Figure 3.

Effects of MFG-E8 on neutrophil recruitment in mice with pristane-induced lupus and human SLE. (a) BMDNs tracking assay. Recruited PKH67⁺ Mfge8^−/− BMDNs and PKH26⁺ WT BMDNs into lung and peritoneum were detected by flow cytometry (n=6 mice per group). (b) Mfge8^−/− and WT BMDNs for migration assay in vitro with or without rmMIP-2 (1 ng/ml) stimulation. Representative images of migrated BMDNs were shown, original magnification × 100. More than five random microscopic fields per well were counted. (c) In pristane-induced early inflammation (16 h), after pretreatment with rmMFG-E8 (20 μg/kg), infiltrated PMN in the lung and peritoneum from Mfge8^−/− and WT mice were determined by flow cytometry (n=6–8 mice per group). (d) Mfge8^−/− and WT BMDNs for migration assay in vitro with or without rmMFG-E8 (500 ng/ml) treatment, when rmMIP-2 (1 ng/ml) was added as chemotactic stimulus, were determined. (e) The surface expression of CXCR2 on Mfge8^−/− and WT BMDNs were analyzed by flow cytometry, and the average mean fluorescence intensities (MFI) for CXCR2 were shown. (f) PMN ratio in PBCs and their surface CXCR2 expression from 36 healthy controls and 52 SLE patients were measured by flow cytometry and the average MFI for CXCR2 were calculated. (g) After treatment with 500 ng/ml rhMFG-E8, surface expression of CXCR2 on isolated neutrophils from SLE patients were determined (n=12 individuals per group). (h) After pretreatment with rmMFG-E8 (20 μg/kg), surface expression of CXCR2 on BMDNs from WT mice, which were exposed to pristane for 16 h were detected by flow cytometry (n=6 mice per group). For all experiments, data are presented as means±S.E.M., *P<0.05, **P<0.01,***P<0.001; ns, not significant