IgE Immune Complexes Mitigate Eosinophilic Immune Responses through NLRC4 Inflammasome

Figure 2

Ova-IgE ICs increased FcεR2 surface expression and eosinophil granule contents, but did not affect the degranulation. EoL-1 human eosinophils were primed with TLR2 ligand PAM3CSK4 (1 µg/ml) and treated with IgE (10 µg)-Ova (1 µg) ICs. (a) Surface expression of FcεR1α and FcεR2. (b) CD63 and CD69 on EoL-1 eosinophils were determined by flow cytometry. EoL-1 cells were gated based on their size and granularity using FSC-H/SSC-H by removing debris and doublet cells using FSC-A/FSC-H. Single cells were subgated using FcεR1α, FcεR2, CD63, and CD69. Percentages within the gates indicate the proportion of these receptor expressions in EoL-1 cell population. Values represent the mean ± SD and are representative of two separate experiments. (c) MMP2, MMP9, TIMP1, TIMP2, EDN, and ECP mRNA expressions by real-time qPCR after Ova-IgE ICs treatment in TLR2-primed EoL-1 human eosinophils. Values represent the mean ± SD and are representative of two separate experiments. Student’s t-test shows the significant difference between stimulated and nonstimulated cells. (d) Western blot and corresponding densitometry analyses of cell lysates (30 µg). MMP2, MMP9, TIMP1, and TIMP2 protein expressions were immunoblotted. Values represent the mean ± SD and are representative of three separate experiments. (e) Pro- and active MMP9 enzyme activity from the supernatants of the stimulated and nonstimulated cells was measured by gelatin zymography assay. Lane 1: Human recombinant MMP9 (hrMM9) (10 ng) was used as a positive control.

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