relationship between the increase in PKC activity and the subsequent release of histamine. Two PKC inhibitors, staurosporine (0.1 to 1 nM) and sphingosine (25 to 50 microM), inhibited anti-IgE induced release, yet, potentiated the release of mediators after a challenge with 1 microM f-met peptide. Both stimuli led to an increase in the intracellular Ca2+ levels that correlated well with the release of histamine, however, the anti-IgE-induced responses were typically only 50% of those required to give equivalent histamine release when f-met peptide initiated release. Pharmacologic evidence suggested that the up-regulation of PKC was required for a full IgE-mediated Ca2+ response and that PKC contributed to the elevated Ca2+ levels that persist for up to 15 min after the addition of anti-IgE. In contrast, the PKC inhibitor, staurosporine, did not affect the initial increase in Ca2+ after the addition of f-met peptide but reduced the rate at which Ca2+ was removed from the cytosol. Experiments with the phorbol ester, PMA, suggested that substantial degranulation can occur in the absence of any increase in intracellular Ca2+. The addition of 10 ng/ml PMA 10 min before the addition of f-met peptide did not affect the magnitude of the initial Ca2+ transient but increased the rate at which Ca2+ levels returned to a stable baseline. Similar pretreatment with PMA almost completely abolished the anti-IgE antibody-induced Ca2+ response. These experiments, together with other previous data, suggest that the activation of PKC is a prodegranulatory component of the IgE-mediated signal transduction pathway, yet serves principally to modulate the Ca2+ signal when f-met peptide initiates release.     

Unresponsiveness of rat peritoneal mast cells to immunologic reactivation.

Rat peritoneal mast cells cocultured with 3T3 fibroblasts (MC/3T3) were activated with Ag or with anti-IgE antibodies in the presence of Ca2+, and their responsiveness to a second similar challenge was evaluated. MC/3T3 were presensitized with IgE anti-DNP antibodies and activated with DNP-human serum albumin. When these MC/3T3 were reactivated with the same Ag 2 and 6 h later, they released only a minimal percentage of histamine. A gradual recovery of responsiveness was detected during the first 7 days after activation, and a full recovery was attained by days 14 to 21. A similar pattern of unresponsiveness was observed when MC/3T3 were challenged and rechallenged with cercarial Ag after presensitization with anticercarial serum. Activation of MC/3T3 with one Ag (DNP-human serum albumin or cercarial) and rechallenge 3 days later with the other Ag did not overcome the state of partial unresponsiveness. Challenging MC/3T3 with anti-IgE led to a subsequent unresponsiveness to rechallenge with the same ligand, regardless of whether or not the cells were presensitized with IgE antibodies. Cross-linkage with anti-IgE resulted in a more intense and prolonged state of unresponsiveness in comparison with that observed with Ag. When MC/3T3 activated with anti-IgE were rechallenged with various IgE-independent agents they released a percentage of histamine comparable to that of control cultures challenged with these secretagogues for the first time. MC/3T3 partially resynthesized their histamine content during the two-week period after activation. Our results suggest that MC undergo a temporary state of "physiologic" unresponsiveness after immunologic activation in the presence of calcium ions.     

Ionophore A-23187 induced histamine release from rat mast cells and rat basophil leukemia (RBL-1) cells.

Ionophore A-23187 releases histamine from normal mast cells apparently by promoting Ca++ influx (Foreman et al, Nature 245: 249, 1973). In our hands at concentrations of greater than 0.2 mug/ml release occurs in 1 to 2 min, is blocked by metabolic inhibitors, and is unaccompanied by cytotoxicity (trypan-blue uptake, lactic dehydrogenase (LDH) release). At higher doses (0.5 mug/ml) histamine release is followed by significant cytotoxicity, but again Ca++ is required. In parallel studies, we examined cultured rat basophilic leukemia (RBL-1) cells. These cells, which apparently have normal surface receptors for IgE, contained approximately 700 ng histamine/10(6) cells but did not release histamine when IgE-mediated release was looked for. They do not respond to doses of ionophore which would be expected to give non-cytotoxic histamine release. At higher doses histamine release is preceded by progressive LDH release: LDH release is 75% complete at 5 min whereas 10 min are required for 75% maximal histamine release. This reaction requires Ca++ and is temperature dependent but is not inhibited by metabolic poisons (2-deoxyglucose, dinitrophenol, CN-). These studies suggest that either Ca++ does not enter into these cells normally or that one or more mechanisms which are ordinarily triggered by the changes in Ca++ flow are unresponsive in the RBL-1 cells. These studies also underline the importance of ruling out cytotoxicity in ionophore-induced phenomena.     

