In vitro analysis of factors influencing the ratio of IgE to IgG1

Abstract

Background: IgE-mediated food allergy is a common disease affecting up to one tenth of the population, especially in industrialized nations, and provoking symptoms that range from mild skin manifestations to life-threatening anaphylaxis. Curative and preventive treatment is very limited, creating a danger for the patients and an economic burden for the patients and their stakeholders. Therefore, an advanced knowledge of immunological mechanisms which could serve as a future treatment target is necessary. A promising target is the ratio of the immunoglobulins IgE and IgG4 in humans, or respectively IgE and IgG1 in mice. Several studies describe that sensitized but tolerant patients presented a higher ratio of food-specific IgG4 to IgE as opposed to food allergic patients. Hence, it is of great interest to understand which factors influence the ratio of these immunoglobulins, contributing to a more allergy-releasing or allergy-protective immune response.

Objective: This thesis aims to evaluate the impact of B cell receptor (BCR) signaling strength, differential IL-4 and glucose levels on the ratio of membrane-bound and secreted IgE and IgG1 in murine B cell cultures.

Methods: The experiments are based on an in vitro B cell culture system that mimics a T cell-dependent germinal center reaction: splenic B cells from BALB/c mice were co-cultured with cells from the feeder cell line 40 LB and stimulated with cytokines and, if applicable, F(ab’)2 fragments of anti-Ig antibodies, among others. The frequencies of IgE- and IgG1-expressing cells were evaluated by flow cytometry, the secretion of these antibodies by ELISA and the class switch recombination generating these antibodies by semiquantitative analysis of switch circles.

Results: Calcium flux experiments revealed that F(ab’)2 fragments of anti-IgM and anti-Ig kappa antibodies could serve as a model for different BCR signaling strengths. Flow cytometric analysis of IgE+ and IgG1+ B cells and plasmablasts showed that stimulation with both F(ab’)2 fragments decreased the frequencies of these isotype-switched cells. The ratio of IgE+ to IgG1+ B cells diminished with increased BCR signaling which was similar for BCR stimulation with F(ab’)2 fragments of anti-IgM and anti-Ig kappa antibodies. In contrast, increasing concentrations of IL-4 or glucose in cell cultures raised the ratio of IgE/IgG1.

Conclusion: In our germinal center model, the stimulation of the BCR with F(ab’)2 fragments of anti-Ig antibodies selectively inhibited the formation of IgE+ B cells and plasmablasts more strongly than that of IgG1+ cells. The strength of this inhibition corresponded to the BCR signaling strength. Semiquantitative analysis of switch circles showed that indirect class switching from IgM to IgG1 to IgE was the principal mechanism of IgE+ B cell generation in our cell culture system. IL-4 and glucose levels seemed to influence the IgE/IgG1 ratio, too. All in all, these findings suggest that complex B cell-extrinsic environmental stimuli differently influence the generation of IgG1- vs. IgE-switched cells, even though Th2-cytokines, especially IL-4, promote switching to both isotypes.