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.     

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.     

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.     

Down-regulation of human basophil IgE and FC epsilon RI alpha surface densities and mediator release by anti-IgE-infusions is reversible in vitro and in vivo

Previously, infusions of an anti-IgE mAb (rhumAb-E25) in subjects decreased serum IgE levels, basophil IgE and FcepsilonRIalpha surface density, and polyclonal anti-IgE and Ag-induced basophil histamine release responses. We hypothesized that these effects would be reversed in vivo by discontinuation of infusions and in vitro by exposing basophils to IgE. Subjects received rhumAb-E25 biweekly for 46 wk. Blood samples taken 0-52 wk after rhumAb-E25 were analyzed for serum IgE and basophil expression of IgE, FcepsilonRIalpha, and CD32. Basophil numbers were unaffected by infusions. Eight weeks after infusions, free IgE levels rose in vivo but did not reach baseline. Basophil IgE and FcepsilonRIalpha rose in parallel with free IgE while CD32 was stable. FcepsilonRI densities, measured by acid elution, returned to 80% of baseline, whereas histamine release responses returned to baseline. Basophils cultured with or without IgE or IgG were analyzed for expression of IgE, FcepsilonRIalpha, and CD32. By 7 days with IgE, expression of IgE and FcepsilonRIalpha rose significantly, whereas cultures without IgE declined. IgE culture did not effect CD32. IgG culture did not effect expression of any marker. The present results strongly suggest that free IgE levels regulate FcepsilonRIalpha expression on basophils.     

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.     

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.     

Does improvement management of atopic dermatitis influence the appearance of respiratory allergic diseases? A follow-up study

Background

Flavonoids, a large group of polyphenolic metabolites derived from plants have received a great deal of attention over the last several decades for their properties in inflammation and allergy. Quercetin, the most abundant of plant flavonoids, exerts a modulatory action at nanomolar concentrations on human basophils. As this mechanism needs to be elucidated, in this study we focused the possible signal transduction pathways which may be affected by this compound. Methods: K2-EDTA derived leukocyte buffy coats enriched in basophil granulocytes were treated with different concentrations of quercetin and triggered with anti-IgE, fMLP, the calcium ionophore A23187 and the phorbol ester PMA in different experimental conditions. Basophils were captured in a flow cytometry analysis as CD123bright/HLADRnon expressing cells and fluorescence values of the activation markers CD63-FITC or CD203c-PE were used to produce dose response curves. The same population was assayed for histamine release.

Results

Quercetin inhibited the expression of CD63 and CD203c and the histamine release in basophils activated with anti-IgE or with the ionophore: the IC50 in the anti-IgE model was higher than in the ionophore model and the effects were more pronounced for CD63 than for CD203c. Nanomolar concentrations of quercetin were able to prime both markers expression and histamine release in the fMLP activation model while no effect of quercetin was observed when basophils were activated with PMA. The specific phosphoinositide-3 kinase (PI3K) inhibitor wortmannin exhibited the same behavior of quercetin in anti-IgE and fMLP activation, thus suggesting a role for PI3K involvement in the priming mechanism.

Conclusions

These results rule out a possible role of protein kinase C in the complex response of basophil to quercetin, while indirectly suggest PI3K as the major intracellular target of this compound also in human basophils.     

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.     

Upregulated expression of substance P in basophils of the patients with chronic spontaneous urticaria: induction of histamine release and basophil accumulation by substance P

Human basophils have been implicated in the pathogenesis of chronic spontaneous urticaria (CSU), and substance P (SP) is a possible candidate as histamine-releasing factor in some patients with CSU. However, little is known of relationship between basophils and SP in CSU. In the present study, we investigated expression of SP and NK1R on basophils from patients with CSU, and influence of SP on basophil functions by using flow cytometry analysis, basophil challenge, and mouse sensitization model techniques. The results showed that plasma SP level and basophil numbers in CSU patients were higher than that in HC subject. The percentages of SP+ and NK1R+ basophils were markedly elevated in CSU blood in comparison with HC blood. Once added, SP induced up to 41.2 % net histamine release from basophils of CSU patients, which was comparable with that provoked by anti-IgE, and fMLP. It appeared that SP induced dramatic increase in blood basophil numbers of mice following peritoneal injection. Ovalbumin (OVA)-sensitized mice had much more SP+ and NK1R+ basophils in blood than non-sensitized mice. In conclusion, the elevated plasma concentration of SP, upregulated expression of SP and NK1R on basophils, and the ability of SP in induction of basophil degranulation and accumulation indicate strongly that SP is most likely a potent proinflammatory mediator, which contributes greatly to the pathogenesis of CSU through basophils. Inhibitors of SP and blockers of NK1R are likely useful agents for treatment of CSU.     