Anti-human IgG causes basophil histamine release by acting on IgG-IgE complexes bound to IgE receptors.

We have reexamined the ability of anti-human IgG antibodies to induce histamine release from human basophils. A panel of purified murine mAbs with International Union of Immunological Societies-documented specificity for each of the four subclasses of human IgG was used. Of the 24 allergic subjects studied, the basophils of 75% (18/24) released greater than 10% histamine to one or more anti-IgG1-4 mAb, whereas none of the 13 nonatopic donor's basophils released histamine after stimulation with optimal amounts of anti-IgG mAb. The basophils of 85% (11/13) of the nonatopic donors did respond to anti-IgE challenge, as did 92% (22/24) of the atopic donor cells. Histamine release was induced most frequently by anti-IgG3, and 10/18 anti-IgG responder cells released histamine with mAb specific for two or more different subclass specificities. The rank order for induction of histamine release was anti-IgG3 greater than anti-IgG2 greater than IgG1 greater than anti-IgG4. As in our previous study using polyclonal anti-IgG, 100- to 300-micrograms/ml quantities of the anti-IgG mAb were required for maximal histamine release, about 1000-fold higher than those for comparable release with anti-human IgE. Specificity studies using both immunoassays and inhibition studies with IgE myeloma protein indicated that anti-IgG induced histamine release was not caused by cross-reactivity with IgE. Ig receptors were opened by lactic acid treatment so that the cells could be passively sensitized. Neither IgE myeloma nor IgG myeloma (up to 15 mg/ml) proteins could restore the response to anti-IgG mAb. However, sera from individuals with leukocytes that released histamine upon challenge with anti-IgG mAb could passively sensitize acid-treated leukocytes from both anti-IgG responder and nonresponder donors for an anti-IgG response. The only anti-IgG mAb that induced release from these passively sensitized cells were those to which the serum donor was responsive. Sera from non-IgG responders could not restore an anti-IgG response. These data led to the hypothesis that the IgG specific mAb were binding to IgG-IgE complexes that were attached to the basophil through IgE bound to the IgE receptor. This was shown to be correct because passive sensitization to anti-IgG could be blocked by previous exposure of the basophils to IgE. We conclude that anti-IgG-induced release occurs as a result of binding to IgG anti-IgE antibodies and cross-linking of the IgE receptors on basophils.     

Measurement of IgE on human basophils: relation to serum IgE and anti-IgE-induced histamine release.

The number of IgE molecules bound to human basophils was calculated from direct measurements of the IgE dissociated after exposing leukocytes to pH 3.7 acetate buffer in the cold. In 18 donors studied, cell-bound IgE ranged from 4000 to 500,000 molecules/basophil and correlated with the serum IgE concentration (r = 0.89, p less than 0.001) which ranged from 5 to 3,000 ng/ml. Sensitivity of these cells to anti-IgE was tested to explore the relationship between cell-bound IgE and the concentration of anti-IgE required for histamine release. Cells from some nonatopic donors (4000 to 100,000 IgE molecules/basophil) were as sensitive as cells from allergic donors (100,00 to 500,000 IgE molecules/basophil). Moreover, cells from donors having approximately the same cell-bound IgE concentration varied widely in their sensitivity to anti-IgE. We conclude that an intrinsic property of human basophils ("releasability") is an important parameter in determing mediator release.     

