Page 122 - 2021_10-Haematologica-web

G. Zuchtriegel et al.
required for the induction of neutrophil responses. Moreover, we found that extravasation of classical/inflammatory monocytes remained unaffected by VN deficiency collectively indicating that VN particu- larly mediates the trafficking of neutrophils to inflamed tissue. This might be explained by higher expression of the scavenger receptor LRP-1 (which serves as a receptor of the VN binding partner PAI-1) on activated neutrophils as compared to activated classical monocytes, hence extending previous observations on the involvement of VN in leukocyte recruitment under inflammatory condi- tions.34-38
With respect to the deposition of VN on the surface of postcapillary venules in inflamed tissue, we hypothesized that this glycoprotein contributes to the regulation of intravascular interactions of neutrophils in the extravasa- tion process of these immune cells. Here, we show that VN deficiency does not alter the rolling behavior of neu- trophils in inflamed postcapillary venules, suggesting that VN does not influence the ‚selectin-dependent phase‘ of the neutrophil extravasation process. Most interestingly, however, the average adherence time of a neutrophil to the endothelial surface was significantly lower in VN-/- mice than in WT controls. Furthermore, adoptively trans- ferred neutrophils from WT donor mice accumulated less efficiently in the inflamed tissue of VN-/- recipient mice as compared to neutrophils isolated from VN-/- or WT donor mice transferred into WT recipient animals. Hence, our findings clearly demonstrate that endothelially deposited VN stabilizes intravascular adherence of neutrophils on the microvascular endothelium in inflamed tissue. To our knowledge, this is the first description of a protein specif- ically regulating this critical step in the extravasation cas- cade of neutrophils. Further, our observations indicate that stable intravascular adherence of neutrophils is pre- requisite for (and does not interfere with) the subsequent transmigration of these immune cells into the perivascu- lar space, a process that is facilitated by sequential het- erophilic (e.g., between neutrophil LFA-1/CD11a and endothelial ICAM-2/CD102 or JAM-A as well as between neutrophil Mac-1/CD11b and ICAM-2/CD102 or JAM-C) and homophilic (between neutrophil and endothelial PECAM-1/CD31 or CD99) molecular interactions between neutrophils and endothelial cells as well as by endothelial molecules such as VE-cadherin, ESAM, or CD99L2.11-14
In addition to interactions with endothelial cells, the interplay between leukocytes and platelets considerably contributes to the extravasation of leukocytes.39-42 In this context, platelets have recently been demonstrated to guide intravascularly crawling neutrophils and mono- cytes to their site of transmigration into the interstitial tis- sue.19 Since VN is able to bind to platelets through αvb3 and αIIbb3 integrins via its RGD motif43,44 these cellular blood components might also participate in VN-depen- dent neutrophil trafficking. In further experiments, how- ever, VN deficiency neither altered intravascular adher- ence of platelets nor interactions of intravascularly adher- ent platelets and neutrophils. In line with these observa- tions, platelet depletion did also not significantly change intravascular adhesion times of neutrophils in inflamed tissue collectively indicating that platelets do not con- tribute to the stabilization of neutrophil adherence in the microvasculature.
Besides platelet integrins, integrins expressed on the sur-
face of leukocytes including αvb3, αvb5, αvb1, αLb2, and αMb2 represent potential interaction partners of VN.3-5 Similar to our results for platelet integrins, however, blockade of these integrins did not significantly alter the average time of neutrophils resting on the endothelial sur- face of postcapillary venules in the inflamed mouse cre- master muscle.
Beyond their established role in fibrinolysis, the com- ponents of the fibrinolytic system are increasingly recog- nized as mediators of immune cell migration.20-24 Recently, PAI-1 has been implicated in leukocyte trafficking to the site of inflammation by regulating intravascular adher- ence and (subsequent) transmigration of these immune cells.29 Since VN is capable of binding to PAI-1 via its somatomedin B domain (thereby extending the half-life of this protease inhibitor in fibrinolysis,3-5) heteromeriza- tion of VN with PAI-1 might promote intravascular adher- ence of neutrophils to the endothelium in inflamed tissue. In order to prove this hypothesis, we reconstituted PAI-1- deficient animals with different PAI-1 mutant proteins. Substitution with active stable PAI-1 or a PAI-1 mutant protein lacking anti-protease activity completely rescued the adhesion and extravasation defect of neutrophils observed in PAI-1-deficient mice. Substitution of PAI-1- deficient animals with a PAI-1 mutant protein lacking its VN binding domain, however, significantly diminished the average intravascular adhesion time of neutrophils as compared to active stable PAI-1-substituted mice resem- bling our observations in VN-deficient animals. Conversely, substitution of PAI-1-deficient mice with this non-VN-binding PAI-1 mutant protein induced short, but not firm adherence of neutrophils to the inflamed vessel wall which might be due to VN-independent binding of PAI-1 to its receptor LRP-1.29 Finally, substitution of VN- deficient animals with VN-PAI-1 heteromer protein com- pletely rescued the adhesion defect of neutrophils observed in animals lacking VN. Consequently, complex formation of VN with PAI-1 is needed for the stabiliza- tion of intravascular adherence of neutrophils in the acute inflammatory response.
Intravascular firm adherence of leukocytes to microvas- cular endothelial cells is facilitated by interactions between endothelially expressed members of the immunoglobulin superfamily (e.g., ICAM-1/CD54, VCAM-1/CD106) and leukocyte b2 integrins in higher affinity conformations.11-14 We therefore proposed that VN-PAI-1 heteromers stabilize intravascular adhesion of neutrophils in the inflamed microvasculature by activat- ing neutrophil b2 integrins. In our experiments, endothe- lial-bound VN and PAI-1 were identified to co-localize with intravascularly adherent neutrophils pointing to interactions of complexes of VN and PAI-1 with adhering neutrophils in the inflamed venular microvasculature. Importantly, however, exposure of neutrophils to the VN- PAI-1 complex (but not to VN, PAI-1, and uPA alone or to the VN-uPA complex) induced a significant increase in the expression of CD11a/LFA-1 and – to a lesser degree – of CD11b/Mac-1 on their cell surface, but did not further promote affinity changes in these b2 integrins. Although the induction of conformational changes in integrins enhances their affinity for their individual binding part- ners, the formation of multiple bonds to multivalent sub- strates in a process termed ‘integrin clustering’ is thought to be prerequisite for sustained cell adhesion. In leuko- cyte adhesion, integrins initially form transient microclus-
2650
haematologica | 2021; 106(10)