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.     

Identification of histamine releasing factor(s) in the late phase of cutaneous IgE-mediated reactions

We have shown that fluids collected from antigen-challenged skin blisters during the late phase reaction cause the release of substantial amounts of histamine (means = 42%, n = 14) from human basophils in vitro. Control fluids collected either during the immediate phase or from an unchallenged blister released less than or equal to 10% histamine from both basophils and lung mast cells. Late phase blister fluids induced low levels of histamine release from human lung cells (means = 11%, n = 4) that were slightly but not significantly greater than levels induced by control blister fluids. The characteristics of basophil release were similar to IgE-mediated stimuli in dose dependence, calcium and temperature requirements, and kinetics. The IgE dependence of the late phase blister fluid was demonstrated by desensitization of the basophils to anti-IgE, which obviated the response to anti-IgE and blister fluid but did not affect a non-IgE-mediated stimulus. Removal of the cell surface IgE with lactic acid also abolished the response to both anti-IgE and late phase blister fluid. Incubation of the "stripped" cells with serum containing IgE myeloma restored the response to anti-IgE but failed to affect response to late phase blister fluid. The characteristics of release obtained with this factor closely resemble those of an IgE-dependent histamine releasing factor from cultured macrophages previously described by our group.     

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.     

The cell surface glycoprotein Mac-1 (CD11b/CD18) mediates neutrophil adhesion and modulates degranulation independently of its quantitative cell surface expression

It has previously been shown that during degranulation Mac-1 (CD11b/CD18)--a glycoprotein that plays a central role in neutrophil adhesion-is up-regulated on PMN surfaces. It has been assumed that this quantitative change in adhesion Ag expression on the cell surface would in turn lead to increased cellular adhesiveness. In contrast, we found that at an incubation temperature of 16 degrees C, stimulated neutrophil adhesion to plastic tissue culture dishes in the presence of FMLP (2.5 x 10(-6) M), TNF (10 ng/ml), or PAF (1 x 10(-4) M) occurred without cellular degranulation or Mac-1 surface up-regulation as measured cytofluorometrically. As shown by functional inhibition studies employing monoclonal antibodies 60.3 (anti-CD18) and 60.1 (anti-CD11b), adhesion at 16 degrees C, where no CD11b/CD18 up-regulation was seen, is mediated by CD11b/CD18 just as it is at 37 degrees C, where degranulation and CD11b/CD18 up-regulation could be demonstrated. The physiologic importance of these findings was underscored by experiments done on endothelial monolayers, which showed that PMN association with endothelial cells is absolutely independent from the quantitative up-regulation of Mac-1 on PMN surfaces. When neutrophils were stimulated at 37 degrees C by endotoxin, an agent that does not induce aggregation (a form of intercellular adhesion), Mac-1 surface expression increased only after cells had become adherent, whereas cells held in suspension to prevent cell-substrate adhesion neither degranulated nor up-regulated their Mac-1 surface expression. Thus, not only is adherence independent of degranulation and Mac-1 cell surface up-regulation, but both degranulation and Mac-1 surface up-regulation appear to depend on the process of adhesion. Correspondingly, incubation of neutrophils with antibodies 60.1 and 60.3 inhibited not only adhesion of cells stimulated with FMLP at 37 degrees C but degranulation as well. These results indicate that Mac-1 influences degranulation as well as it controls adhesion not by its mere quantity on the cell surface, but rather by an yet undefined molecular modulation.     

Differential response of human basophil activation markers: a multi-parameter flow cytometry approach

Background

Basophils are circulating cells involved in hypersensitivity reactions and allergy but many aspects of their activation, including the sensitivity to external triggering factors and the molecular aspects of cell responses, are still to be focused. In this context, polychromatic flow cytometry (PFC) is a proper tool to investigate basophil function, as it allows to distinguish the expression of several membrane markers upon activation in multiple experimental conditions.

Methods

Cell suspensions were prepared from leukocyte buffy coat of K2-EDTA anticoagulated blood specimens; about 1500-2500 cellular events for each tested sample, gated in the lymphocyte CD45dim area and then electronically purified as HLADRnon expressing/CD123bright, were identified as basophilic cells. Basophil activation with fMLP, anti-IgE and calcium ionophore A23187 was evaluated by studying up-regulation of the indicated membrane markers with a two-laser six-color PFC protocol.

