Comparative effect of recombinant IL-1, -2, -3, -4, and -6, IFN-gamma, granulocyte-macrophage-colony-stimulating factor, tumor necrosis factor-alpha, and histamine-releasing factors on the secretion of histamine from basophils

Most cytokines possess multiple biologic activities. This study was undertaken to investigate the effect of rIL-1 beta, -2, -3, -4 and -6, IFN-gamma, TNF-alpha, and granulocyte-macrophage (GM)-CSF on basophils from 16 donors and the amount of histamine released was compared with that by partially purified mononuclear cell-derived histamine-releasing factor (HRF) and anti-IgE. We found that only IL-3 and GM-CSF at relatively high doses (50 to 500 ng/ml) released small amounts of histamine (3 to 14%) from two allergic donors. In contrast, both HRF and anti-IgE released significant amounts of histamine from all donors. Other cytokines did not release any measurable quantity of histamine. Simultaneous addition of several cytokines to the basophils also failed to release histamine. IL-3, GM-CSF, and IL-1 can also release histamine at lower concentrations (less than 5 ng/ml) when incubated with basophils in the presence of D2O. Basophils from 6 out of 13 allergic donors released histamine in response to IL-3, whereas three donors responded to IL-1 beta and two responded to GM-CSF. The results of this study demonstrated that although IL-3 and GM-CSF release small amounts of histamine only from a select group of allergic patients, mononuclear cell-derived HRF is more potent in their action and release histamine from normals as well as allergic patients.     

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.     

Interleukin-5, interleukin-3, and granulocyte-macrophage colony-stimulating factor cross-compete for binding to cell surface receptors on human eosinophils.

Human interleukin (IL)-5 receptors were characterized by means of binding studies using bioactive 125I-labeled IL-5. Of purified primary myeloid cells, eosinophils and basophils but not neutrophils or monocytes expressed surface receptors for IL-5. Binding studies showed that eosinophils expressed a single class of high affinity receptors (Ka = 1.2 x 10(10) M-1) with the number of receptors being small (less than 1000 receptors/cell) and varying between individuals. Among several cell lines examined only HL-60 cells showed detectable IL-5 receptors which were small in numbers (200 receptors/cell) and also bound 125I-IL-5 with high affinity. The binding of IL-5 was rapid at 37 degrees C while requiring several hours to reach equilibrium at 4 degrees C. Specificity studies revealed that the two other human eosinophilopoietic cytokines IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) inhibited the binding of 125I-IL-5 to eosinophils. No competition was observed by other eosinophil activating or nonactivating cytokines. The inhibition of 125I-IL-5 binding by IL-3 and GM-CSF was partial up to a concentration of competitor of 10(-7) M with GM-CSF consistently being the stronger competitor. Converse experiments using IL-5 as a competitor revealed that this cytokine inhibited the binding of 125I-IL-3 and of 125I-GM-CSF in some but not all the individuals tested, perhaps reflecting eosinophil heterogeneity in vivo. Cross-linking experiments on HL-60 cells demonstrated two IL-5-containing complexes of Mr 150,000 and Mr 80,000 both of which were inhibited by GM-CSF. The competition between IL-5, IL-3, and GM-CSF on the surface of mature eosinophils may represent a unifying mechanism that may help explain the common biological effects of these three eosinophilopoietic cytokines on eosinophil function. This unique pattern of competition may also be beneficial to the host by preventing excessive eosinophil stimulation.     

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.     

HIV-1 envelope gp41 peptides promote migration of human Fc epsilon RI+ cells and inhibit IL-13 synthesis through interaction with formyl peptide receptors

We evaluated the effects of synthetic peptides (2017, 2019, 2020, 2021, 2023, 2027, 2029, 2030, 2031, and 2035) encompassing the structure of HIV-1(MN) envelope gp41 on both chemotaxis of human basophils and the release of preformed mediators (histamine) and of cytokines (IL-13). Peptides 2019 and 2021 were potent basophil chemoattractants, whereas the other peptides examined were ineffective. Preincubation of basophils with FMLP or gp41 2019 resulted in complete desensitization to a subsequent challenge with homologous stimulus. Incubation of basophils with low concentration (5 x 10(-7) M) of FMLP, which binds with high affinity to N-formyl peptide receptor (FPR), but not to FPR-like 1, did not affect the chemotactic response to a heterologous stimulus (gp41 2019). In contrast, a high concentration (10(-4) M) of FMLP, which binds also to FPR-like 1, significantly reduced the chemotactic response to gp41 2019. The FPR antagonist cyclosporin H inhibited chemotaxis induced by FMLP, but not by gp41 2019. None of these peptides singly induced the release of histamine or cytokines (IL-4 and IL-13) from basophils. However, low concentrations of peptides 2019 and 2021 (10(-8)-10(-6) M) inhibited histamine release from basophils challenged with FMLP but not the secretion caused by anti-IgE and gp120. Preincubation of basophils with peptides 2019 and 2021 inhibited the expression of both IL-13 mRNA, and the FMLP-induced release of IL-13 from basophils. These data highlight the complexity of the interactions between viral and bacterial peptides with FPR subtypes on human basophils.     

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.     

