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.     

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.     

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)     

Human lung macrophage-derived histamine-releasing activity is due to IgE-dependent factors

Human lung macrophages obtained from surgical specimens spontaneously secreted a factor(s) (which we term macrophage factor) during 24-hr culture that induced calcium-dependent histamine release from human basophils and lung mast cells. Macrophage factor induced noncytotoxic histamine release from purified (85%) basophils. The kinetics of release were relatively slow and similar to that of anti-IgE. We performed a series of experiments to test the IgE dependence of macrophage factor-induced release. Preincubation of basophils with anti-IgE in calcium-free medium resulted in complete desensitization to macrophage factor-induced histamine release (i.e., when calcium and macrophage factor were added to the basophils, no histamine release occurred), and preincubation with macrophage factor in calcium-free medium resulted in partial desensitization to anti-IgE-induced histamine release. Pretreatment of basophils with pH 3.9 lactic acid buffer, which dissociates basophil IgE from its receptors, markedly reduced the capacity of basophils to release histamine in response to macrophage factor. Basophils that were incubated with IgE myeloma (but not with IgG) after lactic acid treatment partially or completely regained their capacity to release histamine in response to macrophage factor. Fluid-phase IgE myeloma (15 micrograms/ml) (but not IgG) inhibited basophil histamine release induced by two macrophage-derived supernatants, whereas IgE myeloma (200 micrograms/ml) did not inhibit release due to other supernatants. IgE-affinity columns removed the histamine-releasing activity of five macrophage-derived supernatants, and IgG-affinity columns had similar effects. However, neither affinity column removed the histamine-releasing activity of three other macrophage-derived supernatants. On Sephadex G-75 chromatography, nearly all of the histamine-releasing activity migrated as single peak with an apparent m.w. of 18,000. These results suggest that, although macrophage factor are heterogeneous, they are related, as they are a IgE-dependent factors that induce histamine release by interacting with cell surface IgE. These macrophage factors may be responsible for stimulation of basophil/mast cell mediator release in chronic allergic reactions.     

Activation of human basophils through the IL-8 receptor.

IL-8 and its structural analogs derived from blood platelets have been proposed as stimuli of IgE-independent basophil activation. In order to clarify the mechanism of action of these peptides, we examined the effects of pure IL-8, connective tissue-activating peptide III (CTAP-III), neutrophil-activating peptide 2 (NAP-2), and platelet factor 4 (PF-4) on blood basophils with and without pretreatment by IL-3, which modulates mediator release. After pretreatment with IL-3, significant histamine release was observed with 10(-8) M and 10(-7) M IL-8 and 10(-7) M NAP-2, but not with the other peptides. At higher concentrations (10(-6) M), however, all IL-8 analogs, as well as the unrelated cationic peptides poly-D-lysine, histone VS, and lysozyme, induced histamine release to variable degrees. Binding and competition studies with [125I]IL-8 revealed specific IL-8R on basophils from a patient with chronic myelogenous leukemia and normal individuals. From 3500 to 9600 receptors with a mean Kd value of 0.15 nM were found on average per chronic myelogenous leukemia and normal basophil, respectively. NAP-2 weakly competed for IL-8 binding. IL-8 and, to a lesser extent, NAP-2 led to a transient rise of cytosolic free calcium concentration ([Ca2+]i), which was independent of a preexposure to IL-3. IL-8 prevented the [Ca2+]i rise induced by NAP-2, but did not influence [Ca2+]i responses to other agonists, e.g. C5a, C3a, or platelet-activating factor. IL-8 induced [Ca2+]i changes and histamine release in IL-3-primed basophils were pertussis toxin sensitive. CTAP-III or PF-4 did not compete for IL-8 binding, did not induce [Ca2+]i changes, and did not influence the [Ca2+]i response to IL-8 and NAP-2. This study shows that IL-8 and NAP-2 activate human basophils by a receptor-mediated mechanism similar to that operating in neutrophils. At high concentrations histamine release can also be induced by cationic peptides by a mechanism that does not involve the IL-8R, and probably depends on cationic interactions.     

Platelet-activating factor induces mediator release by human basophils primed with IL-3, granulocyte-macrophage colony-stimulating factor, or IL-5.

