Recovery from desensitization of IgE-dependent responses in human lung mast cells.

BACKGROUND: Clinical desensitization and oral food immunotherapy are therapeutic interventions that allow individuals who react adversely to an allergen (drug or food) to be made tolerant to the allergen. However, tolerance is brief, and allergen hypersensitivity can recur within days following allergen withdrawal. OBJECTIVE: We hypothesize that the reason these treatments are temporary reflects rapid recovery of mast cells from a desensitized state. We sought to test this. METHODS: Desensitization of IgE-mediated histamine release from human lung mast cells was explored by methods that partially replicate the pattern of treatment during clinical desensitization. Specific and non-specific desensitization and changes in surface IgE were examined following desensitization. Recovery from desensitization was also studied. RESULTS: Desensitization of mast cell responses was readily induced with concentrations of antigen or anti-IgE that were suboptimal for secretion. There was little or no non-specific desensitization when lung mast cells were exposed to antigens. There was no loss of cell surface IgE following desensitization. Removing the desensitizing stimulus from the media following desensitization allowed the cells to recover with half-point of recovery of ~1.5 days and complete recovery after 5 days. Both the functional response and histamine content recovered within this time frame. The recovery appeared possible because both antigens and anti-IgE dissociated rapidly from cells after washing to remove excess stimulus. CONCLUSIONS AND CLINICAL RELEVANCE: Human lung mast cells readily recover from a desensitized state following removal of desensitizing antigen. This finding provides a potential explanation for the ephemeral nature of clinical desensitization.

Cat allergen-induced blood basophil reactivity in vitro predicts acute human nasal allergen challenge responses in vivo.

BACKGROUND: Basophil histamine release (BHR) to allergen has been used as a confirmatory test to support the clinical diagnosis of allergic disease. OBJECTIVE: Among subjects reporting respiratory cat allergy, we hypothesized that cat-induced BHR in vitro would predict nasal allergen challenge (NAC) response in that same individual. We therefore compared the magnitude of cat allergen-induced BHR to NAC outcome and serological measures of cat-specific IgE and the ratio of cat-specific IgE to total IgE. METHODS: Forty-two subjects with a history of cat allergy, positive cat puncture skin test (PST) and detectable cat-specific IgE (> 0.1 kAU/L, ImmunoCap) participated with consent. Subjects were grouped as positive or negative cat allergen-induced BHR, with a positive result defined as the release of ≥ 20% of the total cellular histamine content. The majority of subjects also underwent a NAC with a positive result defined as ≥ 5 total sneezes. RESULTS: Subjects with a positive compared with a negative cat allergen BHR had higher cat-specific IgE levels at 5.40 ± 1.24 kAU/L (n=25) vs. 1.55 ± 0.73 kAU/L (n=17, P=0.01) as well as a higher cat-specific IgE/total IgE ratio [6.1 ± 1.4% (n=25) vs. 1.6 ± 0.9% (n=17, P=0.01)]. Of the 31 subjects who underwent a NAC, a positive NAC was observed in 78% (18/23) with a positive cat allergen BHR compared with 37% (3/8) with a negative cat allergen BHR, giving a positive predictive value of 78% and a negative predictive value of 63%. The diagnostic sensitivity and specificity of a positive BHR to predict a positive NAC was 86% and 50%, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: A positive cat allergen-induced BHR is associated with higher cat-specific IgE levels, a higher cat-specific to total IgE ratio and is predictive of a positive cat-induced NAC [ClinicalTrials.gov NCT00604786].

Characterization of syk expression in human lung mast cells: relationship with function.