IgE enhances Fc epsilon receptor I expression and IgE-dependent release of histamine and lipid mediators from human umbilical cord blood-derived mast cells: synergistic effect of IL-4 and IgE on human mast cell Fc epsilon receptor I expression and mediator release

We investigated the effects of IgE versus IL-4 on Fc epsilon RI surface expression in differentiated human mast cells derived in vitro from umbilical cord blood mononuclear cells. We found that IgE (at 5 micrograms/ml) much more strikingly enhanced surface expression of Fc epsilon RI than did IL-4 (at 0.1-100 ng/ml); similar results were also obtained with differentiated mouse mast cells. However, IL-4 acted synergistically with IgE to enhance Fc epsilon RI expression in these umbilical cord blood-derived human mast cells, as well as in mouse peritoneal mast cells derived from IL-4-/- or IL-4+/+ mice. We also found that: 1) IgE-dependent enhancement of Fc epsilon RI expression was associated with a significantly enhanced ability of these human mast cells to secrete histamine, PGD2, and leukotriene C4 upon subsequent passive sensitization with IgE and challenge with anti-IgE; 2) preincubation with IL-4 enhanced IgE-dependent mediator secretion in these cells even in the absence of significant effects on Fc epsilon RI surface expression; 3) when used together with IgE, IL-4 enhanced IgE-dependent mediator secretion in human mast cells to levels greater than those observed in cells that had been preincubated with IgE alone; and 4) batches of human mast cells generated in vitro from umbilical cord blood cells derived from different donors exhibited differences in the magnitude and pattern of histamine and lipid mediator release in response to anti-IgE challenge, both under baseline conditions and after preincubation with IgE and/or IL-4.     

Corticosteroids inhibit prostaglandin release from perfused mesenteric blood vessels of rabbit and from perfused lungs of sensitized guinea pig.

Infusion of norephinephrine (NE) (1 - 3 mug/ml/min) into the isolated mesenteric vascular preparation of rabbit resulted in a rise in perfusion pressure, which was associated with the release of prostaglandin E-like substance (PGE) at a concentration of 2.81 +/- 0.65 ng/ml in terms of PGE2. Indomethacin (3 mug/ml) abolished the NE-induced release of PGE. Arachidonic acid (0.2 mug/ml) in the presence of indomethacin did not restore the NE-induced release of PGE. Hydrocortisone (10 - 30 mug/ml) and dexamethasone (2 - 5 mug/ml) also inhibited the NE-induced release of PGE. The inhibitory action of both corticosteroids was abolished by arachidonic acid (0.2 mug/ml). Antigen-induced release of a prostaglandin-like substance (PGs) (43.1 +/- 3.8 ng/ml in terms of PGE2 and a rabbit aorta contracting substance (RCS) from perfused lungs of sensitized guinea pigs was completely abolished by indomethacin (5 mug/ml) or by hydrocortisone (100 mug/ml). Indomethacin, however, increased histamine release up to 280% of the control level, which was 470 +/- 54 ng/ml, while hydrocortisone diminished histamine release down to 30% of the control level. A superimposed infusion of arachidonic acid (1 mug/ml) into the pulmonary artery reversed the hydrocortisone-induced blockade of the release of RCS and PGs. It may be concluded that corticosteroids neither inhibit prostaglandin synthetase nor influence prostaglandin transport through the membranes but they do impair the availability of the substrate for the enzyme.     

Effect of NCDC, a protease inhibitor, on histamine release from rat peritoneal mast cells induced by anti-IgE.

NCDC dose-dependently inhibited histamine release from rat peritoneal mast cells induced by anti-IgE. Moreover, NCDC inhibited Ca(2+)-mobilization from intracellular Ca(2+)-stores as well as histamine release in mast cells activated by anti IgE, the effect on both of these phenomena being closely correlated. Anti-IgE induced a rapid increase in IP3 production from phosphoinositides in mast cells, with its production in 15 sec, followed to baseline levels within 1 min. Anti-IgE stimulated PLC activity on mast cells membrane preparation. NCDC dose-dependently inhibited the generation of IP3. These results suggest that the inhibitory effect of NCDC on the release of histamine induced by anti-IgE is due to, in part at least, the inhibition of PI-specific PLC and that the inhibitory effects of NCDC are involved in intracellular calcium store.     

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.