Exposure to monomeric IgE in vitro markedly upregulated the ability of mature mouse peritoneal mast cells or mouse BMCMCs or cloned mast cells to bind IgE. Two separate lines of evidence indicate that this response largely, if not entirely, reflected the increased surface expression of FcεRI. First, while mouse mast cells also express FcγRII/ III (17
), which can bind IgE immune complexes (18
), virtually all of the binding of monomeric IgE to mouse mast cells that is detectable under the conditions used in our experiments reflects binding of the ligand to a single class of high affinity binding sites, i.e., FcεRI (18
). Second, we used anti-IgE to immunoprecipitate surface-bound IgE, and associated IgE receptors, from lysates of BMCMCs that had been first incubated with or without IgE at 5 μg/ml for 21 h and then exposed briefly to excess IgE just before recovery for flow cytometry and Western blot analysis. We found that, in comparison to aliquots of the same mast cell population that had been incubated without IgE, mast cells that had been incubated for 21 h with IgE exhibited an ~14-fold increase in IgE binding by flow cytometry and an ~17-fold increase in IgE- and cell surface–associated FcεRI β chain by Western blot analysis (Fig. B
). Thus, the IgEinduced increase in IgE binding that was detected in BMCMCs by flow cytometry was directly proportional to the amount of FcεRI β chain obtained from anti-IgE immunoprecipitates of the same cells.
The IgE-induced changes in levels of mast cell FcεRI surface expression that were detected in our experiments reflect the operation of two processes, which can be distinguished by their sensitivity to inhibition by cycloheximide. In confirmation of the findings of Furuichi et al. with RBL cells (14
), we found that the earliest component of the IgEdependent increase in BMCMC FcεRI expression (which occurred within 3 h of addition of IgE) was largely resistant to inhibition by cycloheximide. By contrast, virtually all of the subsequent sustained, and striking, upregulation of FcεRI expression in these cells was ablated by cycloheximide. The most straightforward interpretation of these findings, which we favor, is that the initial, cycloheximide-insensitive, component of the response reflects IgE-dependent suppression of the loss of preformed FcεRI expressed on the cell surface (14
), whereas the sustained, cycloheximide-sensitive component is more dependent on protein synthesis, e.g., of new receptors.
To address the in vivo relevance of our findings, we performed experiments in normal mice and mice that had been rendered genetically IgE deficient. IgE −/− mice expressed baseline levels of FcεRI that were 83% reduced compared with those of normal (IgE +/+) mice. Furthermore, both IgE −/− and normal mice exhibited significant increases in the levels of peritoneal mast cell FcεRI expression after i.v. treatment with IgE. These findings demonstrate that IgE is a major regulator of mouse mast cell FcεRI expression in vivo. Indeed, analysis of data from the IgE-treated IgE −/− mice and the IgE +/+ mice that had been treated with IgE, IgG2a, or PBS revealed a striking positive correlation between the log10
of the serum IgE concentration at sacrifice and the log10
of the molecules of equivalent soluble fluorochrome (MESF) values for FcεRI expression by peritoneal mast cells from the same mice (R = 0.871, P
<0.0001). Thus, our findings in mice directly support the general form of the hypothesis that was originally proposed by Malveaux et al. with respect to human basophils (13
), namely, that circulating levels of IgE can influence levels of effector cell surface expression of FcεRI.