BACKGROUND: Previous studies indicate that the protein tyrosine kinase, syk, is critical in transducing FcɛRI-mediated signals. In human basophils, 'releasability' has been linked to the extent of syk expression. Human lung mast cells, like basophils, are also found to be variably responsive to IgE-dependent activation. OBJECTIVE: The aim of the present study was to determine whether the wide variability in human lung mast cell responses, following IgE-dependent activation, has a relationship with syk expression. METHODS: Mast cells were isolated from human lung tissue and 'releasability' was determined by activating the cells with a maximal releasing concentration of anti-IgE. Syk levels in mast cells were determined by immunoblotting and flow cytometry. RESULTS: Histamine release from mast cells, challenged with a maximal releasing concentration of anti-IgE, ranged from 0% to 69% (mean±SEM, 24±2%, n=53). A proportion of these preparations (nine out of 53) released very low levels of histamine (5%) in response to anti-IgE. Flow cytometry of a subset of preparations indicated that a weak response to anti-IgE was not related to a lack of surface IgE. Immunoblotting and flow cytometry studies demonstrated that, compared with mononuclear cells, human lung mast cells express low and variable levels of syk. However, there was no correlation between syk expression and mast cell releasability. Nonetheless, a number of putative inhibitors of syk including NVP-QAB205 (EC50, 0.2 µm) effectively attenuated the IgE-dependent release of histamine from mast cells. CONCLUSION AND CLINICAL RELEVANCE: These studies indicate that although syk may play an important role in mediating degranulation, the relative level of syk expression does not govern human lung mast cell releasability. Identification of the mechanisms that govern IgE-dependent activation of human lung mast cells is likely to be of wider clinical significance, given the central role that mast cells play in the development of allergic asthma.

Localizing a control region in the pathway to leukotriene C(4) secretion following stimulation of human basophils with anti-IgE antibody.

Mediator release from human basophils is a self-limited process, but down-regulation of the signaling cascades leading to secretion of leukotriene C(4) (LTC(4)) is controlled independently of the pathway leading to IL-4 secretion. In the current studies, we have explored the regulation of upstream signaling events leading to activation of extracellular signal-related kinases (ERKs; previously shown to be required for LTC(4) generation) in human basophils. IgE-, but not FMLP-mediated activation, induced sustained tyrosine phosphorylation of syk, of shc, and an association of shc to the Grb2/son of sevenless 2 complex. In contrast, IgE-mediated activation resulted in transient activation of p21(ras) and mitogen-activated protein/ERK kinase 1, which were kinetically associated with phosphorylation of ERKs. The canonical Shc/Grb2/son of sevenless pathway to activation of p21(ras) is therefore sustained, while p21(ras) activity is not. We have previously shown that phosphatidylinositol 3 kinase activity is required for p21(ras) activity and, in the current studies, we show that of the p85-sensitive forms of p110 possible, basophils express only p110 delta and that there are no changes in association between p21(ras) and p110 delta in stimulated basophils. We used the generation of phospho-Akt as a marker of the presence of phosphatidylinositol-3,4,5-trisphosphate and found that phospho-Akt is transient on a time scale consistent with p21(ras) activity. On the basis of information obtained in these and other studies, we localize down-regulation of IgE-mediated LTC(4) secretion to a region of the signaling cascade antecedent to p21(ras) activation, downstream of phosphatidylinositol 3 kinase activity and probably involving regulation of phosphatidylinositol-3,4,5-trisphosphate levels.

Piceatannol is an effective inhibitor of IgE-mediated secretion from human basophils but is neither selective for this receptor nor acts on syk kinase at concentrations where mediator release inhibition occurs.

BACKGROUND: Syk kinase is probably an early necessary tyrosine kinase involved in IgE-mediated secretion from human basophils. Causal testing of the role of syk kinase in the secretion requires a selective pharmacological agent. Piceatannol has previously been used to demonstrate the causal role of syk in secretion but its selectively has recently come into question. OBJECTIVE: To determine whether piceatannol inhibits IgE-mediated signalling events in a manner consistent with its putative inhibitory effects on syk kinase and at concentrations relevant to its inhibition of mediator release. METHODS: Human basophils were examined for the effects of piceatannol on mediator release or various signalling steps. RESULTS: We show that while piceatannol has an IC50 for inhibition of IgE-mediated histamine release of 3-5 microm, these same concentrations inhibit secretion of phorbol 12-myristate 13-acetate (PMA)-induced histamine release (as previously shown) and leukotriene C (LTC)4 release induced by fMLP. Concentrations of piceatannol up to 100 microm also did not inhibit IgE-mediated phosphorylation of shc, a immediate downstream target of syk kinase. Similar concentrations also did not inhibit IgE-mediated cytosolic calcium elevations, another downstream signal thought to be dependent on syk kinase. In contrast, piceatannol did modify the cytosolic calcium response that follows stimulation with formyl methionyl-leucyl-phenylalanine (fMLP). CONCLUSION: Taken together with published studies using other cell types, we conclude that piceatannol does not inhibit secretion from human basophils by inhibiting the activity of syk kinase.

