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G. Zuchtriegel et al.
significantly vary between WT and VN-/- mice (Figure 3A). In contrast, intravascular firm adherence of neutrophils was significantly diminished in VN-/- mice animals as compared to WT animals (Figure 3A). Conversely, anti- body-mediated depletion of platelets (by >95%) signifi- cantly reduced the number of intravascularly adherent (>30 s) and transmigrated neutrophils in the field of view, but did not significantly change intravascular adhesion times of these immune cells in the microvasculature of postischemic tissue (Figure 3B). These data confirm previ- ous observations documenting that intravascularly adher- ent neutrophils do not stop crawling in the microvascula- ture and start their transmigration (which results in less firmly adhered and transmigrated leukocytes quantified in the field of view) until they are captured by intravascularly adherent platelets.19
In addition to αvb3 and αIIbb3 integrins, VN is able to interact with αvb5, αvb1, αLb2/LFA-1/CD11a, and αMb2/Mac-1/CD11b integrins.3-5 In further in vivo microscopy experiments, we therefore sought to evaluate the effect of these binding partners of VN on the stabi- lization of neutrophil intravascular adhesion. In the postischemic mouse cremaster muscle, antibody block- ade of αv or αIIbb3 integrins did not significantly alter intravascular adhesion of neutrophils to the endothelium. Although antibody blockade of αLb2/LFA-1/CD11a or of αMb2/Mac-1/CD11b as well as of the endothelial b2 inter- action partner ICAM-1/CD54 (Figure 3C) significantly reduced numbers of intravascularly firmly adhered (>30 s) neutrophils, average adhesion times of neutrophils in the microvasculature were not significantly changed (Figure 3D). These data indicate that b2 integrins and ICAM- 1/CD54 are already involved in the induction of neu- trophil intravascular adherence.
Effect of heteromerization of vitronectin with PAI-1 on neutrophil trafficking
PAI-1 is another binding partner of VN that is also involved in neutrophil trafficking.20-24 We here confirm that murine PAI-1 (Online Supplementary Figure S2) binds to VN, but not to fibronectin (FN) (Online Supplementary Figure S3A), ultimately forming VN-PAI-1 heteromers (Online Supplementary Figure S3B) as evidenced by (sandwich) enzyme-linked immunosorbent assay (ELISA) analyses. Moreover, VN-PAI-1 heteromers were found in the periph- eral blood of unstimulated mice whose levels slightly increased upon induction of systemic inflammation (Online Supplementary Figure S4). This increase in circulating VN- PAI-1 heteromers might be due to the release of VN and PAI-1 from the liver, of PAI-1 by the microvascular endothelium, as well as of VN, PAI-1, and pre-formed com- plexes of the single proteins by activated platelets28 in the acute phase of the inflammatory response. Immunostaining and confocal laser scanning microscopy on cremasteric tissue whole mounts further revealed that VN, PAI-1, and adherent neutrophils co-localize on the postischemic venular endothelium (Figure 4A). In order to evaluate the role of complex formation of VN with PAI-1 for the stabilization of neutrophil intravascular adhesion, we performed in vivo microscopy experiments in the postischemic cremaster muscle of PAI-1-deficient mice which received different PAI-1 mutant proteins (Figure 4B). Substitution of PAI-1-deficient mice with active stable PAI- 1 (CPAI) or a PAI-1 mutant protein lacking anti-protease activity (PAI-RR) resulted in significantly higher numbers
of intravascularly firmly adherent (>30 s) neutrophils, sig- nificantly lower numbers of intravascularly shortly adhered (<30 s) neutrophils as well as higher average adhe- sion times of adherent neutrophils as compared to PAI-1- deficient mice substituted by a PAI-1 mutant protein lack- ing its VN binding domain (PAI-QR). In this context, sub- stitution of PAI-deficient mice with PAI-QR induced short adhesion, but not of firm adherence of neutrophils to the microvascular endothelium as compared to vehicle-treated PAI-1-deficient animals (which might be due to PAI-1 bind- ing to its receptor LRP-129 without prior interaction of PAI- 1 and VN). Further, substitution of VN-/- mice with VN-PAI- 1 protein rescued the adhesion defect arising from VN defi- ciency (Online Supplementary Figure S5). These results strengthen our concept that the interaction of VN and PAI- 1 is critical for the stabilization of intravascular adhesion of neutrophils.
In autoperfused flow chambers coated with CD62E/E- selectin and ICAM-1/CD54, an additional coating with VN- PAI-1 (Online Supplementary Figure S3) did not significantly alter rolling and adherence of neutrophils, whereas an addi- tional coating with the chemokine CXCL1/KC induced a significant elevation in numbers of adherent neutrophils (Online Supplementary Figure S4C). These data suggest that chemokines, but not VN-PAI-1 heteromers, are critical for the induction of intravascular adherence of neutrophils.
Effect of vitronectin-plasminogen activator inhibitor-1 heteromers on activation of b2 integrins
in neutrophils
In inflamed tissue, intravascular firm adherence of neu- trophils to the microvascular endothelium is facilitated by interactions of endothelial members of the immunoglobu- lin superfamily (e.g., ICAM-1/CD54) and neutrophil b2 integrins in higher affinity conformation.16,17 Employing multi-channel flow cytometry, exposure to VN-PAI-1, but not to VN, uPA, PAI-1, or VN-uPA significantly enhanced the fluorescence signal for the b2 integrins CD11a/LFA-1 and – to a lesser degree – CD11b/Mac-1 on the surface of neutrophils as compared to unstimulated controls (Online Supplementary Figures S5A and S6).
As a measure of conformational changes of b2 integrins, binding of their interaction partner ICAM-1/CD54 to neu- trophils was analyzed in a next step. Similarly to our pre- vious results for expression of b2 integrins, binding of ICAM-1/CD54 to neutrophils was significantly increased upon exposure to VN-PAI-1, but not upon exposure to VN, uPA, PAI-1, or VN-uPA, as compared to unstimulated con- trols (Figure 5B). Application of receptor associated protein (RAP; blocking members of the LDL receptor family), application of blocking anti-LRP-1 antibodies, or inhibitors of p38 (but not of JNK or ERK1/2) mitogen-activated pro- tein kinases (MAPK) almost completely abolished VN-PAI- 1-elicited ICAM-1/CD54 binding.
In order to specifically evaluate the effect of VN-PAI-1 on conformational changes of b2 integrins, conformation- specific antibodies for the detection of the intermediate (KIM127) or high-affinity conformation (mAb 24) of b2 integrins (which are only available for human integrins) were used (Figure 5C). Upon exposure of VN-PAI-1 to human neutrophils, however, binding of ‘KIM127’ and ‘mAb 24’ was not significantly altered as compared to basal antibody binding in unstimulated controls whereas exposure to the chemokine CXCL1/KC significantly increased ‘KIM127’ and ‘mAb 24’ binding. Collectively,
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haematologica | 2021; 106(10)