Figure 2
Figure 2. G-CSF–induced IL-17A secretion is independent of Th1/Th2 cytokines and IL-21 dependent. (A) IL-17A and (B) IL-17F in culture supernatants generated by splenocytes from naive or G-CSF–mobilized WT or respective knockout B6 mice stimulated with CD3 for 72 hours. (C) IL-17A in culture supernatants generated by splenocytes from naive or G-CSF–mobilized WT or BALB/c.IL-4−/− mice stimulated with CD3 for 72 hours. Splenocytes from individual animals were cultured separately (n = 3-7 individual animals/condition pooled from 2 or 3 replicate experiments). Data represent mean ± SEM. *P < .05, **P < .01, naive vs G-CSF–treated. (D-E) IL-17A in culture supernatants generated by splenocytes from naive or G-CSF–mobilized (D) WT, Jα18−/− or (E) γδ−/− B6 mice (n = 5-10 per group from replicate experiments) stimulated with CD3 for 72 hours. NS indicates not significant. (F) TGF-β1 in culture supernatants generated by splenocytes from naive or G-CSF–mobilized BALB/c.WT mice stimulated with CD3 for 72 hours. (G) Real-time polymerase chain reaction (PCR) analysis of IL-6 mRNA expression (left) by nonstimulated splenocytes from naive or G-CSF–treated B6 or BALB/c mice. Data represent pooled data from duplicate experiments with n = 3 or 4 mice/group. IL-6 protein secretion (right) by splenocytes stimulated with CD3 for 72 hours from naive or G-CSF–treated B6 or BALB/c mice. Histogram represents pooled data (n = 9-11 individual animals/condition) from 3 replicate experiments. *P < .05, **P < .01, ***P < .001 for marked groups. (H) IL-17A in culture supernatants generated by splenocytes from naive or G-CSF–mobilized WT, IL-6−/− or (I) IL-12/23p40−/− or (J) B6.IL-21R−/− mice stimulated with CD3 for 72 hours. Histograms represent combined data (n = 7-10 individual animals/condition) pooled from replicate experiments. (K) Real-time PCR analysis of IL-21 mRNA expression in freshly isolated splenocytes (T0) or those cultured for 24 hours with CD3 (T24) from naive (white bars) or G-CSF–treated (black bars) B6 mice. Splenocytes from individual animals were cultured separately. (Left graph) Combined data (n = 3 or 4 individual animals/condition) from replicate experiments. (Right graph) IL-21 mRNA expression levels in FACS-sorted cell subsets from T24 cultures represented as combined data (n = 3-6 individual animals/condition) from replicate experiments. Data are mean ± SEM. (L) Real-time PCR analysis of IL-21 mRNA expression in splenocytes cultured for 24 hours with CD3 from WT or IL-17A−/− donors. Combined data (n = 4 or 5 individual animals/condition) from replicate experiments.

G-CSF–induced IL-17A secretion is independent of Th1/Th2 cytokines and IL-21 dependent. (A) IL-17A and (B) IL-17F in culture supernatants generated by splenocytes from naive or G-CSF–mobilized WT or respective knockout B6 mice stimulated with CD3 for 72 hours. (C) IL-17A in culture supernatants generated by splenocytes from naive or G-CSF–mobilized WT or BALB/c.IL-4−/− mice stimulated with CD3 for 72 hours. Splenocytes from individual animals were cultured separately (n = 3-7 individual animals/condition pooled from 2 or 3 replicate experiments). Data represent mean ± SEM. *P < .05, **P < .01, naive vs G-CSF–treated. (D-E) IL-17A in culture supernatants generated by splenocytes from naive or G-CSF–mobilized (D) WT, Jα18−/− or (E) γδ−/− B6 mice (n = 5-10 per group from replicate experiments) stimulated with CD3 for 72 hours. NS indicates not significant. (F) TGF-β1 in culture supernatants generated by splenocytes from naive or G-CSF–mobilized BALB/c.WT mice stimulated with CD3 for 72 hours. (G) Real-time polymerase chain reaction (PCR) analysis of IL-6 mRNA expression (left) by nonstimulated splenocytes from naive or G-CSF–treated B6 or BALB/c mice. Data represent pooled data from duplicate experiments with n = 3 or 4 mice/group. IL-6 protein secretion (right) by splenocytes stimulated with CD3 for 72 hours from naive or G-CSF–treated B6 or BALB/c mice. Histogram represents pooled data (n = 9-11 individual animals/condition) from 3 replicate experiments. *P < .05, **P < .01, ***P < .001 for marked groups. (H) IL-17A in culture supernatants generated by splenocytes from naive or G-CSF–mobilized WT, IL-6−/− or (I) IL-12/23p40−/− or (J) B6.IL-21R−/− mice stimulated with CD3 for 72 hours. Histograms represent combined data (n = 7-10 individual animals/condition) pooled from replicate experiments. (K) Real-time PCR analysis of IL-21 mRNA expression in freshly isolated splenocytes (T0) or those cultured for 24 hours with CD3 (T24) from naive (white bars) or G-CSF–treated (black bars) B6 mice. Splenocytes from individual animals were cultured separately. (Left graph) Combined data (n = 3 or 4 individual animals/condition) from replicate experiments. (Right graph) IL-21 mRNA expression levels in FACS-sorted cell subsets from T24 cultures represented as combined data (n = 3-6 individual animals/condition) from replicate experiments. Data are mean ± SEM. (L) Real-time PCR analysis of IL-21 mRNA expression in splenocytes cultured for 24 hours with CD3 from WT or IL-17A−/− donors. Combined data (n = 4 or 5 individual animals/condition) from replicate experiments.

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