Results

Following stimulation, CD63, CD13, CD45 and the ectoenzyme CD203c up-regulated their membrane expression, while CD69 did not; CD63 expression occurred immediately (within 60 sec) but only in a minority of basophils, even at optimal agonist doses (in 33% and 14% of basophils, following fMLP and anti-IgE stimulation respectively). CD203c up-regulation occurred in the whole basophil population, even in CD63non expressing cells. Dose-dependence curves revealed CD203c as a more sensitive marker than CD63, in response to fMLP but not in response to anti-IgE and to calcium ionophore.

Conclusion

Use of polychromatic flow cytometry allowed efficient basophil electronic purification and identification of different behaviors of the major activation markers. The simultaneous use of two markers of activation and careful choice of activator are essential steps for reliable assessment of human basophil functions.     

Specific inhibition of phorbol ester and DiC8-induced histamine release from human basophils by the protein kinase C inhibitors, H-7 and H-9

The release of histamine and other inflammatory mediators from human basophils is triggered by numerous stimuli, including chemical, physical and receptor-mediated activators. Several mechanisms of cell activation including protein kinase C activation have been proposed to operate in these cells. We used phorbol ester and DiC8 to induce histamine release from human basophils and the protein kinase C inhibitors H-7 and H-9 to inhibit this release. Both DiC8 and TPA induced histamine release were inhibited by H-7 (ID 50 = 37 mcM) and H-9 (IC 50 = 20 mcM). However, anti-IgE, fmlp and A23187-induced histamine release were unaffected. In contrast, the calmodulin antagonists W-7 and perphenazine effectively inhibited histamine release by all five stimuli. Therefore, different biochemical pathways appear to be critical for basophil activation depending on the nature of the stimulus used.     

Cyclosporin A rapidly inhibits mediator release from human basophils presumably by interacting with cyclophilin.

We have examined the effects of cyclosporin A (CsA) and a series of CsA analogs that bind with decreasing affinity to cyclophilin, to evaluate the involvement of this protein in the release of preformed (histamine) and de novo synthesized (peptide leukotriene C4; LTC4) mediators of inflammatory reactions from human basophils. CsA (8 to 800 nM) concentration-dependently inhibited (5 to 60%) histamine release from peripheral blood basophils challenged with anti-IgE. CsA was more potent (92.6 +/- 1.8 vs 59.1 +/- 4.5%; p less than 0.001) and, at low concentrations, more effective when the channel-operated influx of Ca2+ was bypassed by the ionophore A23187 (IC40 = 24.1 +/- 3.9 vs 105.5 +/- 22.2 nM; p less than 0.05). CsA had no effect on the release of histamine caused by phorbol myristate and bryostatin 1 that activate different isoforms of protein kinase C. Inhibition of histamine release from basophils challenged with anti-IgE was not abolished by washing (three times) the cells before anti-IgE challenge. CsA also inhibited the de novo synthesis of LTC4 from basophils challenged with anti-IgE. The inhibitory effect of CsA was very rapid, and the drug, added from 1 to 10 min during the reaction, inhibited the ongoing release of histamine caused by anti-IgE and by A23187. The experiments with CsA analogs (CsG, CsC, CsD, and CsH) showed that CsH, which has an extremely low affinity for cyclophilin, has no effect on basophil mediator release. In addition, there is a significant correlation between the concentrations of CsA, G, C, and D that inhibited by 30% the histamine release induced by anti-IgE (r = 0.99; p less than 0.001) and by A23187 (r = 0.87; p less than 0.001) and their affinity for cyclophilin.     

Generation and release of platelet-activating factor (PAF) from enriched preparations of rabbit basophils; failure of human basophils to release PAF

Rabbit mononuclear cells containing up to 20% basophils and uncontaminated by neutrophils release PAF when stimulated with goat antiserum to rabbit IgE. The amount of PAF detected was a function of basophil concentration but decreased on a per cell basis at high basophil or high total cell concentrations. Calcium ionophore A23187, but not protein A, C5a, or FMLP, initiated rabbit basophil degranulation and PAF release. By contrast, extensive studies using a variety of human leukocyte preparations failed to demonstrate the release of significant levels of PAF from human basophils by IgE-dependent or -independent mechanisms. These results suggest that cells other than the peripheral blood basophil (e.g., the neutrophil) may act as the primary site of PAF production in man.