![]() Representative scatter plots show similar analysis performed on cells elicited from wild-type mice after 6 hours of stimulation with SES in combination with sgp130 (150 ng per mouse) or IFN-γ –/– mice after stimulation with SES in combination with HYPER-IL-6 (40 ng per mouse). ( a) Annexin V/PI staining of leukocytes. Modulation of IL-6 signaling in vivo affects apoptotic clearance of PMN in SES-induced peritoneal inflammation. After 1 hour, mice were sacrificed and IL-6 was quantified (* P < 0.05 versus PBS alone ** P < 0.05, a significant increase versus the additive value of IL-1β plus IFN-γ alone). ( e) Wild-type mice were intraperitoneally administered with PBS, IL-1β (100 ng per mouse), IFN-γ (0.5 U per mouse), or a combination of IL-1β and IFN-γ at these doses. Results are representative of three separate experiments performed with HPMC from different donors. ( d) Composition of the NF-κB complex was determined by supershift using antibodies against p50 (lane 2) and p65 (lane 3). At the indicated intervals, nuclear extracts were prepared and NF-κB activation was monitored by EMSA. ( c) HPMC were stimulated with medium alone (lane 1), HYPER-IL-6 (500 pg/ml, lane 2), IFN-γ (100 U/ml, lane 3), IL-1β (100 pg/ml, lane 4), or a combination of IL-1β and IFN-γ (lane 5). Representative results for three separate experiments are shown. ( b) Northern blot analysis of total RNA isolated from HPMC incubated for 3 hours with medium and IL-1β (100 pg/ml) in the presence or absence of IFN-γ (100 U/ml). At specific time points, IL-6 levels were quantified using ELISA (* P < 0.05, a significant increase versus the additive value of IL-1β plus IFN-γ alone). Growth-arrested HPMC were ( a) incubated with medium alone or IL-1β (100 pg/ml) in the presence or absence of IFN-γ (100 U/ml). Regulation of IL-6 production by IL-1β and IFN-γ in vitro and in vivo. These data emphasize a pivotal role for IFN-gamma in regulating innate immunity through control of both the recruitment and clearance phases of PMN trafficking. Examination of the leukocyte infiltrate from IFN-gamma-/-, IL-6-/-, and wild-type mice showed that apoptosis was aberrant in the absence of IFN-gamma and IL-6 as a result of impaired sIL-6R signaling. ![]() Although HYPER-IL-6 attenuated PMN influx in IFN-gamma-/- mice, IFN-gamma had no effect on PMN infiltration in IL-6-/- mice. To test whether local IL-6 signaling modulated PMN recruitment, inflammation was induced in IFN-gamma-/- and IL-6-/- mice and cytokine signaling adapted by intraperitoneal sIL-6R-IL-6 fusion protein (HYPER-IL-6) or IFN-gamma. Reconstitution of IFN-gamma signaling restored the rate of PMN infiltration and IL-6 levels and was accompanied by normalization of PMN-activating CXC chemokine expression. This defect in PMN recruitment was also associated with the suppressed intraperitoneal expression of IL-1beta and IL-6. Induction of peritoneal inflammation in IFN-gamma-deficient (IFN-gamma-/-) mice emphasized that the initial rate of PMN recruitment was impaired. We now report that IFN-gamma controls PMN infiltration and modulates IL-6 signaling through its soluble receptor (sIL-6R) to promote their apoptosis and clearance. Regulated recruitment and clearance of neutrophils (PMN) is the hallmark of competent host defense and resolution of inflammation.
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