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.

Expression and modulation of FcepsilonRIalpha and FcepsilonRIbeta in human blood basophils.

BACKGROUND: The IgE receptor (FcepsilonRI) may exist as a tetramer (alphabetagamma2) or a trimer (alphagamma2) because FcepsilonRIbeta is dispensable for membrane expression of FcepsilonRIalpha. FcepsilonRIbeta amplifies signaling of FcepsilonRI so that regulation of FcepsilonRIalpha:beta stoichiometry would affect cellular responsiveness. OBJECTIVE: We examined basophils from a variety of donors for differences in their expression of FcepsilonRIalpha and FcepsilonRIbeta protein. METHODS: Enriched blood basophils were assessed at baseline and after IL-3 culture for FcepsilonRIalpha and FcepsilonRIbeta protein by Western blotting, surface FcepsilonRIalpha by flow cytometry, and FcepsilonRIbeta mRNA by real-time PCR. Basophil functional response was measured by allergen-triggered histamine release. RESULTS: For the FcepsilonRIalpha subunit, 2 protein bands with molecular weights of 50 kd and 60 kd were identified by Western blots. The 60-kd band correlated to surface-expressed FcepsilonRIalpha detected by flow cytometry (Spearman R = 0.78, P <.01). Surface FcepsilonRIalpha also correlated with FcepsilonRIbeta protein (Spearman R = 0.92, P <.01). FcepsilonRIbeta protein levels increased disproportionately with higher surface FcepsilonRIalpha expression. The ratio of FcepsilonRIbeta to FcepsilonRIalpha varied 10-fold among donors and correlated with surface FcepsilonRIalpha. Basophil 50-kd alpha protein levels were similar despite a 10-fold range in surface FcepsilonRIalpha expression, implying stores of this protein such as those found in eosinophils. Unlike eosinophils, the basophil 50-kd protein was lost with culture and was absent from supernatants. Levels of beta protein and mRNA were enhanced by IL-3 culture, whereas FcepsilonRIalpha expression (by flow cytometry and 60 kd) was not. CONCLUSION: These findings demonstrate variable stoichiometry of FcepsilonRIalpha:beta in whole cells and that this stoichiometry can be altered by IL-3 culture. With the assumption that all detected beta protein is surface expressed, these findings suggest a variable stoichiometry for FcepsilonRIalpha:beta that is also related to FcepsilonRIalpha surface expression.

The tyrosine kinases p53/56lyn and p72syk are differentially expressed at the protein level but not at the messenger RNA level in nonreleasing human basophils.

Within the general population, individuals can be found whose basophils do not secrete after stimulation through the immunoglobulin (Ig) E receptor. In this study we compared two groups of donors, those whose basophils responded with 65+/-16% histamine release to an optimal concentration of anti-IgE antibody and those whose basophil response was not statistically different from nonstimulated release (1+/-1%). We show that these so-called nonreleasing basophils have at least 10-fold lower expression of the tyrosine kinases, lyn and syk, but normal expression of the tyrosine kinase Btk when compared with the panel of releasing basophils. Indeed, maximum histamine release correlated with expression of both syk (Spearman rank correlation coefficient [Rs] = 0.98) and lyn (Rs = 0.93). In contrast, equivalent levels of messenger RNA (mRNA) for lyn and syk kinase were found for both groups. By sequencing a critical region in the syk mRNA, our results also demonstrate that the frame shift mutation in syk leading to a premature stop codon which has been observed in other cell types is not present in nonreleasing human basophils. Our results suggest that there may be translational or post-translational regulatory mechanisms specific to the expression of two important FcepsilonRI-associated signaling elements in basophils.

