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Blood, Vol. 108, Issue 13, 4214-4222, December 15, 2006
CD157 plays a pivotal role in neutrophil transendothelial migration
Blood Ortolan et al.
108: 4214
Supplemental materials for: Ortolan et al, Vol 108, Issue 13, 4214-4222
Files in this Data Supplement:
- Figure S1. Confocal microscopy analysis of CD157 expression on HUVECs (PDF, 2.31 MB) -
Confluent HUVEC monolayers were stained, and the expression of CD157, CD54, and CD31 was analyzed by laser-scanning confocal microscopy. Cells were imaged using a ×60 oil immersion objective (1.4 NA; bar represents 30 µm). Sections at basal (0 µm), intermediate (1.25 µm), and apical (2.5 µm) levels are shown.
- Figure S2. Quantification of neutrophil trajectory length (PDF, 489 KB) -
PMNs were seeded on confluent HUVEC monolayers, and images were captured every 10 seconds using real-time microscopy. The routes of transmigrating neutrophils treated with anti-CD157, an isotype-matched or anti-CD31 mAb, were measured using the TrackIt! software (Olympus Biosystems). To measure the trajectory length of each migrating neutrophil, the cell centroid was tracked from the moment of cell-to-cell contact to the beginning of diapedesis. The trajectory lengths are plotted. *P < .001.
- Table S1. Quantification of localization of PMNs on the apical side of HUVEC (JPG, 52.5 KB)
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PMNs from healthy donors were labeled with CFSE, incubated with anti-CD157, anti-CD31, or anti-HLA class I mAb, and seeded on TNF- −activated HUVEC monolayers grown on collagen. After 1 hour, samples were stained with silver nitrate to identify endothelial cell junctions and observed simultaneously by DIC and fluorescence confocal microscopy.
- Video S1. Live imaging of PMN migration on TNF-α–activated HUVECs (MOV, 1.07 MB)
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PMNs were seeded on confluent HUVEC monolayers, and images were captured every 10 seconds. On the basis of their migratory properties, PMNs can be divided into three subsets: (1) PMNs able to transmigrate, (2) PMNs unable to transmigrate for the entire assay, and (3) static PMNs that adhere to the apical surface of the HUVECs, extend some pseudopods, but do not polarize and do not migrate.
In this video, PMNs adhere to the apical surface of the HUVECs and transmigrate underneath the HUVEC monolayer within 5 minutes. Several transmigrated cells reach the field during the time of observation. Transmigrated PMNs appear phase dark and are flatter and larger than untransmigrated cells. One PMN reaches the field of observation after ∼16 minutes and walks on the apical surface of the HUVECs without transmigrating by the end of the assay. One PMN (bottom right corner of the field) does not polarize and is unable to migrate.
- Video S2. Live imaging of PMN migration on TNF-α–activated HUVECs in the presence of anti-CD157 mAb (MOV, 1.07 MB)
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The majority of PMNs treated with anti-CD157 mAb migrate on the surface of HUVECs but do not transmigrate. They display a migratory phenotype with a very dynamic leading edge showing continuous and rapid extension and retraction of pseudopods. They cross over several junctions without perceiving their presence. One PMN crossing the HUVEC monolayer and some static cells can also be observed in this movie.
- Video S3. Live imaging of PMN migration on TNF-α–activated HUVECs in the presence of anti-CD18 mAb (MOV, 823 KB)
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PMNs treated with anti-CD18 mAb do not adhere firmly to HUVECs. They display a polarized phenotype, adhere to the HUVECs only through the uropod but not with the cell body, and wave in several different directions. No transmigrating cells are appreciable.
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