Page 114 - 2021_10-Haematologica-web

G. Zuchtriegel et al.
lonephritis, or rheumatic disease.6-10 The functional role of VN under these pathological conditions, however, remains largely unclear.
The recruitment of white blood cells (leukocytes) from the microvasculature to the site of inflammation is a fun- damental process in the immune response.11-14 In inflamed tissue, circulating leukocytes become captured and start to roll on the microvascular endothelium in a selectin-dependent manner.15 This triggers the intermedi- ate affinity conformation of b2 integrins on the surface of rolling leukocytes which allows them to further slow down in the bloodstream.16,17 Subsequently, interactions of chemokines presented on the microvascular endothe- lium with their cognate chemokine receptors on rolling leukocytes as well as of endothelial E-selectin/CD62E with leukocyte P-selectin glycoprotein ligand-1 (PSGL- 1/CD168) are supposed to initiate the full activation of leukocyte integrins ultimately facilitating intravascular adhesion of these immune cells to the endothelial sur- face.15,18 After stabilizing their adhesion, leukocytes intravascularly crawl to sites of adherent platelets, from where they finally extravasate to the perivascular tissue and migrate to their target destination.19 Whereas a vari- ety of adhesion and signaling molecules have been char- acterized to control distinct steps of this highly complex process, the mechanisms underlying the stabilization of leukocyte adhesion to the microvascular endothelium are still poorly understood.
Fibrinolysis is an elementary biological process that maintains blood perfusion by preventing clot formation in the vasculature. Plasmin is the principal effector pro- tease in the fibrinolytic system, which is activated by tis- sue-plasminogen activator (tPA) and – to a lesser degree – by urokinase-type plasminogen activator (uPA). The activity of these serine proteases is tightly controlled by plasminogen activator inhibitor-1 (PAI-1). Besides these well-known fibrinolytic properties, it has become evi- dent that the components of the fibrinolytic system also considerably contribute to different biological processes such as immune cell trafficking.20-24 With respect to the distinct interactive properties of VN and the substantial involvement of the various binding partners of this gly- coprotein in immune cell responses, we hypothesize that VN is critical for leukocyte recruitment to the site of inflammation.
Methods
A detailed description of the methods employed in this study is included in the Online Supplementary Appendix.
All experiments were performed according to German legis- lation for the protection of animals and approved by the local government authorities (Regierung von Oberbayern).
Six hours after intra-peritoneal injection of the chemokines CXCL1 or CCL2, leukocyte recruitment to the peritoneal cavity was studied by flow cytometry in wild-type (WT) or VN-defi- cient mice. The single steps of the neutrophil (visualized by flu- orescence-labeled anti-Ly-6G monoclonal antibodies) extrava- sation process were analyzed by in vivo microscopy in the cre- master muscle of anesthetized mice deficient for distinct pro- teins or receiving different inhibitors/blocking antibodies. Integrin activation/trafficking was assessed in neutrophils from WT mice by flow cytometry and spinning disc confocal microscopy.
Results
Distribution of vitronectin in inflamed tissue
Under a variety of inflammatory conditions, enhanced tissue levels of VN have been observed.6-10 The exact dis- tribution patterns of this glycoprotein in inflamed tissue, however, remained unclear. Employing immunostaining and confocal laser scanning microscopy on tissue whole mounts of the mouse cremaster muscle, VN was barely detected in unstimulated tissue (Figure 1A). In the acute inflammatory response upon sterile (ischemia-reperfu- sion [I/R]; 30/120 minutes [min]) injury, however, VN was found to be deposited on the luminal surface of postcap- illary venules. This microvascular deposition of VN was nearly absent upon enzymatic degradation of gly- cosaminoglycans (GAG).
Role of vitronectin for myeloid leukocyte trafficking
In order to characterize the role of VN for myeloid leukocyte recruitment to the site of inflammation, we ini- tially used a peritoneal leukocyte trafficking assay. As identified by multi-channel flow cytometry analyses of the peritoneal lavage fluid, 6 hours of intraperitoneal stimulation with the chemokines CXCL1/KC or CCL2/MCP-1 induced a significant increase in numbers of neutrophils (CD45+ CD11b+ Gr-1high CD115low) and clas- sical/inflammatory monocytes (CD45+ CD11b+ Gr-1high CD115high; Figure 1B), but not of non-classical monocytes (CD45+ CD11b+ Gr-1low CD115high; data not shown) recruit- ed to the peritoneal cavity of WT mice as compared to unstimulated controls. This increase in numbers of neu- trophils was almost completely abolished in VN+/- or VN- /- mice, whereas the recruitment of classical/inflammatory monocytes remained unaffected by VN deficiency. In this context, we found that expression of the low density lipoprotein-related receptor protein-1 (LRP-1), which serves as a receptor for the VN binding partner PAI-1,25-27 was higher on the surface of activated murine neutrophils (mean fluorescence intensity 1,023.0±231.4) than on acti- vated classical monocytes (mean fluorescence intensity 473.0±128.8). Moreover, LRP-1 was identified to be expressed heterogeneously in murine neutrophils (Online Supplementary Figure S1).
Role of vitronectin for intravascular interactions of neutrophils
In order to further decipher the role of VN in the extravasation process of neutrophils, we performed multi-channel in vivo microscopy on the mouse cremaster muscle. In these experiments, we observed that Ly-6G+ neutrophils in VN-/- mice are unable to stabilize their adhesion to the endothelial surface of postcapillary venules in inflamed tissue, whereas neutrophils in WT mice properly adhered to the microvascular endothelium (Figure 2A; Online Supplemtary Videos S1 and S2). Accordingly, the quantitative analysis of these events revealed no significant differences in the numbers of rolling neutrophils or in the rolling velocity of neutrophils between WT and VN-/- mice animals upon I/R (30/120 min) or after 6 hours of intrascrotal stimulation with CXCL1/KC or CCL2/MCP-1 (Figure 2A). In contrast, the average number of neutrophils adhering to the vessel wall of postcapillary venules for more than 30 seconds (s) was significantly lower in VN-/- mice than in WT controls, whereas the average number of neutrophils adhering for
2642
haematologica | 2021; 106(10)