Human recombinant histamine-releasing factor activates human eosinophils and the eosinophilic cell line, AML14-3D10.

The human recombinant histamine-releasing factor (HrHRF) was previously shown to induce histamine release from human basophils from a subset of donors. The ability of HrHRF to directly induce histamine release from only certain basophils was thought to involve interaction between HrHRF and a particular kind of IgE, termed IgE(+), on the surface of these cells. Recent studies disproved the hypothesis that the IgE molecule or its high-affinity receptor, FcepsilonRI, is involved in secretion of histamine and cytokines by basophils stimulated with HrHRF. Rather, data suggest that HrHRF is a cytokine that stimulates basophils by binding to a cell-surface structure other than the IgE molecule. This report describes the effects of HrHRF on another inflammatory cell type: eosinophils from mildly allergic donors. In purified eosinophils primed with granulocyte-macrophage colony-stimulating factor, both tumor necrosis factor alpha (TNF-alpha) and HrHRF induced increased secretion of interleukin (IL) 8. In addition, both HrHRF and IL-5 enhanced secretion of IL-8 stimulated by TNF-alpha. Secretion of IL-8 reached a plateau level in less than 24 hours, was inhibited by cycloheximide, and required the presence of HrHRF throughout the culture period. In some eosinophil preparations, HrHRF induced calcium mobilization that was inhibited by pertussis toxin. Additionally, HrHRF caused secretion of IL-8 from the human eosinophilic cell line, AML14-3D10, which does not possess the alpha chain of FcepsilonRI. These data provide evidence that HrHRF contributes to activation of eosinophils and thus suggest an additional role for HrHRF in the pathophysiologic mechanisms of allergic disease.

Phosphatidylinositol-3 kinase regulates p21ras activation during IgE-mediated stimulation of human basophils.

Cross-linking of IgE or a bacterial product (f-Met-Leu-Phe; FMLP) induces the release of leukotriene C4 (LTC4) and histamine in human basophils. However, the signaling mechanisms in human basophils are only partially understood. It has been demonstrated that extracellular signal-regulated kinases (ERK1/2) specifically regulate the pathway for LTC4 generation, but not for histamine release and interleukin-4 production. More recent studies have suggested that tyrosine kinase (syk)-mediated phosphorylation of shc is responsible for the ras-ERK cascade via the formation of shc-Grb2-Sos2 following stimulation with anti-IgE antibody, but not FMLP, in human basophils. However, while characterizing the role of phosphatidylinositol (PI)-3 kinase in signaling pathways leading to basophil mediator release, it was noted that this pathway might also regulate p21ras activation. Anti-IgE antibody, but not FMLP, resulted in phosphorylation of p85 (regulatory subunit of PI3 kinase), suggesting activation of PI3 kinase. Inhibition of PI3 kinase by selective inhibitor (LY294002) abolished anti-IgE antibody- but not FMLP-induced phosphorylation of MEK1 (MAPK kinase/ERK kinase) and ERKs while inhibiting LTC4 generation as well as histamine release. IgE-mediated activation of ras (upstream of MEK-ERK) was also inhibited. But, further upstream, phosphorylation of syk and of shc and inducible association between shc and Grb2 were not affected. Furthermore, the IgE-mediated cytosolic calcium response ([Ca(++)](i)) was also diminished. These results suggest that functional responses may be dependent on the activity of PI3 kinase, which regulates at least 2 important signaling pathways: by regulating activation of ras for the MEK-ERK pathway and the increase in [Ca(++)](i).

The relationship between serum IgE and surface levels of FcepsilonR on human leukocytes in various diseases: correlation of expression with FcepsilonRI on basophils but not on monocytes or eosinophils.

