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.

Regulation of IgE-mediated signalling in human basophils by CD32b and its role in Syk down-regulation: basic mechanisms in allergic disease.

BACKGROUND: CD32b has been previously demonstrated to modulate IgE-mediated secretion from human basophils. However, exploration of the implications of this regulation has been limited. One unstudied area is whether regulation of signalling by CD32 also alters some of the phenotypic changes induced by IgE-mediated activation. The reported character of CD32-mediated signal transduction is not clear for human basophils and the two primary mechanisms considered important in this reaction predict different long-term outcomes, notably predicting different outcomes for down-regulation of syk expression. OBJECTIVE: Syk expression was considered a unique point of phenotypic control in human basophils and the role of CD32b in its regulation is explored in this study. However, initial pilot studies discovered that IL-3 could markedly up-regulate CD32 expression and first describing the consequences of this up-regulation became an additional focus of this study. METHODS: Human basophils were examined for the changes in IgE-mediated signalling during simultaneous engagement of CD32b. RESULTS: Preliminary experiments noted that CD32b could be up-regulated by IL-3 (3- to 12-fold). Both natural variation and induced up-regulation of CD32b modulated the efficacy of this receptor to inhibit IgE-mediated release. Signalling induced by engagement of CD32b (lyn, syk, SHP-1, or SHIP1 phosphorylation) was more consistent with a mode of action involving SHIP1 rather than SHP-1. IgE-mediated down-regulation of syk expression was not altered by co-engagement of CD32b, a result also consistent with a SHIP1-dependent mechanism of inhibition. CONCLUSIONS: Taken together these results suggest that the combined action of IgE and IgG could generate a natural mechanism, whereby the significant variation in syk expression in allergic subjects occurs without necessarily also inducing mediator release.

Subthreshold desensitization of human basophils re-capitulates the loss of Syk and FcεRI expression characterized by other methods of desensitization.

BACKGROUND: Clinical desensitization of patients to drugs involves progressive exposure to escalating doses of drug over a period of 24 h. In prior studies, this method was re-capitulated in vitro to also demonstrate loss of mast cell or basophil responsiveness. However, most signalling studies of human basophils have identified changes in signalling by using other methods of inducing cellular desensitization. OBJECTIVE: This study examined two well-described endpoints of basophil desensitization, loss of syk or FcεRI expression, under conditions of subthreshold desensitization. METHODS: The loss of FcεRI and syk was examined in human basophils. RESULTS: It was shown that both loss of syk and FcεRI/IgE occurred during an escalating series of stimulation (anti-IgE Ab) and that expression loss occurred despite the presence of little histamine release. If basophils were first cultured for 3 days in 10 ng/mL IL-3, the concentration-dependence of histamine release shifted to 100-fold lower concentrations of stimulus. However, loss of syk did not show any change in its EC50 while loss of FcεRI also shifted 100-fold. From the perspective of early signal element activation, the marked shift in the EC50 for histamine release was not accompanied by similar shifts in the EC50s for several signalling elements. The EC50s for phospho-Src, phospho-SHIP1, phospho-Syk, or phospho-Cbl did not change while the EC50s for phospho-Erk and the cytosolic calcium response did shift 100-fold. CONCLUSIONS: These studies show that under normal conditions, subthreshold desensitization leads to loss of two critical signalling molecules (FcεRI and syk) but under at least one condition, treatment with IL-3, it is possible to markedly blunt the loss of syk, but not FcεRI, while executing a proper subthreshold titration. These data also suggest that IL-3 modifies only the sensitivity of signalling elements that are downstream of syk activation.

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.

Expression of CD203c and CD63 in human basophils: relationship to differential regulation of piecemeal and anaphylactic degranulation processes.

BACKGROUND: Activation of human basophils results in the release of many different mediators and the expression of new cell surface proteins. The markers CD63 and CD203c have been used in recent years to assess basophil activation but there have been many studies that demonstrate that expression of these markers can be dissociated from histamine release. OBJECTIVE: To determine the signal transduction requirements for CD203c and CD63 expression. METHODS: The current study began by exploring the dependency of CD203c and CD63 expression on protein kinase C (PKC) using known selective inhibitors of PKC. RESULTS: Between 30 and 300 nm, Ro-31-8220 and bisindoylmaleimide II (Bis II) had no effect on formyl-met-leu-phe- or anti-IgE-induced CD63 or CD203c but enhanced IgE-mediated expression of CD63 by an average of 15-fold at concentrations >1 microm. These results led to the suggestion that these inhibitors altered the normal pathways of degranulation (by a non-PKC dependent mechanism), shifting the normal presence of piecemeal degranulation to the process termed anaphylactic degranulation (AND). Morphological studies demonstrated that concentrations of Ro-31-8220 and Bis II>1 mum dramatically increased the presence of degranulation sacs, a morphological feature of AND. CONCLUSION: It is proposed that CD63 expression results from only the AND form of histamine release.

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.

IgE-regulated loss, not IgE-regulated synthesis, controls expression of FcepsilonRI in human basophils.

