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.

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.

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].

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.

Single-cell analysis of Ca++ changes in human lung mast cells: graded vs. all-or-nothing elevations after IgE-mediated stimulation.

Human lung mast cells were examined by digital video microscopy for changes in cytosolic free ionized calcium [( Ca++]i) after stimulation with anti-IgE antibody or specific antigens. These studies sought to determine whether the mast cell response resembled a graded or an all-or-nothing process. Preliminary experiments indicated that labeling mast cells with fura-2 did not alter their response to IgE-mediated stimulation. Subsequent experiments established that an IgE-mediated stimulus evoked an elevation of [Ca++]i from a baseline value of 85 nM to an average of 190 nM (range 60-450 nM, n = 23), with an average histamine release of 26%. There was a good correlation (Rs = 0.67) between the average net [Ca++]i change and the subsequent histamine release (regression equation: %HR = 0.189[net(Ca)-52]). [Ca++]i elevations were found to precede histamine release (t1/2 for [Ca++]i of 35 s vs. t1/2 for histamine release of 110 s). Single-cell analysis found that even for very low values of histamine release, nearly all cells demonstrated a [Ca++]i response. However, this response was markedly heterogeneous, ranging from no response to responses two to three times the mean. Comparative studies of mast cells stimulated under optimal and suboptimal conditions established that there was a graded [Ca++]i response dependent on the strength of the stimulus. An all-or-nothing reaction for the [Ca++]i response was ruled out.

Differences in the behavior of cytoplasmic granules and lipid bodies during human lung mast cell degranulation.

We used a morphometric and autoradiographic approach to analyze changes in specific cytoplasmic granules and cytoplasmic lipid bodies associated with human lung mast cell degranulation. Mast cells were dissociated from lung tissue by enzymatic digestion and were then enriched to purities of up to 99% by countercurrent centrifugation elutriation and recovery from columns containing specific antigen bound to Sepharose 6 MB. Degranulation was induced by goat anti-IgE. At various intervals after stimulation, parallel aliquots of cells were recovered for determination of histamine release or were fixed for transmission electron microscopy. We found that lipid bodies, electron-dense structures that lack unit membranes, were present in both control and stimulated mast cells. Autoradiographic analysis showed that lipid bodies represented the major repository of 3H-label derived from [3H]arachidonic acid taken up from the external milieu. By contrast, the specific cytoplasmic granules contained no detectable 3H-label. In addition, lipid bodies occurred in intimate association with degranulation channels during mast cell activation, but the total volume of lipid bodies did not change during the 20 min after stimulation with anti-IgE. This result stands in striking contrast to the behavior of specific cytoplasmic granules, the great majority of which (77% according to aggregate volume) exhibited ultrastructural alterations during the first 20 min of mast cell activation. These observations establish that mast cell cytoplasmic granules and cytoplasmic lipid bodies are distinct organelles that differ in ultrastructure, biochemistry, and behavior during mast cell activation.

Generation of leukotrienes by purified human lung mast cells.

Although mediator release from mast cells and basophils plays a central role in the pathogenesis of human allergic disease, biochemical studies have been restricted to rat peritoneal mast cells and basophilic leukemia cells because they could be easily purified. We have used two new techniques of cell separation to purify human lung mast cells to 98% homogeneity. Lung cell suspensions were obtained by dispersion of chopped lung tissue with proteolytic enzymes. Mast cells were then purified from the suspensions by countercurrent centrifugal elutriation and affinity chromatography. The purified mast cells released both histamine and slow-reacting substance of anaphylaxis (SRS-A) (leukotriene C and D) during stimulation with goat anti-human IgE antibody. Moreover, these preparations were able to generate significant quantities of SRS-A (32 +/- 7 x 10(-17) LTD mole-equivalents/mast cell) at all stages of purification, indicating that a secondary cell is not necessary for the antigen-induced release of SRS.