BACKGROUND: Expression of receptors for IgE (FcepsilonR) have been mainly studied on mast cells and blood basophils in the context of allergic disease. Some reports have noted limited expression of FcepsilonR on other leukocytes, including blood monocytes and eosinophils in certain patients. An association between human blood basophil expression of FcepsilonRIalpha and serum IgE has been noted among allergic subjects. OBJECTIVE: Recent evidence supports regulation of FcepsilonRIalpha by free IgE on both mast cells and basophils. We hypothesized that this relationship would exist across an extremely wide range of IgE levels for human basophils, irrespective of underlying disease. We further examined whether a similar relationship existed between serum IgE and FcepsilonRIalpha or FcepsilonRII (CD23) expression on monocytes and eosinophils in these same subjects. METHODS: Blood was obtained from nonallergic subjects (n = 3) and subjects with allergic asthma (n = 5), atopic dermatitis (n = 3), hypereosinophilic syndromes (n = 7), hyper-IgE syndrome (n = 6), helminth infestation (n = 6), or IgE myeloma (n = 1). Levels of serum IgE were determined by using RIA and ranged from 3 to 4.7 mg/mL. Levels of cell surface FcepsilonRIalpha, FcepsilonRII, and IgE were measured by using immunofluorescence and flow cytometry. RESULTS: Basophil surface IgE density and FcepsilonRIalpha expression correlated with serum IgE levels (r = 0. 67 and r = 0.46, respectively; P <.01; n = 31) regardless of the disease state. In contrast, monocyte FcepsilonRIalpha expression did not correlate with serum IgE (r = 0.09, P >.5, n = 29), and low-level eosinophil FcepsilonRIalpha expression was only detected in a single asthmatic subject. CD23 expression was not detected on basophils or eosinophils, except for the eosinophils from the donor with IgE myeloma. CD23 was present on monocytes from some donors but did not correlate with serum IgE levels. CONCLUSIONS: In a variety of disease states, FcepsilonRIalpha expression by basophils, but not monocytes or eosinophils, correlated with serum IgE levels across a 6-log range of IgE. These data support the concept of in vivo regulation of FcepsilonRIalpha on basophils by serum IgE and further demonstrate that this is independent of allergic disease per se.

Dual phase priming by IL-3 for leukotriene C4 generation in human basophils: difference in characteristics between acute and late priming effects.

Previous studies have suggested that enhancement of mediator release from human basophils by IL-3 occurs in at least two phases, and the current studies further characterize the signaling changes that accompany these two phases of the basophil in response to IL-3. The test stimulus for these studies was anaphylatoxin split product of C component (C5a), which does not induce leukotriene C4 release without prior IL-3 treatment. Functionally, IL-3 priming occurs after 5 min, disappears by 2 h, and returns by 18 h. In contrast, the kinetics of cytosolic phospholipase A2 (cPLA2) and extracellular signal-regulated kinase (ERK1/2) phosphorylation, induced by IL-3, do not show the second rise by 18 h. The kinetics of cPLA2 and ERK1/2 phosphorylation following stimulation with C5a are the same for cells that were not treated with IL-3 as for those treated for 18 h, i.e., a lag in phosphorylation of cPLA2 and ERK1/2 lasting 30 s before its eventual rise. Previous studies showed that a 5-min treatment with IL-3 induced little change in the C5a-induced cytosolic calcium response, while 24 h of treatment resulted in a marked and sustained cytosolic calcium elevation during the C5a-induced response. The first phase of the IL-3 priming effect (5-15 min of treatment) was unaffected by cycloheximide, while the second phase (18 h) was inhibited. These data suggest that early IL-3 priming results from preconditioning cPLA2, i.e., causing its phosphorylation, while late priming results from a qualitative change in the cytosolic calcium response.

Intracellular expression and release of Fc epsilon RI alpha by human eosinophils.

