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. Author manuscript; available in PMC: 2013 Jun 1.
Published in final edited form as: J Immunol. 2012 Apr 27;188(11):5655–5664. doi: 10.4049/jimmunol.1102330

FIGURE 4.

FIGURE 4

Essential roles of CXCR2 on enhanced accumulation of neutrophils during CLP-induced sepsis in CRTH2−/− mice. A, At 24 h after five-puncture CLP surgery, peritoneal exudate cells were collected by peritoneal lavage with 3 ml PBS. The number of peritoneal cells is expressed per peritoneal cavity. B and C, Peripheral neutrophils were purified from peripheral blood 4 h after sham or five-puncture CLP surgery as described in the methods section. The mRNA expression of CRTH2 (Gpr44) (B) and PPAR-gamma (Pparg) (C) in peripheral neutrophils were measured by qRT-PCR. N.D., not detected. D and E, The level of CXCL1 (D) and CXCL2 (E) in peritoneal lavage fluid at 24 h after sham or five-puncture CLP surgery was measured by ELISA. Data expressed as mean ± SEM. n = 5–6 in each group. *, p < 0.05, compared with WT group. These results are representative of two different experiments. F-H, Peripheral neutrophils were purified from peripheral blood 2 h after five-puncture CLP surgery as described in Materials and Methods. CXCR2 mRNA levels in peripheral neutrophils were measured by qRT-PCR (F). Data are expressed as mean ± SEM. n = 4 in each group. The level of CXCR2 in peripheral neutrophils was measured by flow cytometry (G–I). Representative flow cytometry data in Gr1 high positive gated cells (neutrophils) is shown (G). The percentage of CXCR2 positive cells in Gr1 high positive cells (H) and the mean fluorescence intensity (MFI) of CXCR2 is shown (I). Data of (H) and (I) expressed as mean ± SEM. n = 5–6 in each group. #, p < 0.05, compared with sham-operated group. *, p < 0.05, compared with WT group. J, Peripheral PMNs were purified from peripheral blood 2 h after CLP surgery. Chemotaxis assay was performed by using CXCL2 (30 ng/ml), as the ligand, or not (control cells) in a Boyden chamber. Data are expressed as percentage of migrated cells in total cells. n = 4 in each group. *, p < 0.05, compared with CLP-operated WT mice. K, Neutrophils were purified from peripheral blood 2 h after five-puncture CLP surgery. The histogram data of GRK2 positive cells in Gr1 high positive cells are shown. The data shown are representative of three independent experiments.