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. 2012 Mar 15;185(6):628–636. doi: 10.1164/rccm.201108-1533OC

Figure 4.

Figure 4.

Disruption of CCL5–CXCL4 heteromer formation abrogates neutrophil recruitment in LPS-induced acute lung injury. (A) Correlation of leukocyte counts and CCL5–CXCL4 heteromers in bronchoalveolar lavage (BAL) fluid from patients with acute lung injury and adult respiratory distress syndrome. (B) Quantification of CCL5–CXCL4 heteromers in supernatants of homogenates of lungs from mice exposed to LPS and having received antiplatelet serum (50 μl) or MKEY (50 μg). n = 3 for each bar. Statistical significance was tested using Kruskal-Wallis test with Dunn post hoc test. *Indicates significant difference to all other groups. (C) Isolated neutrophils were perfused over human umbilical vein endothelial cells (HUVEC) treated with tumor necrosis factor (TNF) (50 ng/ml, 12 h). In addition, recombinant CCL5 and CXCL4 were complexed and immobilized on HUVEC. Thereafter, neutrophils were perfused in presence or absence of MKEY. n = 8 for each bar. Statistical significance was tested using one-way analysis of variance (ANOVA) with Dunnett post hoc test. *Indicates significant difference compared with HUVEC treated with CCL5–CXCL4 in absence of MKEY. (D–F) Mice were treated with MKEY (50 μg) 1 hour before or after LPS inhalation, scrambled MKEY (sMKEY, 50 μg), antibodies to CCL5 and CXCL4, or platelet-depleting serum. Four hours after LPS inhalation, mice were killed. (D) Displayed are intravascular (top), interstitial (middle), and alveolar neutrophil counts (bottom). (E) Protein concentration (top), fluorescein isothiocyanate (FITC)–dextran clearance (middle), and elastase (bottom, uniform bars) and myeloperoxidase (MPO) activity (bottom, hatched bars) in BAL fluids. n = 8–10 for each bar. Statistical significance was tested using one-way ANOVA with Dunnett post hoc test. *Indicates significant difference compared with mice receiving LPS. (F) Representative photographs of histologic (left) and scanning electron analyses (right) of mice receiving phosphate-buffered saline (ctrl) or LPS in presence or absence of MKEY. Scale bars indicate 50 μm for scanning electron microscopy and 250 μm for histology. Quantification of histologic lung sections (bottom). n = 8–10 for each bar. Statistical significance was tested using one-way ANOVA with Dunnett post hoc test. *Indicates significant difference compared with LPS-treated animals.