Although Fc epsilon R have been detected on human eosinophils, levels varied from moderate to extremely low or undetectable depending on the donor and methods used. We have attempted to resolve the conflicting data by measuring levels of IgE, Fc epsilon RI, and Fc epsilon RII in or on human eosinophils from a variety of donors (n = 26) and late-phase bronchoalveolar lavage fluids (n = 5). Our results demonstrated little or no cell surface IgE or IgE receptors as analyzed by immunofluorescence and flow cytometry. Culture of eosinophils for up to 11 days in the presence or absence of IgE and/or IL-4 (conditions that enhance Fc epsilon R on other cells) failed to induce any detectable surface Fc epsilon R. However, immunoprecipitation and Western blot analysis of eosinophil lysates using mAb specific for Fc epsilon RI alpha showed a distinct band of approximately 50 kDa, similar to that found in basophils. Western blotting also showed the presence of FcR gamma-chain, but no Fc epsilon RI beta. Surface biotinylation followed by immunoprecipitation again failed to detect surface Fc epsilon RI alpha, although surface FcR gamma was easily detected. Since we were able to detect intracellular Fc epsilon RI alpha, we examined its release from eosinophils. Immunoprecipitation and Western blotting demonstrated the release of Fc epsilon RI alpha into the supernatant of cultured eosinophils, peaking at approximately 48 h. We conclude that eosinophils possess a sizable intracellular pool of Fc epsilon RI alpha that is available for release, with undetectable surface levels in a variety of subjects, including those with eosinophilia and elevated serum IgE. The biological relevance of this soluble form of Fc epsilon RI alpha remains to be determined.

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.

Mechanisms and pharmacologic control of basophil-derived IL-4 and IL-13.

Human basophils represent a major source of interleukin (IL) 4 and 13 protein, cytokines which share several biological activities central to the pathogenesis of allergic inflammation. Studies show that the production of these cytokines differs with respect to mode of activation and the time course of their secretion. Pharmacologic evidence indicates that IL-4 generation, which is most prominent with IgE-dependent activation, is mediated through a calcineurin-dependent pathway. In contrast, multiple pathways seem evident in the regulation of IL-13 in basophils.

Pharmacologic regulation of histamine release by the human recombinant histamine-releasing factor.

BACKGROUND: The recently cloned human recombinant IgE-dependent histamine releasing factor (HrHRF) was initially thought to stimulate histamine release from human basophils from a subpopulation of allergic donors by interacting with the IgE molecules on the surface of these cells. Additional data suggest that HrHRF exerts its biologic effects by binding to a distinct cell surface structure and not to IgE. OBJECTIVE: To address the hypothesis that the HrHRF signaling pathway is distinct from the classical high-affinity IgE receptor (FcepsilonRI) pathway, we used pharmacologic agents known to affect basophil histamine release. METHODS: In this report we compared the effect of staurosporine, Bis II, Gö 6976, rottlerin, and pertussis toxin on histamine release from human basophils mediated by the following stimuli: HrHRF, polyclonal human anti-IgE antibody, and antigen, as well as the IgE-independent stimulus, FMLP. RESULTS: None of these modulators, except rottlerin, could differentiate histamine release induced by anti-IgE or antigen from that induced by HrHRF. Rottlerin enhanced HrHRF-mediated histamine release and dose dependently blocked FMLP-mediated release without affecting basophil activation by either anti-IgE or antigen. CONCLUSION: These data suggest a unique signaling pathway for HrHRF and thus strengthen the hypothesis that HrHRF binds to a specific receptor other than IgE.

The human recombinant histamine releasing factor: functional evidence that it does not bind to the IgE molecule.

