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Vitronectin stabilizes leukocyte adhesion
ters on the surface of these immune cells that confluent into large focal adhesions. This leads to the establishment of complex clusters of adhesion and signaling molecules that trigger outside-in signaling, thus representing a criti- cal event in post-adhesion strengthening.45-47 Here, we demonstrate that VN-PAI-1 heteromers substantially reg- ulate the clustering of b2 integrins on the surface of neu- trophils. With respect to our in vivo microscopy observa- tions, these findings conversely suggest that the stabiliza- tion of neutrophil intravascular adherence particularly requires integrin clustering on these immune cells rather than the induction of conformational changes of inte- grins. Inactive integrins exhibit a bent-closed conforma- tion, not extended (E−) and not high affinity (H−), and are thus unable to bind a ligand. In contrast, fully activated integrins (E+H+) bind to ICAM-1/CD54 expressed on opposing cells in trans (e.g., supporting neutrophil arrest). Integrins transitioning from the resting (E−H−) to the fully activated (E+H+) conformation through E+H− or E-H+ con- formations, however, bind to ICAM-1/CD54 on the same cell in cis. In (intravascularly rolling) neutrophils, this tran- sition process prohibits the arrest of these immune cells on the microvascular endothelium.48 Recently, not only E+H+ integrins, but already E-H+ and E+H- integrins were found to form clusters on the surface of neutrophils dur- ing cell activation.49 Since endothelially presented VN- PAI-1 heteromers specifically mediate the stabilization of neutrophil adherence on the microvascular endothelium, these protein complexes are thought to promote the bind- ing of clustering integrins in trans.
Importantly, however, we are not able to exclude that binding of VN to PAI-1 concomitantly interferes with the inhibitory action of PAI-1 on tissue plasminogen activa- tor (tPA), a proteolytic enzyme that has also been demonstrated to promote leukocyte trafficking to the site of inflammation.50 Furthermore, previous in vitro
studies proposed a mechanism in which uPA cleaves the RGD motif of VN thereby reducing pro-adhesive proper- ties of VN in an uPAR-dependent manner. Binding of PAI-1 to uPA in this protein complex effectively inter- feres with these events and subsequently restores the pro-adhesive properties of VN.51 In addition, binding of VN to uPAR has been shown to induce integrin signaling in cells by transmitting a mechanical stimulus to the inte- grin through the plasma membrane ultimately facilitat- ing cell adhesion.52 Since uPAR has recently been demon- strated to be dispensable for the extravasation of neu- trophils in the acute inflammatory response (at least in the model systems employed in the present study),53 we cannot clearly state to which extent these uPAR-depen- dent mechanisms contribute to VN-dependent stabiliza- tion of intravascular adherence of neutrophils in the inflamed microvasculature. Along the same line, fibrin(ogen) has been described to facilitate interactions between leukocyte integrins and endothelial ICAM- 1/CD54.54 In this context, however, fibrin(ogen) has been identified to specifically promote the transmigration of neutrophils to postischemic tissue.50
In order to restore homeostasis, VN-PAI-1 heteromers are cleared from circulation through the scavenger recep- tor LRP-1.25-27 Recently, we have demonstrated that PAI- 1-dependent leukocyte responses in sterile injury are mediated via this receptor protein and subsequent MAPK-dependent intracellular signaling events.29 VN- PAI-1 heteromer-dependent activation of neutrophil b2 integrins might therefore also rely on such molecular processes. In line with this hypothesis, we show that VN-PAI-1 heteromer-elicited surface trafficking of b2 integrins in neutrophils requires LRP-1 and p38 (but not JNK or ERK1/2) MAPK-dependent signaling events. Noteworthy, also selectin- and integrin-triggered signals contribute to the activation of p38 MAPK15 collectively
Figure 7. Graphical synopsis. In inflamed tissue, heteromers of vitronectin (VN) and plasminogen activator inhibitor-1 (PAI-1) are deposited on the activated venular endothelium via glycosaminoglycans (GAG). Subsequently, this protein complex interacts with LRP-1 on intravascularly adhering neutrophils and induces p38 MAPK- dependent intracellular signaling pathways. As a consequence of these events, b2 integrins form clusters on the surface neutrophils ultimately allowing these immune cells to stabilize their adhesion via endothelial ICAM-1/CD54 in trans.
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