Modulation of human basophil histamine release by hemopoietic growth factors

To determine what kinds of CSF modulate human basophil function, recombinant or purified hemopoietic growth factors were tested for the effect on histamine release from basophils. Both granulocyte (G)-macrophage (M) CSF and IL-3 markedly enhanced histamine release upon stimulation with anti-IgE in a dose-dependent manner (maximal enhancement 25.5% by GM-CSF and 30.8% by IL-3 as expressed as percent increase against total cellular histamine content), whereas G-CSF, M-CSF, and IL-4 had no effect. Enhancing action of these factors was still observed in the highly enriched basophil population, suggesting that this action was not via contaminating cells. Enhancement of histamine release by both factors was not necessarily IgE mediated, because they also amplified histamine release upon stimulation with FMLP and ionophore A23187. The enhancement by both factors was temperature dependent, and took place rapidly and reached plateau levels in 15 min. GM-CSF and IL-3 achieved the similar plateau level of augmentation and no additive effects were observed between them. This finding suggests that they enhance histamine release by sharing the same pathway in the release reaction.     

Human monocytes generate basophil histamine-releasing activities

Human peripheral blood monocytes generated activities during 24-h culture that were capable of triggering histamine release from 17 of 18 human basophil donors. Monocytes and their in vitro transformed macrophages continued to elaborate these basophil histamine-releasing activities for at least 3 wk in culture. In the 18 basophil donors tested, maximum histamine release induced by monocyte supernatants was 33.8 +/- 5.9% (mean +/- SEM) of total basophil histamine content; optimum anti-IgE-induced release was 38.8 +/- 6.2%. Basophil histamine release in response to monocyte activities was optimal at 37 degrees C and at calcium concentrations of 2 to 5 mM. Release was greater than 90% complete 1 min after challenge and was inhibited by anti-allergic drugs. The mechanism of release appeared to be independent of IgE binding. Gel filtration of supernatants derived from both day 1 (monocyte stage) and day 14 (macrophage stage) cultures demonstrated activity peaks with approximate m.w. of 12,000 and 30,000. In contrast to the marked responsiveness of basophils, only 2 of 10 human lung mast cell preparations responded; release in those preparations was low: 3% and 13% histamine release, respectively. Thus, monocytes produce potent histamine-releasing activities with differential actions on basophils and mast cells.     

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.     

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.     

Comparative effects of basophil-directed growth factors

IL-3, IL-5, and GM-CSF exert various overlapping functions in basophils. We investigated the receptor expression profiles and concentration-dependent effects of IL-3, IL-5, and GM-CSF on several basophil functions in comparison with their effects on eosinophils. The order of the receptor expression levels was IL-3Ralpha>IL-5Ralpha>GM-CSFRalpha in basophils and IL-5Ralpha>or=GM-CSFRalpha>IL-3Ralpha in eosinophils. Compared with eosinophils, basophils expressed a much higher level of IL-3Ralpha and similar levels of IL-5Ralpha and GM-CSFRalpha. The order of potency was IL-3>IL-5=GM-CSF for degranulation, survival, and CD11b expression in basophils, and IL-5=GM-CSF>or=IL-3 for survival and CD11b expression in eosinophils. However, IL-3 induced CD69 expression preferentially in basophils. Our results indicate that IL-3 is the most potent activator of human basophils, and that the rank order of potency of hemopoietic growth factors virtually corresponded to their receptor expression levels in both cell types.     

Differences in functional consequences and signal transduction induced by IL-3, IL-5, and nerve growth factor in human basophils

Previous studies have indicated a redundancy in the effects of the cytokines, IL-3, IL-5, and nerve growth factor (NGF) on acute priming of human basophils. In the current study, we have examined the effects of these three cytokines on 18-h priming for leukotriene C4 generation, their ability to induce Fc(epsilon)RIbeta mRNA expression, or their ability to sustain basophil viability in culture. We also examine a variety of the signaling steps that accompany activation with these cytokines. In contrast with the ability of IL-3 to alter secretagogue-mediated cytosolic calcium responses following 18-h cultures, 18-h treatment with IL-5 or NGF did not affect C5a-induced leukotriene C4 generation or alter C5a-induced intracellular Ca2+ concentration elevations. IL-3 and IL-5, but not NGF, induced Fc(epsilon)RIbeta mRNA expression and all three improved basophil viability in culture with a ranking of IL-3 > IL-5 > or = NGF. All three cytokines acutely activated the extracellular signal-regulated kinase pathway and the signaling elements that preceded extracellular signal-regulated kinase and cytosolic phospholipase A2 phosphorylation, consistent with their redundant ability to acutely prime basophils. However, only IL-3 and IL-5 induced Janus kinase 2 and STAT5 phosphorylation. This pattern of signal element activation among the three cytokines most closely matched their ability to induce expression of Fc(epsilon)RIbeta mRNA. Induction of the sustained calcium signaling that follows overnight priming with IL-3 appeared to be related to the strength of the early signals activated by these cytokines but the relevant pathway required was not identified. None of the signaling patterns matched the ability of the cytokines to promote basophil survival.     

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.     

The functional basis of granulocyte-macrophage colony stimulating factor, interleukin-3 and interleukin-5 receptor activation, basic and clinical implications

The cytokines granulocyte-macrophage colony stimulating factor, interleukin-3 and interleukin-5 have overlapping activities on cells expressing their receptors. This is explained by their sharing a receptor signal transduction subunit, beta c. This communal signaling subunit is also required for high affinity binding of all three cytokines. Therapeutic approaches attempting to interfere or modulate haemopoietic cells using cytokines or their analogues can in some instances be limited due to functional redundancy amongst cytokines using shared receptor signaling subunits. Therefore, a better approach would be to develop therapeutics against the shared subunit. Studies examining the GM-CSF, IL-3 and IL-5 receptors have identified the key events leading to functional receptor activation. With this knowledge, it is now possible to identify new targets for the development of a new class of antagonist that blocks the biological activity of all the cytokines utilizing beta c. This approach may be extended to other receptor systems such as IL-4 and IL-13 where receptor activation is dependent on a common signaling and binding subunit.     

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.     

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.     

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.     

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.