MPO provokes neutrophil recruitment in vivo. (A) Representative liver sections of WT and Mpo^−/− mice after 90 minutes of hepatic ischemia with subsequent 20 hours of reperfusion are shown (PMN = red-brown; ×200, captured with Zeiss AxioCam HRc mounted on a Zeiss Axioskop with AxioVision 3.1. Tonal value correction, brightness, and contrast were adjusted with Adobe Photoshop CS3 extended 10.0). (B) PMN were quantified in liver sections of WT or Mpo^−/− mice on hepatic ischemia and reperfusion (hpf = high-power field; magnification ×600; sham n = 5-8, treated n = 9-10, one-way ANOVA P < .0001). (C) Number of adherent leukocytes in postcapillary venules of the cremaster muscle of WT (19 vessels in 4 mice) and Mpo^−/− mice (18 vessels in 4 mice) stimulated with TNF-α was assessed by intravital microscopy. (D) Number of perivascular PMN in TNF-α stimulated cremaster muscle of WT (n = 4) and Mpo^−/− mice (n = 4) was evaluated in Giemsa-stained whole mounts. (E) MPO-immunoreactivity (αMPO, green, Alexa Fluor 488) on intraportal injection of HSA or human MPO is shown in hepatic sections of WT mice (blue = Dapi, original magnification ×100, captured with Retiga 1300 CCD camera mounted on Leica DMLB fluorescence microscope by iVision version 4.0, processed as described in panel A). (F) Hepatic PMN-infiltration was quantified on intraportal injection of HSA, active MPO (MPO), inactive MPO M243T and Q91T, or ABAH-inactivated MPO (n = 3-8, hpf = high-power field, magnification ×600, one-way ANOVA P < .01). (G) Number of adherent leukocytes in cremaster muscle postcapillary venules was assessed before (basal, white bars) and after (post injection, black bars) injection of saline solution (16 vessels in 5 mice), active (15 vessels in 3 mice) or inactive MPO Q91T (12 vessels in 3 mice) into the carotid artery of WT mice. (H) Mean diameter of PMN adherent to the endothelium of cremaster muscle venules in mice on injection of saline, MPO, or Q91T MPO to the carotid artery or intrascrotal TNF-α application (500 ng) and a representative image of vessel-adherent PMN in MPO and TNF-α–treated mice are shown (magnification ×400; intravital microscopy was performed with an Olympus BX51 microscope with a CF8/1 CCD-camera [Kappa]), recorded on S-VHS recorder and digitalized with MetaMorph software [Molecular Devices]). (I) Intraluminal staining of MPO (green) and negatively charged glycocalyx residues (Alcian blue) in WT mice on MPO injection of the carotid artery (MPO i.a.) or control (ctrl). Scale bars, 30 μm; fluorescence images captured and processed as described in panel E and bright-field images as described in panel A. Bars represent means; error bars, SEM. *P < .05, **P < .01, ***P < .001.