The phospholipid platelet-activating factor (PAF) is a potent cell-derived bioactive molecule thought to be involved in diverse inflammatory processes. It has been shown that PAF can activate different leukocyte types and platelets, particularly in synergy with other agonists. In this study we examined the effect of PAF upon the release of histamine and leukotriene (LT) C4 by basophils when added alone and in combination with different agonists and cytokines. PAF by itself did neither induce histamine release nor the generation of LTC4 by basophils. However, basophils primed by the hematopoietic growth factors (hGF) IL-3, granulocyte-macrophage (GM)-CSF, or IL-5 (10 ng/ml) released preformed and de novo synthesized mediators in response to PAF at 10 to 100 nM concentrations. The extent of mediator release by hGF primed basophils in response to PAF was similar to that induced by an optimal concentration of monoclonal anti-IgE. Thus, similar to NAP-1/IL-8 and C3a, PAF efficiently stimulates mediator release in hGF-primed basophils only. However, PAF was clearly a more potent trigger of LTC4 formation in IL-3-primed cells than NAP-1/IL-8 or C3a. When PAF was used as a second trigger, the priming effect of IL-5 was less than that of IL-3 or GM-CSF, whereas the response for other IgE-independent agonists (i.e., C5a or FMLP) was augmented equally by all three hGF. IL-1 beta-pretreated basophils released minimal amounts of histamine in response to PAF. Neither TNF-alpha nor PAF, nor the combination thereof, was able to induce basophil mediator release. The efficiency of the different cytokines to prime for PAF responsiveness was strikingly similar to their capacity to enhance anti-IgE-induced mediator release. Similar to other IgE-independent agonists, the kinetic of mediator release in response to PAF was very rapid. PAF pretreatment of basophils did not enhance mediator release in response to diverse agonists, such as C5a and FMLP, in contrast to the capacity of PAF to augment the response of other leukocyte types to appropriate stimuli. Thus, depending on the presence of IL-3, GM-CSF, or IL-5, PAF is a potent basophil agonist capable of inducing histamine release as well as de novo synthesis of LTC4.     

IL-8 inhibits histamine release from human basophils induced by histamine-releasing factors, connective tissue activating peptide III, and IL-3.

We have previously purified and partially characterized histamine releasing factors (HRF), which were derived from a mixture of human mononuclear cells and platelets. We now report the effect of IL-8 upon HRF-, connective tissue activating peptide III (CTAP III)-, and IL-3-induced histamine release from human basophils. We determined that IL-8 itself, at concentrations between 10(-7) to 10(-11) M, does not release histamine from basophils, although positive results are observed in two of 26 subjects at 10(-7) M. Unfractionated (crude) HRF released histamine in 25 of 26 donors, in the range of 6.7% to 100% of total basophil histamine stores. When basophils were preincubated with IL-8 (10(-7) to 10(-11) M) for 5 min, followed by a 40-min incubation with HRF, histamine release was significantly inhibited in 20 of 25 donors. Inhibition was observed at as little as 10(-11) M IL-8, with maximal inhibition being attained at 10(-9) M. HRF-containing supernatants contain a mixture of different histamine-releasing moieties. To better define which factor(s) may be inhibited by IL-8, fractionated supernatants, purified CTAP III, and IL-3 were studied. Histamine release produced by two different HRF-containing chromatographic fractions (HRFvoid and HRFpeak 2) and purified CTAP-III (5 micrograms/ml) was inhibited by IL-8 in 10 of 12 donors, three of three donors, and seven of 10 donors, respectively. IL-3 (5000 U/ml)-dependent histamine release was inhibited by IL-8 in all subjects tested. In contrast, histamine release by anti-IgE and FMLP was not affected by IL-8. Thus, IL-8 appears to be an inhibitor of cytokine-like molecules that induce histamine release and may represent the previously described 8-kDa histamine release inhibitory factor present in mononuclear cell supernatants.     

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.     

IL-3 augments adhesiveness for endothelium and CD11b expression in human basophils but not neutrophils.

