Immune Netw.  2013 Jun;13(3):102-106. 10.4110/in.2013.13.3.102.

Extravasating Neutrophil-derived Microparticles Preserve Vascular Barrier Function in Inflamed Tissue

• Lim K
• Sumagin R
• Hyun YM

• KMID: 2150773
• DOI: http://doi.org/10.4110/in.2013.13.3.102

Abstract

Emerging evidence suggests that gap formation and opening of the endothelial junctions during leukocyte extravasation is actively controlled to maintain the integrity of the vascular barrier. While the role for endothelial cells to this process has been well defined, it is not clear whether leukocytes are also actively contributing to endothelial barrier function. We have recently showed that extravasating leukocytes deposit microparticles on the subendothelium during the late stages of extravasation, which is LFA-1 dependent. Using multiphotonintravital microscopy (MP-IVM) of mouse cremaster muscle vessels in the current work, we show that microparticle formation and deposition maintains the integrity of the microvascular barrier during leukocyte extravasation. Inhibition of neutrophil-derived microparticle formation resulted in dramatically increased vascular leakage. These findings suggest that deposition of microparticles during neutrophil extravasation is essential for maintaining endothelial barrier function and may result in temporal difference between neutrophil extravasation and an increase in vascular leakage.

Keyword

Vascular barrier function; Leukocyte extravasation; Neutrophils; Monocytes; Microparticles

MeSH Terms

Animals
Endothelial Cells
Leukocytes
Lymphocyte Function-Associated Antigen-1
Mice
Microscopy
Monocytes
Muscles
Neutrophils
Lymphocyte Function-Associated Antigen-1

Figure

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  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 or the CD11a blocking Ab and in unstimulated cremaster. The ratio of CD18+ microparticles over CD31+ cells was calculated. MP, microparticles. *p=0.0004. **p=0.0006. (B) Vascular leakage was measured and calculated using MP-IVM from CXCL2-stimulated venules in the superfusion of control IgG or CD11a blocking Ab. Vascular leakage was averaged over five points measured outside of the venules every 30 sec based on the initial Texas Red intensity. *p<0.0001. a.u., Arbitrary unit. (C) A representative image of vascular leakage (Texas Red dextran intensity) after extravascular inhibition of LFA-1 showed significant increase in fluorescence compared to IgG. The still images were taken at 30 min of treatment with CXCL2 and the superfusion of the IgG or CD11a blocking Ab. CD18-mCFP mice were i.v. injected with Texas Red dextran, and the cremaster venules were imaged. Red: Texas Red dextran, Cyan: CD18+ leukocytes. (D & E) The cremaster venules of CD18-mCFP mice were stimulated with CXCL2 (1 nM), and the adherent cells in the vasculature were then counted before (pre-Ab: white) and 30 min after (post-Ab: black) superfusion with either the CD11a blocking Ab or control IgG (D). The extravasating leukocytes were counted in the interstitial regions surrounding stimulated venules with LFA-1 Ab or control IgG added to the superfusate (E). The number of extravasating cells was calculated per 100 µm for 30 min.

• 

  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 or IgG control. Vascular leakage was averaged over 5 points measured outside of the venules every 30 sec based on the initial Texas Red intensity. *p<0.05, **p<0.005. The vascular leakage was then compared under each condition 30 min after stimulation. a.u., Arbitrary unit. Unstim., Unstimulated; No depl., No depletion; Neu. depl., Neutrophil depletion by i.v. injection of anti-Ly-6G Ab; Infl. Mo. depl., Inflammatory monocyte depletion in CCR2 KO mice; Neu./Infl. Mo. depl., Neutrophil and inflammatory monocyte depletion by i.v. injection of anti-Ly-6G/Ly-6C (Gr1) Ab; Mo. depl., Monocyte depletion by i.v. injection of clodronate liposomes.

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