Evidence that IgE molecules mediate a spectrum of effects on mast cell survival and activation via aggregation of the FcepsilonRI

Abstract

We demonstrate that binding of different IgE molecules (IgEs) to their receptor, FcepsilonRI, induces a spectrum of activation events in the absence of a specific antigen and provide evidence that such activation reflects aggregation of FcepsilonRI. Highly cytokinergic IgEs can efficiently induce production of cytokines and render mast cells resistant to apoptosis in an autocrine fashion, whereas poorly cytokinergic IgEs induce these effects inefficiently. Highly cytokinergic IgEs seem to induce more extensive FcepsilonRI aggregation than do poorly cytokinergic IgEs, which leads to stronger mast cell activation and survival effects. These effects of both types of IgEs require Syk tyrosine kinase and can be inhibited by FcepsilonRI disaggregation with monovalent hapten. In hybridoma-transplanted mice, mucosal mast cell numbers correlate with serum IgE levels. Therefore, survival effects of IgE could contribute to the pathogenesis of allergic disease.

Fig. 1.

In vitro effects of HC and PC IgEs. (A) BMCMC were incubated with the indicated IgEs (5 μg/ml) for 8 h before measurement of IL-6 in culture supernatants. (B) Mixed cultures of WT and FcεRI α^–/– BMCMC were incubated with 10 μg/ml SPE-7 or H1 DNP-ε-206 IgE for 3 days without IL-3 or other growth factors before flow cytometric analysis for FcεRI and annexin V. ○, Predicted survival values for WT cells; ▵, predicted survival values for FcεRI α^–/– cells. These values are based on the assumption that each type of cell does not affect the survival of the other. Actual results are indicated by filled (WT) or open (FcεRI α^–/–) bars. Asterisks indicate differences that are statistically significant (P < 0.05) from the predicted values. (C) WT BMCMC were incubated with or without IL-3, or with the indicated IgE without IL-3, for 3 days before flow cytometric analysis of cell survival.

Fig. 2.

Biological outcomes of IgE binding to mast cells in the absence of added growth factors. (A) BMCMC were incubated with the indicated IgEs for 24 h before analysis of cellular content of histamine. (B) BMCMC were incubated with the indicated IgEs for 50 min before analysis of histamine released into culture supernatants. (C) Comparison of kinetics of histamine release induced by 5 μg/ml SPE-7 IgE and that induced from H1 DNP-ε-206 IgE-sensitized BMCMC after stimulation (Stim.) with 100 ng/ml DNP₂₁ HSA. (D) Leukotriene release into culture supernatants from BMCMC incubated with the indicated IgEs for 50 min. (E) Internalization of FcεRI. BMCMC were incubated with the indicated concentrations of H1 DNP-ε-206 or SPE-7 IgE for 24 h before analysis of FcεRI expression. (F) DNA synthesis. BMCMC were incubated with the indicated IgEs for 24 h in the presence of [³H]thymidine for the last 6 h. (G) Effects of low-level cross-linking vs. IgE binding. BMCMC were incubated with the indicated IgEs (5 μg/ml) in the presence or absence of 1 or 10 ng/ml DNP₂₁ HSA for 3 days before survival assays.

Fig. 3.

Inhibition of IgE-induced mast cell cytokine secretion, survival, and signaling by monovalent hapten. (A) BMCMC were incubated with the indicated IgEs (5 μg/ml) in the presence of the indicated concentrations of hapten for 8 h before measurement of IL-6. (B) BMCMC were incubated with the indicated IgEs (5 μg/ml) in the presence of the indicated concentrations of hapten, but without IL-3, for 3 days before survival assays. (C) BMCMC were incubated with the indicated IgEs (5 μg/ml) in the presence of the indicated concentrations of DNP-lysine for 15 min. For comparison, cells were stimulated with 100 ng/ml stem cell factor (SCF) for 15 min. Cell lysates were subjected to immunoblotting with anti-phospho-ERK (pERK), anti-phospho-p38 (pp38), or anti-phospho-Akt (pAkt). No Stim, incubation with PBS.

Fig. 4.

Syk is required for HC IgE-induced cytokine production and IgE-induced survival. (A) Lyn^–/– or syk^–/– BMCMC and control (WT) cells were incubated with the indicated IgEs for 8 h before measurement of IL-6. For comparison, cells were sensitized overnight with 0.5 μg/ml 206 IgE and stimulated with 100 ng/ml DNP₂₁ HSA for 8 h. (B) Lyn^–/– or syk^–/– BMCMC were incubated with the indicated IgEs (5 μg/ml) but without IL-3 for 3 days before survival assays.

Fig. 5.

Effects of IgE on mast cell numbers in vivo. Hybridoma cells secreting H1 DNP-ε-206 or H1 DNP-ε-26 IgE and hybridoma cells secreting anti-DNP IgG2b or PBS were inoculated i.p. into CAF1/J mice. Mice were killed 2 weeks later. Serum IgE and IgG2b levels were measured by ELISA, and mast cells were counted in various tissues. (A) Data for the stomach are shown for two of three separate experiments performed. (Left) n = 6–8. (Right) n = 4. Asterisks indicate statistical significance; P < 0.05 vs. values in PBS-injected mice. (B) Correlation coefficients for mast cell numbers in the stomach mucosa and serum Ig from the experiment shown on the right in A were 0.534 (P = 0.0077) for serum IgE and 0.243 (P = 0.2675) for serum IgG2b.