Regulation of Syk kinase and FcRbeta expression in human basophils during treatment with omalizumab

Abstract

Background: In human basophils from different subjects, maximum IgE-mediated histamine release and the level of Syk protein expression correlate well. Recent studies suggest that in some patients treated with omalizumab, the response to stimulation with anti-IgE antibody increases. In unrelated studies there is also evidence that the composition of FcepsilonRI in basophils differs among subjects. This observation raised the possibility that the stoichiometry of FcRbeta/FcepsilonRIalpha is not fixed to a 1:1 ratio and might be modifiable during changes in the basophil's environment.

Objective: We sought to determine whether treatment with omalizumab results in increases in Syk expression and anti-IgE-mediated histamine release and disproportionately alters the relative presence of FcRbeta and FcepsilonRIalpha.

Method: Syk, FcepsilonRIalpha, and FcRbeta expression was monitored during the treatment of subjects with omalizumab.

Results: Treatment with omalizumab reduced histamine release from peripheral blood leukocytes stimulated with cat allergen in vitro, but histamine release stimulated with anti-IgE antibody increased 2-fold. Expression of Syk increased 1.86-fold. There was no change in the expression of c-Cbl, a signaling element that is sensitive to the presence of IL-3, and no increase in response to formyl-met-leu-phe (tripeptide), a response that also increases in the presence of IL-3. There was a 60% decrease in the FcRbeta/FcepsilonRIalpha ratio in patients treated with omalizumab.

Conclusions: In the context of previous studies, these studies provide support for a proposal that Syk expression is modulated in vivo through an IgE-dependent mechanism and that the ratio of FcepsilonRI alpha and beta subunits in basophils is influenced by factors extrinsic to the cell.

Figure 1. Increases in syk expression and anti-IgE mediated histamine release in subjects treated with omalizumab

(A) Measurement of histamine release induced with an optimal concentration of anti-IgE antibody on pre-treatment (Day 0, BSL), 4–6 weeks after the start of treatment (MID), and 15 weeks (ca. 104 days) after the start of treatment (FINAL) for subjects treated with omalizumab (n=12). (B) Measurement of syk expression in basophils by flow cytometry for the same group of subjects in panel A. (C) Measurement of histamine release induced with anti-IgE antibody in the placebo group (n=4). (D) Measurement of syk expression by flow cytometry in the placebo group. Spontaneous histamine release averaged 3 ± 0.3%. It was not different between groups and did not change during the course of the study.

Figure 2. Kinetics of the increase in anti-IgE Ab-induced histamine release and syk expression

The data is expressed relative to the pre-treatment response or levels of syk expression (n=6); (^◯) histamine release induced by anti-IgE antibody and (^●) syk expression measured by flow cytometry. Both kinetic curves, taken as a whole, are statistically different than no change (1.0)(p<0.0001).

Figure 3. Effect of omalizumab treatment on other IL-3 sensitive characteristics of human basophils

(A) The ratio of the measured expression of c-cbl in purified basophils (as assessed by quantitative Western blots) or the ratio of percent histamine release induced by 1 μM FMLP. The ratio is calculated as the endpoint at day 104 (FINAL) divided by the endpoint pre-treatment (day 0, BSL). (B) Expression of cell surface FcεRIα and bound IgE levels on peripheral blood basophils from omalizumab treated or placebo subjects were determined by flow cytometry and by Western blotting. The ratio of expression level at day 104 (FINAL) to pre-treatment (BSL) is plotted (n=12, treated with omalizumab, n=4, treated with placebo agent). (C) The ratio of expression of FcRβ and FcεRIα as determined by Western blot analysis of purified blood basophils from omalizumab treated or placebo subjects (BSL and FINAL). All data are expressed as mean ± SEM.

Figure 4. Absence of change in FcRβ:FcεRIα ratio in vitro

Purified basophils were cultured in RPMI-1640 with IL-3 (10 ng/ml) for 13 days (D13) either without (NS) or with (S) prior lactic acid treatment to remove endogenously bound IgE. After 13 days of culture, cells were counted, lysed, and lysates analyzed by the semi-quantitative Western blot procedure used in the omalizumab study in order to quantitatively assess expression levels. (A) Expression of p60, relative to pre-culture levels (D0) for non-stripped and stripped cells (n=5) (p = 0.12 | H_o = 1.0 for non-stripped (NS) cells and p= 0.0005 | H_o = 1.0 for stripped (S) cells). (B) Ratio of FcRβ:FcεRIα for Day 0 (D0) and non-stripped (NS) and stripped cells (S) on day 13 (D13) (n=5) (p = n.s. for comparison of 3 groups). Errors shown represent SEM.