We have recently showed that leukocytes become extremely elongated and deposit microparticles on the subendothelium during extravasation through inflamed vessels in an LFA-1-dependent manner (5
). Hereby, we hypothesized that leukocyte-derived microparticles play a protective role, and prevent vascular leakage during leukocyte extravasation, by either forming a physical barrier on the subendothelium or inducing signaling to regulate vascular barrier function.
To investigate this hypothesis, we first quantified the number of β2
) containing microparticles formed per endothelial cell of the vasculature in chemokines-stimulated tissue. The number of CD18+
microparticles isolated from chemokines-stimulated cremaster tissue was normalized to the total number of CD31+
cells isolated from the same tissue. Superfusion of LFA-1 blocking Ab over the extravascular regions of the cremaster muscle dramatically decreased microparticle formation, compared to control IgG superfusion (). The physical properties of microparticles derived from migrating leukocytes were determined to be <1 µm using flow cytometry (Fig. S1
). Therefore, we further examined the effects of microparticle formation on vascular barrier during CXCL2-induced neutrophil migration across the vascular wall. We calculated the vascular leakage in exteriorized mouse cremaster venules by measuring Texas Red fluorescence intensity in the interstitial area adjacent to blood vessels of CD18-mCFP mice, which were i.v. injected with Texas Red dextran. Inhibition of the LFA-dependent microparticle formation by superfusion of the LFA-1 blocking Ab significantly increased dextran leakage (~4.2 fold), compared to the unstimulated condition (). We confirmed that both intravascular leukocyte adhesion and transendothelial migration were not affected by extravascular inhibition of LFA-1 function (). These findings suggest that the observed increase in dextran leakage under this condition was specifically due to decreased formation and deposition of microparticles on the subendothelium.
Figure 1 Microparticles derived from extravasating leukocytes regulate vascular leakage in an LFA-1-dependent manner. (A) The numbers of CD18+ microparticles and CD31+ cells were determined in chemokines-stimulated cremaster superfused with either the IgG control (more ...)
Neutrophil extravasation is often linked to vascular leakage (16
) leading to tissue injury in inflammatory models (19
). Therefore, we expanded our study to determine which subset of circulating leukocytes was primarily responsible for the protective effect on vascular barrier function through deposition of microparticles during extravasation. To examine this, vascular leakage during leukocyte extravasation (induced by fMLP) was further assessed after specific depletion of both/either intravascular neutrophils and/or monocytes, with and without inhibition of LFA-1 (by extravascular superfusion of LFA-1 blocking Ab). Consistent with previous studies (21
), Ab-mediated neutrophil depletion showed complete preservation of vascular barrier function independently of inhibition of LFA-1 mediated microparticle formation (). These results suggest that neutrophil extravasation is accompanied by increased vascular leakage and further imply that microparticle deposition by extravasating neutrophils affects vascular leakage. In contrast, inhibition of microparticle formation after depletion of intravascular inflammatory monocytes failed to prevent vascular leakage in stimulated vessels (). Consistently, depletion of both neutrophils and inflammatory monocytes also prevented the increase in vascular leakage during leukocyte extravasation (). Therefore, during leukocyte extravasation in inflammation, neutrophilderived microparticles play an important role in maintaining vascular barrier function. Interestingly, depletion of intravascular monocytes using clodronate liposomes also prevented vascular leakage regardless of extravascular inhibition of LFA-1 or not (). As monocytes have been shown to facilitate neutrophil trafficking (12
), it is possible that the clodronate liposome-mediated depletion of monocytes may decrease vascular leakage by inhibiting transendothelial migration of neutrophils leading to reduced microparticle formation by extravasating neutrophils. In conclusion, these findings provide new evidence for a functional role of neutrophil-derived microparticles in preserving endothelial barrier integrity during leukocyte extravasation.
Figure 2 Microparticles derived from extravasating neutrophils regulate vascular barrier function. Vascular leakage was measured using MP-IVM from fMLP-stimulated venules of the leukocyte subset depleted mice after extravascular superfusion of either CD11a Ab (more ...)