Nevertheless, mast cells from IgE −/− mice exhibited detectable, albeit greatly reduced, levels of FcεRI on their surface (Fig. C
), indicating that factors other than IgE must also contribute to the regulation of mouse mast cell FcεRI expression. IL-4 has multiple effects that augment IgE-dependent immune responses in mice; it is required for normal IgE responses (30
) and can also promote the expansion of certain mouse mast cell populations (32
). Moreover, peritoneal mast cells of IL-4 −/− mice exhibit fewer FcεRI than do those of IL-4 +/+ mice (34
). IL-4 can also upregulate levels of mRNA for the FcεRI α chain in human eosinophils (35
). We found that recombinant mouse IL-4 (at 10 ng/ml) slightly enhanced peritoneal mast cell FcεRI expression over that induced by IgE alone, but had little or no effect in the absence of IgE (data not shown). In light of this finding, one may speculate that the reduction in mast cell FcεRI expression in IL-4 −/− mice might reflect, at least in part, the low levels of IgE in these animals (31
One of the most significant aspects of our study is the observation that changes in the concentration of IgE antibody not only can regulate the level of cell surface expression of FcεRI on mouse mast cells, but that this IgE-dependent upregulation of mast cell FcεRI expression is functionally significant. This result was, in some respects, unexpected. In their studies of human basophils derived from patients with different concentrations of circulating IgE, Conroy et al. (11
) detected no significant differences in the histamine release responses induced by different concentrations of antiIgE in basophils from nonatopic donors, which expressed as few as 3,900 IgE molecules/basophil, or allergic donors, which expressed up to 500,000 IgE molecules/basophil. Moreover, basophils from donors whose cells expressed approximately the same amounts of cell-bound IgE varied greatly in their sensitivity to anti-IgE–induced histamine release, perhaps reflecting a wide spectrum of intrinsic releasability of human basophils derived from different individuals (11
By contrast, we found that IgE concentrations can regulate the expression of FcεRI by mouse mast cells within a range that has significant consequences with respect to IgEdependent effector cell function. Compared with mast cells that had been incubated with levels of monomeric IgE similar to those present in the sera of some normal mice (i.e., 0.005 μg/ml), mast cells that had been incubated with concentrations of IgE in the range observed in the sera of parasite-infected mice (i.e., 5.0 μg/ml) exhibited significant mediator release at lower concentrations of antigen and also gave significantly greater release of mediators at given higher concentrations of antigen. Thus, IgE-dependent upregulation of mast cell FcεRI expression significantly enhanced both the sensitivity and the intensity of the secretory response of the cells to antigen challenge.
This effect was especially remarkable with respect to cytokine production. Thus, in comparison with cells that had been incubated for 4 d with IgE at 0.005 μg/ml, BMCMCs that had been incubated for 4 d with IgE at 5.0 μg/ml exhibited ~60% greater maximal release of the preformed mediator, serotonin, but ~6-fold greater release of the cytokine IL-6 (Fig. , B
). The effect of IgE incubation on the ability of BMCMCs to produce IL-4 was even more striking. Substantial IL-4 production in response to antigen challenge was detected in BMCMCs that had been incubated for 4 d with IgE at 5.0 μg/ml, but was not detectable at all in cells that had been incubated for 4 d before antigen challenge either without IgE or with IgE at 0.005 μg/ml. The ELISA assay used for our studies has a lower limit of detection of <5 pg/ml of IL-4. It is possible (and perhaps likely) that different conditions of passive sensitization before antigen challenge, or the use of more sensitive indices of IL-4 production, may have permitted us to detect IgEdependent IL-4 production by BMCMCs that had not been incubated with high concentrations of IgE for 4 d before further passive sensitization and antigen challenge (7
). However, under the conditions employed in our experiments, incubation of BMCMCs with IgE at 5.0 μg/ml for 4 d clearly markedly enhanced the ability of these cells to release IL-4 in response to challenge with IgE and specific antigen.
Taken together, our observations identify a novel and potentially important mechanism for enhancing both the sensitivity and intensity of IgE-dependent immune responses. Indeed, because IL-4 can markedly enhance the generation of IgE responses in mice (30
), it is even possible that IgE-dependent upregulation of FcεRI expression by mouse mast cells, and subsequent enhancement of IgE- and antigen-dependent mast cell IL-4 production, may constitute a previously unrecognized positive feedback loop in the expression of IgE-dependent immune responses.
While the regulation of IgE production is complex, potentially involving multiple genetic and environmental factors, serum levels of IgE typically are greatly increased during parasite infections and are also elevated in most patients with allergic diseases (38
). Our findings suggest that any mechanism that results in the substantial elevation of IgE levels may also result in significantly enhanced IgE-dependent effector cell function. Furthermore, no matter what may be the precise minimum number of cross-linked FcεRI that are required to trigger mast cell or basophil mediator release (43
), cells that express increased numbers of FcεRI on their surface can be sensitized adequately with larger numbers of different IgE species of distinct antigen specificities. Thus, increased expression of FcεRI by mast cells would also permit each cell to be simultaneously sensitized to respond to a larger number of different antigens.
In the context of acquired immune responses to parasites, IgE-dependent enhancement of mast cell FcεRI expression would be expected to benefit the host. Unfortunately, the same mechanisms might also increase the severity of allergic diseases. Our preliminary experiments indicate that IgE can also increase surface expression of FcεRI on in vitro-derived human mast cells (45
). Accordingly, it will be of great interest to assess to what extent IgE-dependent upregulation of FcεRI expression, as here identified in mouse mast cells, also occurs in human FcεRI+
cells, and, if so, to determine whether the ability of IgE to regulate FcεRI can be exploited therapeutically in the management of allergic disorders.