Figure 4.

S100A9 but not platelet TLR4 deficiency increases platelet–neutrophil complexes in the cremaster muscle upon acute inflammation, an observation that is reversed by supplementation of the S100A8/A9 tetramer. (A) The number of platelet–neutrophil complexes in postcapillary venules in the murine cremaster muscle was analysed in WT, S100A9^−/− and TLR4^PF4Cre+ mice via intravital microscopy 2 h after intrascrotal injection of TNFα, and the number of platelets per complex (B) were determined per vessel (n = 21–23). (C) Platelet–neutrophil complexes were assessed manually by the help of Gr-1-AF488 and CD41-BV421 antibody staining (representative images: green = neutrophils, red = platelets; scale bar: 10 µm). Effects of the S100A8/A9 tetramer during acute inflammation were analysed via intravital microscopy of the TNFα-inflamed cremaster and upon K. pneumoniae infection in WT and S100A9^−/− mice. S100A8/A9 tetramer (100 µg) was injected in addition to TNFα and platelet–neutrophil complexes (D,E) were investigated after 2 h. K. pneumoniae-challenged WT and S100A9^−/− mice were injected intraperitoneally with 150 µg S100A8/A9 tetramer immediately after infection, and again after 11 h. Mice were sacrificed after 22 h and neutrophil (PMN) counts in the intravascular (F), interstitial (G) and the alveolar space (H) were determined via flow cytometry (n = 10–12). Data are mean ± SD, * p < 0.05, ** p < 0.005, *** p < 0.001, **** p < 0.0001, ordinary one-way ANOVA or two-way ANOVA.