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Locomotion of monocytes on endothelium is a critical step during extravasation

Abstract

Monocytes, like all leukocytes, undergo a series of sequential steps during extravasation from blood into tissues: tethering, rolling, adhesion and diapedesis. We have discovered an essential step, which we call locomotion, in which the monocyte moves from a site of firm adhesion to the nearest junction to begin diapedesis. Blocking CD11a-CD18 and CD11b-CD18 on human monocytes or adhesion molecules ICAM-1 and ICAM-2 on endothelial cells prevented the monocytes from reaching junctions. The blocked monocytes spun in circles as if they were unable to direct their movement despite being able to adhere and polarize normally. This step fills a gap in the paradigm of extravasation as a multistep process.

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Figure 1: Effects of blocking antibodies on monocyte adhesion and transendothelial migration.
Figure 2: Localization of monocytes away from junctions after blocking of CD11-CD18 integrins or ICAM-1 and ICAM-2.
Figure 3: Diapedesis of monocytes in the absence of antibody blockade on unactivated HUVECs.
Figure 4: Diapedesis of monocytes in the absence of antibody blockade on IL-1β-activated HUVECs.
Figure 5: Montage of a search pirouette after CD18 blockade.
Figure 6: Multiple search pirouettes after ICAM blockade.
Figure 7: Quantification of movement patterns by live microscopy.

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Acknowledgements

We acknowledge R. Liebman for technical support and tissue culture, and L. Pierini for critical review of this manuscript. Supported by National Institutes of Health HL46849 (W.A.M.), HL64774 (W.A.M.), and HL10311 (A.R.S.) and in part by the Charles H. Revson Foundation (A.R.S. and Z.M.).

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Correspondence to Alan R Schenkel.

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Supplementary information

Supplementary Fig. 1 (PDF 90 kb)

Supplementary Video 1

Live imaging of monocytes locomotion during extravasation. Freshly prepared monocytes were co-cultured on confluent HUVEC monolayers and images captured every 20 seconds to make QuicktimeTM movies. In this movie a single monocytes crosses over a endothelial junction then becomes phase dark after reaching a endothelial cell junction and crossing underneath the HUVEC monolayer. (MOV 3524 kb)

Supplementary Video 2

Three monocytes on IL-1b treated HUVEC. Two cells reach the nearest tri-cellular junction and cross underneath the HUVEC monolayer immediately, while a third continues locomotion as the 20 minutes assay ends. (MOV 1113 kb)

Supplementary Video 3

A monocyte when CD18 is blocked spins in one place, a classic pirouette,for the entire 20 minutes assay. (MOV 816 kb)

Supplementary Video 4

On IL-b treated HUVEC, ICAM-1 blockade results in a monocyte performing a pirouette, crossing over a junction,doing a second pirouette, and moving along the apical surface of the endothelium a second time. (MOV 1360 kb)

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Schenkel, A., Mamdouh, Z. & Muller, W. Locomotion of monocytes on endothelium is a critical step during extravasation. Nat Immunol 5, 393–400 (2004). https://doi.org/10.1038/ni1051

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