Effects of dexamethasone on mediator release from human lung fragments and purified human lung mast cells.

Purified human lung mast cells released histamine, leukotrienes, prostaglandin (PG) D2, thromboxane B2 (TxB2), and PGF2 alpha in response to anti-IgE stimulation. Incubation of the cells for 24 h with 10(-6) M dexamethasone, a treatment that inhibits mediator release from human basophils, had no effect on the release of these mediators from mast cells. Dexamethasone treatment of human lung fragments led to little or no inhibition of anti-IgE-induced release of the mast cell-derived mediator, histamine, but produced a significant inhibition of the release of PGE2, PGF2 alpha, and 6-keto-PGF1 alpha. As was the case with purified mast cells, the steroid did not inhibit the release of PGD2 or TxB2 from human lung fragments. Comparison of the quantities of PGD2 and TxB2 produced by purified cells and human lung fragments reveals that the mast cells produce quantities of these metabolites sufficient to account for the entire amount produced by challenged lung fragments. Dexamethasone inhibited spontaneous release from lung fragments of all cyclooxygenase products measured. These results suggest that the human lung parenchymal mast cell phospholipase is not inhibited by dexamethasone, whereas other phospholipase(s) in the lung are inhibited by the steroid. These results may be useful in explaining the resistance of acute allergic reactions, including anaphylaxis, to steroids, despite the potent antiinflammatory activity of steroids on subacute and chronic inflammation, such as in bronchial asthma, which may be initiated by IgE-dependent mechanisms.

Desensitization of IgE-mediated IL-4 release from human basophils.

Previous studies demonstrated marked differences in the time course of interleukin-4 (IL-4) and histamine release, implying different mechanisms of desensitization. To explore this possibility basophils were desensitized with anti-IgE antibody by treating the cells for various periods of time in the absence of extracellular calcium (Ca[e]). The basophil response following addition of Ca(e) was assessed by measuring IL-4 or histamine release by measuring the cytosolic calcium response ([Ca2+]i). The kinetics of desensitization for IL-4 and histamine release did not differ; 50% desensitization occurred at 37+/-8 and 29+/-16 min for IL-4 and histamine release, respectively. The kinetics of desensitization as assessed by the [Ca2+]i response was found to be biphasic; an initial enhancement of the [Ca2+]i response after 5-15 min of desensitization was followed by a rapid decrease so that by 30 min the amount of desensitization was equivalent to that observed for histamine release. Other than this initial period of enhanced calcium response, desensitization kinetics were unexpectedly similar for all measured endpoints. These results suggest that, during stimulation in the absence of Ca(e), there is a down-regulation process that influences all events on a similar time scale. Because the time scales for secretion of histamine, LTC4 or IL-4 or the [Ca2+]i response markedly differ (i.e., stimulation in the presence of Ca(e), these results also indicate that down-regulatory processes differ depending on the presence or absence of Ca(e).

Stimulus-dependent leukotriene release from human basophils: a comparative study of C5a and Fmet-leu-phe.

Previous studies of human basophil mediator release have noted that the bacterial peptide fmet-leu-phe and the anaphylatoxin C5a induce comparable levels of histamine release while only fmet peptide induces leukotriene release. Since 5-lipoxygenase metabolism of arachidonic acid is calcium dependent, we examined the characteristics of the human basophil [Ca++]i response which follows its activation by either fmet peptide or C5a. While the peak [Ca++]i response was essentially identical for these two stimuli, fmet peptide induced a prolonged increase in [Ca++]i while C5a stimulated only a transient increase in [Ca++]i that was essentially over within 2 minutes of adding the stimulus. Simultaneous addition of EDTA with fmet peptide revealed the two phases of the [Ca++]i response and demonstrated that leukotriene release was dependent on an elevated [Ca++]i level in the 2-5 minutes following challenge. Enhancement of leukotriene release induced by C5a by agents such as staurosporine and interleukin-3 also produced a [Ca++]i kinetic curve which resembled fmet peptide. Single cell studies of the [Ca++]i response could detect no subpopulations of cells which responded preferentially to fmet peptide or C5a, eliminating the possibility that the ability of fmet peptide to induce leukotriene was a result of its action on a functionally distinct population of basophils.