A number of natural and recombinant human cytokines have been tested for their ability to activate basophil and neutrophil adhesiveness for human umbilical vein endothelial cells in vitro. Coincubation of basophils and endothelial cell monolayers for 10 min with biologically relevant concentrations of rIL-1, natural IL-2, rIL-4, rIL-5, rIL-6, rIL-8, rGM-CSF, and rIFN-gamma had no effect on basophil adhesiveness. In contrast, rIL-3 induced basophil adhesiveness for endothelial cells (optimal at 1 ng/ml: 144 +/- 18% of control adherence (mean +/- SEM); control basophil binding, 13 +/- 3%, n = 9, p less than or equal to 0.05). This increase in adhesiveness was similar in magnitude to that induced by an optimal concentration of a known potent inducer of basophil adhesiveness (1 microM FMLP, 164 +/- 15% of control adherence, n = 9). Under these experimental conditions, the effects of rIL-3 occurred at concentrations of 0.1 to 30 ng/ml, were partially dependent on calcium, and were not accompanied by histamine release. Fixation experiments demonstrated that the effect of rIL-3 was directed against the basophil rather than the endothelial cell. Neither rIL-3 nor the other cytokines tested had any effect on the adherence of 51Cr-labeled neutrophils, even when tested simultaneously on cells from the same donors. Under experimental conditions that permitted histamine release, no correlation was seen between the ability of rIL-3 (0.3 to 300 ng/ml) to induce histamine release or enhance adhesiveness (n = 8). mAb blocking experiments demonstrated a role for both CD11 and CD18 adherence glycoproteins in basophil adherence induced by rIL-3, and indirect immunofluorescence and flow cytometric analysis revealed that rIL-3 treatment led to rapid and sustained increases in cell surface expression of CD11b antigens on basophils but not neutrophils (e.g., after 10 min: 217 +/- 29 vs 91 +/- 11% of control mean fluorescence intensity, p less than 0.05). However, no correlation was seen between the magnitude of changes in CD11b expression and changes in adhesion when tested simultaneously. These results suggest that local production of IL-3 during allergic reactions in vivo may selectively promote basophil activation, adhesion to endothelium, and recruitment to extravascular sites of inflammation.     

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.     

Recombinant human IL-1 alpha and -1 beta potentiate IgE-mediated histamine release from human basophils

In this study, we have explored the relationship between interleukins and human basophil activation. Previous studies by ourselves and others have found that recombinant human (rh) IL-3 causes histamine release. The ability to release histamine has also been claimed for IL-1 but we cannot confirm this. In experiments with the basophils of 29 donors (excluding one D2O responder), histamine release with 100 ng/ml rhIL-1 alpha was 1.3 +/- 1% (SEM), whereas with rhIL-1 beta, it was 0.8 +/- 1%. Both IL-1 alpha and -1 beta were also used at concentrations of 0.01 to 1000 ng/ml without causing release. Neither increasing the Ca2+ concentration nor adding D2O or cytochalasin B caused IL-1 alpha and -1 beta to become secretagogues. rhIL-1, however, did augment IgE-dependent histamine release. The enhancement was similar with both rhIL-1 alpha and -1 beta, i.e. they were dose-dependent between 0.1 and 3 ng/ml and reached a plateau from 3 to 100 ng/ml. At submaximal histamine release (less than 10%), there was enhancement of three IgE-dependent secretagogues: 125% with goat anti-human IgE (n = 7), 215% with Ag E (n = 10), and 260% with a histamine releasing factor (n = 7). Non-IgE-dependent stimuli (formyl-methionine-leucine-phenylalanine and the ionophore A23187, n = 10) were enhanced less than 5%. rhIL-1-enhancement persisted after cell washing (n = 10). rhIL-1 was active in preparations of 50 to 75% pure basophils in which mononuclear cells were reduced by greater than 95% (n = 4), and mAbH34 to IL-1 beta blocked the enhancement caused by that molecule. We postulate that basophils have an IL-1 receptor which, when occupied, upregulates the response to IgE-related signals. Thus, this work characterizes a second interaction between interleukins and the cells central to the allergic response.     

Very low density lipoproteins (VLDL) trigger the release of histamine from human basophils

Circulating basophils are well established sources of the granule-associated mediator, histamine. The physiological control, however, of histamine release from human basophils is poorly understood. Because histamine may play a role in the transendothelial transport of various compounds, including very low density lipoprotein (VLDL) and its hydrolysis products, we investigated the possibility that VLDL regulates mediator release from basophils. The incubation of VLDL (at physiological concentrations) with basophils (isolated as mixed leukocyte preparations) resulted in a significant release of histamine. Histamine release was dependent on VLDL concentration (half-maximal stimulation occurring at VLDL-protein concentration of 15-20 micrograms/ml), length of incubation (half-maximal release at 5-12 min), temperature (37 degrees C optimum) and required calcium (concentration 0.5-2.0 mM). Furthermore, VLDL-induced histamine release was inhibited by three different mediator-release inhibitors: dimaprit, dibutyryl cAMP and nordihydroguaiaretic acid. Incubation of basophils with LDL or HDL under the same experimental conditions did not result in significant histamine release from basophils. The histamine-secretory response of basophils obtained from different donors varied considerably. Basophils isolated from 28 donors and challenged with 100 micrograms/ml VLDL released 23 +/- 5% of their cellular histamine (mean +/- S.E.; with a range of 0-94%). Desensitization of VLDL-induced histamine release could be accomplished by preincubation of basophils with either VLDL or anti-IgE but not with N-formyl-L-methionyl-L-leucyl-L-phenylalanine. Through the secretion of histamine, a potent vasoactive mediator (and also possibly through granule-associated glycosaminoglycans, stimulants of the enzyme lipoprotein lipase), this novel effect of VLDL may be part of a physiological loop for the regulation of VLDL hydrolysis and lipid transport. This effect of VLDL may also have deleterious consequences, because of the atherogenic properties of histamine.     

