Autoantibodies to IgE and FcεRI and the natural variability of spleen tyrosine kinase expression in basophils

Abstract

Background: Secretion from human basophils and mast cells requires spleen tyrosine kinase (SYK) activity, but SYK expression is highly variable in the general population, and this variability predicts the magnitude of IgE-mediated secretion. One known mechanism of modulating SYK expression in human basophils is aggregation of FcεRI.

Objective: This study examines the possibility that functional autoantibodies are present in a wide variety of subjects and, in particular, subjects whose basophils poorly express SYK. It also tests whether any found antibodies could modulate SYK expression in maturing basophils and whether interaction with FcγRIIb/CD32b modulates the effect.

Methods: An experimental algorithm for classifying the nature of histamine release induced by serum from 3 classes of subjects was developed.

Results: The frequency of functional autoantibodies that produce characteristics concordant with FcεRI-mediated secretion was zero in 34 subjects without chronic spontaneous urticaria (CSU). In patients with CSU, the frequency was lower than expected, approximately 7%. For the 5 of 68 unique sera from patients with CSU tested that contained anti-FcεRI or anti-IgE antibodies, these antibodies were found to induce downregulation of SYK in both peripheral blood basophils and basophils developed from CD34⁺ progenitors. Blocking interaction of these antibodies with CD32b did not alter their ability to downregulate SYK expression.

Conclusions: This study establishes that functional autoantibodies to IgE/FcεRI do not provide a good explanation for the variability in SYK expression in basophils in the general population. They do show that if antibodies with these characteristics are present, they are capable of modulating SYK expression in developing basophils.

Keywords: Fc receptors; Human; allergy; basophil.

Figure 1

Criteria for classification of sera as containing functional auto-antibodies to FceRI or IgE. ‘drugs’ = 3 IgE-pathway-specific drugs, ‘xdesn’ = cross-desensitization with known aggregating anti-IgE Ab, ‘NR’ inability to stimulate basophils of the non-releasing phenotype.

Figure 2

Effect of Ab10523 (blocking Ab for FcgRIIb/CD32b) on sera-induced secretion. Two methods were u sed to demonstrate within the same cell preparations that blocking FcgRIIb/CD32b with Ab10523 (anti-FcgRIIb/CD32b) under known conditions would alter the response to stimuli. These positive controls (positive ctrls, left side of plot) are described in the text. Using similar conditions, the cells were stimulated with sera MM and AB at the concentrations shown in the presence or absence of Ab10523 (anti-FcgRIIb/CD32b). The bars plot the average histamine release for the 4 experiments and the average ratios (response in the presence of Ab10523/response without Ab) for the 4 experiments are shown above the bars.

Figure 3

SYK expression in purified human basophils cultured overnight with serum (see methods) from subjects whose basophils were characterized as non-releasers or releasers. Panel A: Example western blot for SYK expression, NR = serum from non-releaser phenotype, R = serum from releaser phenotype, aIgE = known aggregator 6061P/anti-IgE Ab. The protein p85 (a subunit of PI3K) is used to normalize results (see methods). Panel B: summary analysis of 2 experiments. The serum effects were calculated relative to media control band intensity. Five releaser and 5 non-releaser sera were tested in overnight cultures and the ratios plotted. Panel C: Example western blot analysis for SYK expression, C = media control, A = anti-IgE Ab, As = suboptimal anti-IgE Ab, Cs = control serum (known not to induce secretion), MM, AB, AS = sera known to induce release by the criteria established for FceRI/IgE-mediated release. Overnight cultures as described for panel A. Panel D: average results of the conditions aligned in panel C (n=3, 2 experiments using Western analysis and 1 using flow cytometric analysis).

Figure 4

SYK expression in 15 day culture-derived basophils treated with AS serum ± Ab10523 (anti-FcgRIIb/Cd32b). The various stimuli were added at day 3 of culture and analysis of SYK done by flow cytometry. Panel A; flow cytometry of cells treated with control serum at 1% (serum previously determined to not induce release). Panel B: flow cytometry of cells treated with AS serum at 1% (known to induce release). Panel C: summary results from 2–3 experiments (n=3 for 1% and n=2 for 2% serum), expressed relative to control serum, 22E7 (anti-FceRI Ab) at a concentration previously determined to induce 5% of maximum release. For the AS treated cells, combining results for both 1% and 2% concentrations, FceRI = surface FceRI expression (n=3), Al.Blue = counts of alcian blue positive cells (n=3), Tot.Hist. = total histamine content (n=2).