BACKGROUND: We have previously shown that the human recombinant histamine releasing factor (HrHRF) caused histamine release from a subset of basophils from donors with allergy, and this release seemed to be dependent on the presence of a certain type of IgE, termed IgE+. IgE molecules that did not support HrHRF-induced histamine release were termed IgE-. However, subsequently we demonstrated that HrHRF primes anti-IgE-antibody-induced histamine release from all basophils, irrespective of the type of IgE on the cell surface. OBJECTIVE: Because these data suggested that HrHRF does not exert its biologic effects by binding to IgE, but rather that it interacted with a surface receptor on the basophil, we wanted to obtain functional evidence that HrHRF did or did not bind to the IgE molecule. METHODS: The rat basophilic leukemia cell line (RBL-SX38), which has been transfected to express a functional human FcepsilonRI (alpha-, beta-, and gamma-chains of the receptor) in addition to the normal rat FcepsilonRI, was used. The presence of the human FcepsilonRI receptor enables these cells to be sensitized with human IgE. Cells were passively sensitized with 1000 ng/mL human IgE+ or 1000 ng/mL human IgE- for 60 minutes at 37 degrees C. Unsensitized cells served as a control. After the cells were washed, 1 x l0(5) cells were stimulated in the presence of 1 mmol/L Ca2+ with 0.1 microg/mL anti-IgE, 40 microg/mL HrHRF, or 40 microg/mL mouse recombinant HRF (MrHRF), which has 96% homology to HrHRF. RESULTS: Mean anti-IgE-induced histamine release was 33% +/- 15%, and there was no difference between IgE+ sensitization (32% +/- 12%) and IgE- sensitization (34% +/- 18%). However, in contrast to human basophil experiments, neither HrHRF (0% +/- 0%) nor MrHRF (3% +/- 5%) caused histamine release in RBL cells sensitized with IgE+. In addition, priming the transfected RBL-SX38 cells or the parental cell line, RBL-2H3 cells, with HrHRF or MrHRF did not increase anti-IgE-induced histamine release. CONCLUSION: The results indicate that HrHRF does not bind to IgE, either IgE+ or IgE-. Therefore it appears likely that rHRF signals through its own specific receptor, which is not expressed or functional on RBL-SX38 or RBL-2H3 cells, but which seems to be expressed on basophils of atopic and nonatopic donors.

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

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

Macrophage-derived chemokine induces human eosinophil chemotaxis in a CC chemokine receptor 3- and CC chemokine receptor 4-independent manner.

BACKGROUND: Chemokines are believed to contribute to selective cell recruitment. Macrophage-derived chemokine (MDC) is a CC chemokine that causes chemotaxis of dendritic cells, monocytes, and activated natural killer cells. MDC binds to CC chemokine receptor 4 (CCR4) but not to CCR1, CCR2, CCR3, CCR5, CCR6, or CCR7. OBJECTIVE: Our aim was to determine the in vitro activity of MDC on human eosinophils by using chemotaxis and calcium flux assays. METHODS: Eosinophils were purified from peripheral blood of allergic donors, and chemotactic activity of MDC and other CC chemokines was compared in microchemotaxis chamber assays. The role of CCR3 in these assays was determined by using a CCR3-blocking antibody. Measurements of cytosolic Ca++ mobilization were performed by using fura-2AM labeling, with eosinophils and cell lines transfected with CCR3 or CCR4. Eosinophil expression of CCR3 and CCR4 mRNA was determined by using RT-PCR. RESULTS: MDC (0.1 to 100 nmol/L) caused dose-dependent chemotaxis of purified human eosinophils (maximum approximately 3-fold control). Compared with other CC chemokines, the potency and efficacy for eosinophil chemotaxis were similar for MDC and eotaxin but were less than that observed for RANTES, monocyte chemoattractant protein (MCP)-4, and eotaxin-2. Although MDC can act by means of CCR4, RT-PCR analysis failed to reveal CCR4 mRNA in eosinophils. Effects of MDC on eosinophils was also independent of CCR3, as a blocking mAb to CCR3 failed to inhibit MDC-induced chemotaxis. Furthermore, CCR3-transfected human embryonic kidney cells labeled with Fura-2AM exhibited a rapid rise in intracellular free calcium after stimulation with eotaxin, eotaxin-2, or MCP-4, but not with MDC. Eosinophils cultured for 72 hours in 10 ng/mL IL-5 also demonstrated increased intracellular free calcium after stimulation with eotaxin-2 or MCP-4, but not with up to 100 nmol/L MDC. CONCLUSION: MDC is a CCR3- and CCR4-independent activator of eosinophil chemotaxis, but it does not appear to elicit measurable cytosolic calcium elevations during these responses. MDC appears to act by means of another receptor in addition to CCR4 and may therefore contribute to eosinophil accumulation without working through CCR1 to CCR7.