IgE-dependent regulation of human basophil adherence to vascular endothelium

Mechanisms favoring the recruitment of circulating human basophils to extravascular sites of allergic inflammation are unknown. The basophil secretagogues anti-IgE, and pollen allergens rye grass I and ragweed Ag E (Lol p I and Amb a I) were tested for their ability to promote basophil adherence to umbilical vein endothelial cells. Co-incubation of endothelial cells and basophils with anti-IgE resulted in time and dose-dependent increases in basophil adhesion. These effects were due to activation of the basophil, required both magnesium and calcium, occurred before or in the absence of histamine release, and were seen at concentrations of stimulus below the usual range of secretagogue activity. In contrast, anti-IgE or Ag stimulation of neutrophils, or basophils from donors non-responsive to anti-IgE or Ag with respect to histamine release, had no effect on cell adherence. mAb 60.3, recognizing the CD18 leukocyte adhesion molecule, inhibited anti-IgE-induced enhancement of basophil-endothelial cell binding. Exposure of basophils to low concentrations of Ag in vivo may selectively initiate basophil infiltration into tissue sites of allergic inflammation by enhancing their adherence to endothelium.     

Characteristics of human basophil sulfidopeptide leukotriene release: releasability defined as the ability of the basophil to respond to dimeric cross-links

Human basophils release approximately 90 pmol of LTC4/micrograms histamine when challenged with anti-IgE antibody, but donor to donor variation produces a 1000-fold range of response. There is little conversion to LTC4 to LTE4 in purified preparations of basophils, but conversion to LTE4 does occur if cell densities are high during incubation. Like histamine release, leukotriene release is calcium and temperature dependent and is complete in 20 min, with a t1/2 of approximately 8 min. The process of desensitization also ablates leukotriene release, but there is a distinct two phase process where leukotriene release is enhanced after 5 min of desensitization, whereas histamine release is inhibited and total ablation of leukotriene release occurs only after 45 min of desensitization. Human basophils respond well to stimulation with covalently cross-linked trimeric IgE myeloma but respond poorly to dimeric IgE. This differential sensitivity to the two forms of cross-linked IgE is most exaggerated in the context of leukotriene release, where dimer is 30-fold less efficacious and 100- to 1000-fold less potent than trimer on some donors' basophils. This dichotomy of response is also observed in antigen-challenged cells, where the bivalent hapten, BPO2, also poorly induces leukotriene release in accord with the fact that it predominantly induces dimeric cross-links of penicillin-specific IgE. Anti-IgE dose-response curves reveal a region of dimeric cross-link dominance that may explain the peculiar differences observed in pharmacologic studies of basophil release induced with antigen vs anti-IgE. In addition, there is a continuum of "releasability," where some donors' basophils display no response (histamine or leukotriene release) to dimeric IgE, and others' basophils are essentially equally responsive to both dimeric and trimeric IgE. This releasability difference manifests itself by conferring increased sensitivity to antigenic challenge in those donors' basophils capable of responding to dimeric cross-links such that these donors' basophils are capable of releasing histamine upon antigen challenge while possessing only 50 molecules of cell surface antigen-specific IgE; other dimer-insensitive donors' basophils require 6 to 10-fold greater IgE densities for equal histamine release.     

Calcium-dependent activation of human neutrophils by a synthetic ionophore

Synthetic block copolymers composed of polyoxyethylene and poly-oxypropylene have been demonstrated to possess ionophore activity selective for monovalent cations and to cause histamine release from mouse mast cells and human basophils. We now report calcium-dependent release of granule contents from human neutrophils by the most active of these agents, TI30R2. At a concentration of 100 micrograms/ml (12.5 microM), net lysozyme release ranged from 17-40% after 30 minutes incubation at 37 degrees. Lysozyme release was dose-dependent over stimulus concentrations of 5-50 micrograms/ml (0.625-6.25 microM). Release was dependent upon the presence of extracellular calcium. T130R2 did not induce the release of superoxide anions over 30 minutes of incubation. As T130R2 induces sodium influx into cells, it is likely that a depolarizing influx of sodium ions in the presence of extracellular calcium constitutes a sufficient signal for granule release but not superoxide production by human neutrophils.     

Eotaxin potentiates antigen-dependent basophil IL-4 production.

