F-actin-rich contractile endothelial pores prevent vascular leakage during leukocyte diapedesis through local RhoA signalling

Abstract

During immune surveillance and inflammation, leukocytes exit the vasculature through transient openings in the endothelium without causing plasma leakage. However, the exact mechanisms behind this intriguing phenomenon are still unknown. Here we report that maintenance of endothelial barrier integrity during leukocyte diapedesis requires local endothelial RhoA cycling. Endothelial RhoA depletion in vitro or Rho inhibition in vivo provokes neutrophil-induced vascular leakage that manifests during the physical movement of neutrophils through the endothelial layer. Local RhoA activation initiates the formation of contractile F-actin structures that surround emigrating neutrophils. These structures that surround neutrophil-induced endothelial pores prevent plasma leakage through actomyosin-based pore confinement. Mechanistically, we found that the initiation of RhoA activity involves ICAM-1 and the Rho GEFs Ect2 and LARG. In addition, regulation of actomyosin-based endothelial pore confinement involves ROCK2b, but not ROCK1. Thus, endothelial cells assemble RhoA-controlled contractile F-actin structures around endothelial pores that prevent vascular leakage during leukocyte extravasation.

Leukocyte transendothelial migration: A local affair

“…93 Using a FRET-based DORA RhoA biosensor, we show that RhoA is transiently and locally activated during leukocyte diapedesis and not during the adhesion and crawling phase by inducing Factin-rich rings around the spot where the leukocyte crosses. These rings show asymmetrical phosphorylation of myosin light chain, indicating that these contractile Factin rings serve as elastic straps to limit leakage.…”

“…96,97 In support of a prominent role for ICAM-1, our data show that depletion of ICAM-1 in TNF-a-stimulated endothelial cells resulted in an increase of neutrophil-induced dextran leakage compared to control endothelial cells, although the levels did not reach the levels when RhoA was silenced. 93 PECAM-1 however appeared not to be involved in maintenance of barrier function, as depletion in ECs had no effect on neutrophilinduced dextran leakage and neutrophil transmigration numbers. 93 Homophilic PECAM-1 interactions between ECs and leukocytes are described repeatedly to be important for leukocytes TEM guidance.…”

“…93 PECAM-1 however appeared not to be involved in maintenance of barrier function, as depletion in ECs had no effect on neutrophilinduced dextran leakage and neutrophil transmigration numbers. 93 Homophilic PECAM-1 interactions between ECs and leukocytes are described repeatedly to be important for leukocytes TEM guidance. 98,99 But the fact that we observed no effect of PECAM-1 depletion on both neutrophil TEM efficiency and endothelial barrier function can be explained by the stimulus-dependent role of PECAM-1 in leukocyte diapedesis.…”

“…We recently reported that the GEFs LARG and Ect2 play an important role in maintaining vascular leakage during leukocyte diapedesis. 93 The next section will elaborate on this.…”

“…Our recent data further support this signaling pathway being involved: a GEF screen indicates a role for the RhoAGEFs LARG and Ect2 in this process, activating RhoA that in turn induces myosin-based contraction via ROCK2b and MLCK activation. 93 However, how these F-actin-rich endothelial straps are initiated is not clear. We hypothesize that it all starts with a signaling receptor expressed on the surface of the endothelium that transmits the presence of a transmigrating leukocyte, finally resulting in local RhoA activation.…”