Graded changes in the response of individual human basophils to stimulation: distributional behavior of events temporally coincident with degranulation.

Human basophils, stimulated with either anti-IgE antibody or formyl-methionine-leucine-phenylalanine, were examined by two measures of the cell response that may reflect degranulation. Flow cytometric measurement of either of these two measures, changes in forward scatter intensity (an indirect measure of the basophil size) or changes in the intensity of acridine orange-loaded cells (which labels basophil granules), allowed an assessment of the distribution of single cell responses. With regard to the latter technique, structures that appeared to be basophil granules were shown to metachromatically label with low concentrations of acridine orange, which has little or no effect on histamine release. During stimulation these labeled granules were lost, leading to decreased fluorescence. Changes in either the forward scatter parameter or acridine orange labeling occurred on the same time scale as histamine release, differentiating these measures of the basophil response from early signal transduction events. Challenging basophils with a combination of phorbol myristate acetate and ionomycin caused 100% histamine release and allowed a measurement of the maximum change in forward scatter intensity or loss of acridine orange labeling. The flow cytometric distributions after this treatment were then compared with the distributions obtained by challenging cells with several concentrations of anti-IgE antibody or formyl-methionine-leucine-phenylalanine, which induced various submaximal responses. These flow cytometric distributions demonstrated that single cells could be found in intermediate states of activation, i.e., the response of all cells was graded according to the strength of the stimulus. These studies lead to the general conclusion that all aspects of the basophil response, including those late events in the basophil response we have studied here, as well as early events that we have studied previously, are graded in a continuous manner, according to the magnitude of the stimulus.

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.

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.

Differential effects of cAMP-elevating drugs on stimulus-induced cytosolic calcium changes in human basophils.

Drugs that elevate cAMP levels in human basophils are known to inhibit immunoglobulin E (IgE)-mediated histamine release. We have examined whether cAMP-active agents inhibit the cytosolic Ca2+, [Ca2+]i, response that normally accompanies activation of basophils. As previously described, this [Ca2+]i response is biphasic, one phase dependent on internal sources of calcium and a second later phase dependent on extracellular calcium, as observed in many cell types. Forskolin and rolipram or their combination had no effect on the initial elevation of cytosolic calcium that follows stimulation with anti-IgE antibody. In contrast, the second phase of the IgE-mediated calcium response was inhibited by these agents. For IgE-mediated responses, the relative efficacy of various cAMP active agents (rolipram approximately forskolin < dibutyryl cAMP < forskolin + rolipram) for the inhibition of histamine release and the second-phase calcium response was similar and roughly paralleled the measured increase in basophil cAMP. In contrast, neither the first nor the second phase of the f-Met-Leu-Phe (fMLP)-induced calcium response was inhibited by any of the cAMP-active agents tested. Indeed, at low concentrations of fMLP, a combination of forskolin and rolipram caused slight enhancement of the calcium response. This result was consistent with the observations that these agents had no effect or caused slight enhancement of histamine or leukotriene released induced by fMLP. Similarly, cAMP-active agents caused no inhibition of C5a or phorbol ester (phorbol myristate acetate)-induced histamine release. These observations suggest that inhibition of the phase of the calcium response that is dependent on extracellular calcium could account for the inhibition of histamine release by these agents. However, these studies also suggested that (1) this is effect is not exerted at the level of the inositol trisphosphate (InsP3) receptor or InsP3 metabolism and (2) the mechanisms that maintain the second-phase calcium response are possibly distinct for IgE- and fMLP-mediated reactions because cAMP-active agents inhibited the second-phase response of only one stimulus.