Basophils are a major source of IL-4, which is a critical factor in the generation of allergic inflammation. Eotaxin induces chemotaxis mediated through the CC chemokine receptor 3 (CCR3) present on basophils as well as eosinophils and Th2 cells, thereby promoting cell recruitment. To determine whether eotaxin has other proinflammatory activity, we examined the effect of eotaxin on basophil IL-4 expression by flow cytometry. Eotaxin alone had no effect on basophil IL-4 production, but further increased allergen-stimulated IL-4 expression. Eotaxin also enhanced IL-4 release from purified basophils 2- to 4-fold, as determined by ELISA (p < 0.01). Addition of eotaxin to cultures resulted in a 40-fold left shift in the dose response to Ag. This effect was obtained with physiologic concentrations of eotaxin (10 ng/ml), was abrogated by an Ab to the CCR3 receptor, and was noted with other chemokine ligands of CCR3. Additionally, eotaxin augmented IL-3 priming of basophil IL-4 production in a synergistic manner (p < 0.01). In contrast, no priming was observed with either IL-5 or GM-CSF. These results establish a novel function for eotaxin and other chemokine ligands of CCR3: the potentiation of Ag-mediated IL-4 production in basophils, and suggest a potential nonchemotactic role for CC chemokines in the pathogenesis and amplification of inflammation.     

Neutrophils and mast cells. Comparison of neutrophil-derived histamine-releasing activity with other histamine-releasing factors

Human neutrophil-derived histamine-releasing activity (HRA-N) was partially purified and found to contain a heat-stable 1400 to 2300-Da fraction which caused human basophils and rat basophil leukemia cells (RBL) to degranulate. The capacity of HRA-N to activate basophils was not related to the gender or atopic status of the basophil donor, but was related to anti-IgE responsiveness. Several lines of evidence suggest that HRA-N and anti-IgE induce histamine release through distinctly different mechanisms: 1) the time course of HRA-N- and anti-IgE-induced RBL histamine release are different; 2) HRA-N causes histamine release from RBL with and without surface-bound IgE; 3) lactic acid stripping of IgE from human basophils reduces anti-IgE-induced histamine release, but has no consistent effect on HRA-N-induced histamine release; and 4) passive sensitization of lactic acid-stripped basophils with IgE restores anti-IgE-induced histamine release but not HRA-N-induced histamine release. Several histamine-releasing factors (HRF) were compared with HRA-N. Human nasal HRF (HRF-NW, crude and partially purified fractions of 15 to 30, 3.5 to 9, and less than 3.5 kDa), like HRA-N, caused equal histamine release from both native and IgE-sensitized RBL. However, only the 15- to 30-kDa fraction caused histamine release from human basophils in the doses tested. Mononuclear cell HRF (HRF-M, crude and a partially purified 25 kDa Mr fraction) and platelet HRF (HRF-P, crude preparation) failed to cause histamine release from either native or IgE-sensitized RBL but caused 30 +/- 5.5% and 20 +/- 10% net histamine release from human basophils, respectively. HRA-N and HRF-NW were both stable to boiling. These data, taken together, suggest that the capacity of HRA-N to induce RBL and human basophil histamine release and of HRF-NW to stimulate RBL histamine release is independent of IgE. The data further suggest that HRA-N and HRF-NW can be distinguished by size, and that they both differ from mononuclear cell HRF and platelet HRF. Thus, it appears that inflammatory cells generate a family of distinct HRF.     

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.     

Demonstration of Fcgamma receptors on human basophil granulocytes.

Fcgamma receptors were detected on human basophil granulocytes. The mononuclear cell fraction of human peripheral blood was incubated with heat-aggregated human IgG (HGG) followed by 125I-anti-HGG. Autoradiography of the cells showed that the majority of basophil granulocytes gave a significant number of grains. Basophils were not labeled by preincubation of the same cells with monomeric HGG followed by 125I-anti-HGG. However, the binding of aggregated HGG to basophils was inhibited by the presence of a high concentration of monomeric HGG or its Fc fragment but not by the Fab fragment. Evidence was obtained that Fcgamma receptors are distinct from IgE receptors on the same cells: i) Saturation of basophils with IgE did not affect the binding of aggregated HGG to the cells. ii) Preincubation with and the presence of aggregated HGG failed to affect the binding of 125I-IgE to basophils, or to block passive sensitization of the cells with IgE antibodies. iii) The Fcgamma receptors did not co-cap with IgE receptors. Aggregated HGG failed to induce histamine release from basophils even in the presence of D2O. It was also found that the presence of aggregated HGG on basophils did not modulate IgE-mediated histamine release from the cells.