Expression of the high-affinity receptor on basophils and mast cells is modulated by immunoglobulin E (IgE) antibody. Recent studies have shown that modulation occurs through interaction of IgE with the receptor itself, but the mechanisms underlying this control are not understood. Taking both a theoretical and experimental approach, we examined several competing models that focus on whether there is IgE-regulated loss, IgE-regulated synthesis, or both regulated loss and synthesis of the Fc receptor for IgE (FcepsilonRI). We report that removing IgE from occupied FcepsilonRI resulted in an accelerated loss only in the unoccupied receptor, with no loss of occupied receptors and no loss of total receptors when all receptors were occupied. Together with previous studies, these results establish that there was IgE-regulated loss of receptors. An examination of synthetic rates of FcepsilonRIalpha using pulse-labeling with (35)S-methionine indicated no difference in synthetic rates in the presence or absence of IgE. Similarly, the presence or absence of IgE had no influence on the levels of mRNA for either alpha, beta, or gamma subunits of FcepsilonRI. Using model simulations, we found that regulated-synthesis models could be distinguished from regulated-loss/constant-synthesis models on the basis of the relationship between starting FcepsilonRI densities and changes in density after culture for 1 week in the absence of IgE. Experimental data from this type of study fit a regulated-loss model that did not include regulation of synthesis. Taken together, these results suggest that IgE regulates cell surface expression of FcepsilonRI only by regulating the rate that receptor is lost from the cell surface.

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.

Characteristics of the free cytosolic calcium timelag following IgE-mediated stimulation of human basophils: significance for the nonreleasing basophil phenotype.

These studies examine characteristics of the quiescent period (timelag) of the free cytosolic calcium ([Ca++]i) elevation that follows stimulation of human basophils through the IgE receptor. Previous studies established that the [Ca++]i timelag was sensitive to the rate of ligand binding, but little else is known about this response characteristic. The [Ca++]i timelag could be lengthened using antigenic stimulation that is rapid but only weakly induces secretion: tenfold differences in the "strength" of the stimulus, as assessed by histamine release, are associated with threefold differences in the timelag. Inhibiting p53/56lyn kinase with low concentrations of the specific inhibitor, PP1, lengthened the [Ca++]i timelag dramatically. PP1 was also found to delay the onset of syk phosphorylation and histamine release. Staurosporine and genistein, which are known to inhibit early tyrosine kinases, had, at best, only modest effects on the [Ca++]i timelag. Specific inhibitors of protein kinase C (PKC) had no effect on the [Ca++]i timelag, and direct activation of PKC with PMA had only very modest effects on the timelag. Contrary to expectations, basophils with the so-called nonreleasing phenotype demonstrated an IgE-mediated [Ca++]i response at the single-cell level. However, the length of [Ca++]i timelag in nonreleasing basophils was threefold longer than normally found in releasing basophils. Furthermore, the [Ca++]i response was significantly more asynchronous than in releasing basophils and lacking in a sustained [Ca++]i elevation. These studies indicate that the [Ca++]i timelag following stimulation through the IgE receptor is sensitive to inhibition of lyn kinase but not other agents that have been demonstrated to inhibit early tyrosine kinases previously. However, only one characteristic of the [Ca++]i response phenotype of nonreleasing basophils--the [Ca++]i timelag but not the absence of a sustained [Ca++]i elevation--could be mimicked by inhibition of lyn kinase with PP1.

Pharmacology of IgE-mediated desensitization of human basophils: effects of protein kinase C and src-family kinase inhibitors.

IgE-mediated down-regulation of secretion from basophils and mast cells is an important component of the overall cellular response that determines the ultimate extent of mediator release. The down-regulatory process that occurs during active secretion has also been associated with the methodological phenomenon called desensitization, but the mechanisms underlying desensitization are not understood. A variety of studies have suggested that activation of protein kinase C (PKC) results in down-regulation of IgE-mediated secretion so we have examined the effect of the PKC inhibitors Ro-31-8220 (3-[1-[3-amidinothio)propyl-1H-indol-3-yl]-3-(1-methyl-1H-indol-3- yl) maleimide) and bis-indolylmaleimide II on desensitization in human basophils. At concentrations that have been shown previously to inhibit PKC-mediated functions in basophils completely, these two drugs had no effect on IgE-mediated desensitization. We did find, however, that the src-family kinase inhibitors PP1 [4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine] and PP2 [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3, 4-d]pyrimidine] inhibited desensitization as well as secretion. These data suggest that PKC has little role in down-regulating the IgE-mediated basophil response. However, like the activation signaling cascade, the desensitization process is dependent on the activation of src family kinases.

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.

Functional consequences of FcepsilonRIalpha up-regulation by IgE in human basophils.

These studies examine the functional changes that occur after up-regulation of FcepsilonRIalpha by immunoglobulin E (IgE) for human basophils. Basophils were cultured with and without IgE antibody (PS myeloma IgE or anti-gp120-specific IgE) for 1 week and challenged with anti-IgE, anti-FcepsilonRIalpha, or antigen for histamine and IL-4 secretion. There were no statistically significant changes in their response to anti-IgE or anti-receptor antibodies, as compared with controls incubated for the same period, whereas receptor expression increased an average of 4-fold. There was increased responsiveness to antigenic challenge, most notably at suboptimal concentrations of antigen (gp120 peptide-ovalbumin conjugate). For a 6-fold difference in cell surface density of gp120-specific IgE, there was a 2.2-fold change in antigen potency or 3-fold increases in histamine release at lower antigen concentrations. Similar results were found for secretion of IL-4. Basophil sensitivity, which is a measure of the density of antigen-specific IgE required for 50% of maximal secretion, was used to determine whether up-regulation of FcepsilonRIalpha was coordinated with up-regulation of other components of the IgE-signaling pathway. The results indicated up-regulation of FcepsilonRI is not always accompanied by changes that allow sensitivity to be maintained. These results indicate that functional up-regulation does occur but that its magnitude may be modulated because not all components of the signaling pathway are up-regulated in a balanced manner.

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).