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.

Preliminary characterization of diacylglycerol generation in human basophils: temporal relationship to histamine release and resolution of degranulation.

Purified human basophils were examined for changes in diacylglycerol levels to determine whether the transient nature of a N-formyl-methionyl-leucyl-phenylalanine (fMLP) -stimulated elevation in membrane protein kinase C (PKC) activity could be explained by the transient production of diacylglycerol (DAG). In preliminary experiments total DAG levels were measured by the DAG kinase assay. Although elevations followed stimulation with 1 microM fMLP (basal levels of 15 pmol/10(6) basophils vs. 45 pmol/10(6) basophils at the 3-min time point), there were no detectable changes in the first 60 s of the reaction. Histamine release is typically complete by 30-45 s. Measurement of inositol trisphosphate indicated a rapid increase by 5 s of 2.5 pmol/10(6) basophils. If DAG were produced at similar levels, the DAG kinase assay would not have detected the elevation. Consequently, fMLP-stimulated basophils were examined for changes in 1-stearoyl, 2-arachidonoyl, 3-sn-glycerol (SA-DAG) and 1-oleoyl, 2-arachidonoyl, 3-sn-glycerol by GC-NICIMS (negative ion chemical ionization mass spectroscopy). A 5-s elevation in these two species averaged 2 pmol/10(6) basophils, consistent with the inositol trisphosphate levels and occurring during the period of histamine release. However, a much more pronounced second phase to the SA-DAG response also occurred, mirroring the total DAG levels. This second phase of the DAG response, either total or SA-DAG, was transient on a time scale temporally coincident with the appearance and resolution of degranulation sacs as measured by fluorescence microscopy. These data suggest that there is selective generation of DAG species in the early reaction and the later appearance of DAG may be related to the formation and resolution of granule structures that follow the secretion of histamine.

Graded changes in the response of individual human basophils to stimulation: distributional behavior of early activation events.

These studies examine the distribution of single-cell responses in basophil preparations in the context of four events that may be associated with early activation by anti-immunoglobulin E (IgE) antibody and the bacterial peptide fMet-Leu-Phe (fMLP). In general, we measured the single-cell response distributions after challenge with a concentration of stimulus that resulted in an optimal response and compared this with the distribution that occurred after challenge with suboptimal concentrations of the same stimulus. The elevation in cytosolic calcium, as detected in Fura-2-labeled basophils, after challenge with anti-IgE or fMLP showed graded characteristics in that the distributions were unimodal under conditions of optimal or suboptimal challenge with little skewing from a normal distribution. Similarly, the up-regulation of the cell surface adhesion molecule CD11b, as determined by flow cytometry, showed graded unimodal increases after challenge with anti-IgE antibody at optimal and suboptimal concentrations. In addition, stimulation of basophils led to increased F-actin polymerization. After challenge with an optimal concentration of anti-IgE antibody, the F-actin content of basophils increased to a maximum between 10 and 15 min and returned to near prechallenge levels by 60 min. There was a close correlation between the maximum increase in F-actin content and histamine release regardless of the stimulus; anti-IgE antibody, fMLP, and phorbol ester (PMA) responses lay on the same regression line. The single-cell F-actin polymerization distributions were also unimodal and graded according to the magnitude of the histamine release response. During measurements of the calcium response under the microscope we noted that basophils underwent significant changes in morphology after challenge with any stimulus. These changes were related to both degranulation and nondegranulation events and could be quantitated by a series of image-processing algorithms, which are presented. The kinetics of the morphological change, measured as a change in cell perimeter, paralleled degranulation. Single-cell distributions of the morphologic changes were also unimodal under conditions of both optimal and suboptimal stimulation. Therefore, no evidence of all-or-nothing responses could be observed in the context of these four early activation events. In general, the response distributions resembled normal distributions at both optimal and suboptimal levels of stimulation, which indicated that single basophils responded in a graded manner.