Cathepsin G Controls Arterial But Not Venular Myeloid Cell Recruitment.

Intravital microscopy of the carotid artery, the jugular vein, and cremasteric arterioles and venules in Apoe −/− and CatG-deficient mice ( Apoe −/− Ctsg −/− ) was used to study site-specific myeloid cell behavior after high-fat diet feeding or tumor necrosis factor stimulation. Atherosclerosis development was assessed in aortic root sections after 4 weeks of high-fat diet, whereas lung inflammation was assessed after inhalation of lipopolysaccharide. Endothelial deposition of CatG and CCL5 was quantified in whole-mount preparations using 2-photon and confocal microscopy.

METHODS

An expanded methods section can be found in the online-only Data Supplement . Mice Ctsg −/− 14 and Ccl5 −/− mice 15 were intercrossed with Apoe −/− mice to generate double-mutant mice. Genetically modified mouse strains were backcrossed to C57Bl/6J background for at least 10 generations. All animal experiments were approved by the local ethics committee and performed in accordance with institutional guidelines. Atherosclerosis Studies Mice were fed a high-fat diet (HFD) containing 21% fat (Ssniff) for 4 weeks to induce early atherosclerosis. In a separate set of experiments, Apoe −/− mice were treated with polyclonal immunoglobulin G (IgG) anti-CatG antibodies raised in rabbit (10 µg/mouse, 3×/wk, Biorbyt) or isotype-matching IgG (10 µg/mouse, 3×/wk, Jackson Immuno Research Laboratories) during 4 weeks of HFD feeding. Imaging of Whole-Mount Tissue Apoe −/− mice were fed a HFD or stimulated with tumor necrosis factor (TNF). The carotid artery was explanted and mounted on glass micropipettes. The cremaster muscles of Apoe −/− mice were exteriorized, fixed, and permeabilized. Primary antibodies to CatG (Biorbyt, 10 µg/mL) and CCL5 (R&D, 20 µg/mL) were used to reveal the presence of both molecules.

Adhesion-Strengthening Assay

Adhesion strengthening was analyzed in vitro using IBIDI-Slide IV 0.1 flow chambers (Ibidi). Flow chambers were coated with intercellular adhesion molecule-1 (ICAM1) for neutrophils or vascular cell adhesion molecule-1 (VCAM1) for monocytes, P-selectin, and CatG. Cells were placed into flow chambers and incubated 5 minutes at 37°C. Shear stress was increased from 0.5 to 40 dyn/cm 2 every 30 s using a high-precision syringe pump, and the percentage of remaining cells relative to initial adherent cell number at initial shear stress (0.5 dyn/cm 2 ) was calculated. β 2 Integrin Clustering Under Increasing Shear Stress To study the integrin cluster formation under flow conditions, the cell surface expression of lymphocyte function-associated antigen 1 (LFA1) in neutrophils was analyzed following the adhesion-strengthening assay described above. Before imaging, the cells were stained with a PE-labeled anti-CD11a antibody (2D7, BD Pharmingen) for 15 minutes. Images were acquired with an upright spinning disc confocal microscope.

Intravital Microscopy

Leukocyte-endothelial interactions in the cremaster, the carotid artery, and the jugular vein were analyzed in mice having received HFD or stimulated for 4 hours with 500 ng TNF (intraperitoneally) as described. 6 Antibodies (at 0.5 µg) to Ly6G (1A8, Biolegend), Ly6C (HK1.4, eBioscience), and CD11b (M1/70, eBioscience) were administered to label myeloid cell subsets.

Statistics

All data are expressed as mean±standard error of the mean. Statistical calculations were performed using GraphPad Prism 5 (GraphPad Software Inc.). After calculating for normality by using the D’Agostino Pearson omnibus test, the unpaired Student t test, 1-way, repeated-measures 2-way analysis of variance or nonparametric Mann-Whitney test or Kruskal-Wallis test with post hoc Dunn test were used as appropriate.

Supplementary Material 01