Fig. 2.

Microglia-derived IL-27 causes neutrophil polarization. Cell-specificity of IL-27 and IL-27R expression in the SD rat brain cells (a–d), and the gene expression profile (e, f) in IL-27-modified mouse BM-PMN precursors, and g STAT3/pSTAT3 protein in rat BM-PMN exposed to rIL-27, in vitro. a Photograph of representative gels demonstrating IL-27 p28 and EBI3 mRNA and IL-27Rα mRNA and gp130 mRNA expression in primary rat brain microglia (micro, identified with CD68), neurons (neuro, identified with neurofilament L, NF-L), astrocytes (astro, identified with glial fibrillary acidic protein, GFAP), and oligodendrocytes (oligo, identified with myelin basic protein, MBP) in culture. IL-27 p28 and EBI3 are exclusively expressed by microglia. The gp130 is expressed by all cell types, while IL-27Rα is mainly by astrocytes and neurons. GAPDH was used as an internal control. b, c Representative RT-PCR gels and bar graph of IL-27 p28 and EBI3 mRNA expression in rat brain cells in response to RBC. IL-27 p28 and EBI3 are upregulated in rat microglia (Micro), but not in astrocytes (Astro), oligodendrocytes (Oligo) or neurons (Neuro) at 6 h after exposure to RBC. The data were calculated as mean ± SEM, n = 4–6. *p ≤ 0.05 (using paired t-test), compared with the media control group (without RBC). GAPDH was used as an internal control. d IL-27 p28 protein determined with ELISA in rat microglial culture medium at 6 h after exposure to RBC, LPS, and interferon gamma (IFNγ). *p ≤ 0.05, compared with the control group (n = 3), established using one-way ANOVA followed by Newman–Keuls post-test. e Photograph of RT-PCR gels and f bar graph demonstrating the gene expression profile in the PMNs harvested from mouse BM-PMNs at 24 h after ICH and then cultured in 10% mouse serum in RPMI1640 and treated with recombinant mouse IL-27 (150 pg/ml) or saline for 24 h, in vitro. The analyzed genes included: iNOS, MMP-9, NOX2, Hp, LTF, myeloperoxidase (MPO), neutrophil elastase (ELANE), and GAPDH. Data were expressed as mean ± SEM (n = 3–5). *p ≤ 0.05, compared with the vehicle-treated group, established using paired t-test. g Photos of immune Western blotting of phosphorylated form (pSTAT3) and total STAT3 protein in purified rat BM-PMN in culture at 6 h after incubating in recombinant mouse IL-27 (150 pg/ml) with or without 10 µM STATTIC. GAPDH was used as an internal control