Delay of migrating leukocytes by the basement membrane deposited by endothelial cells in long-term culture.
We investigated the migration of human leukocytes through endothelial cells (EC), and particularly their underlying basement membrane (BM). EC were cultured for 20days on 3μm-pore filters or collagen gels to form a distinct BM, and then treated with tumour necrosis factor-α, interleukin-1β or interferon-γ. Neutrophil migration through the cytokine-treated EC and BM was delayed for 20-day compared to 4-day cultures. The BM alone obstructed chemotaxis of neutrophils, and if fresh EC were briefly cultured on stripped BM, there was again a hold-up in migration. In studies with lymphocytes and monocytes, we could detect little hold-up of migration for 20-day versus 4-day cultures, in either the filter- or gel-based models. Direct microscopic observations showed that BM also held-up neutrophil migration under conditions of flow. Treatment of upper and/or lower compartments of filters with antibodies against integrins, showed that neutrophil migration through the endothelial monolayer was dependent on β(2)-integrins, but not β1- or β(3)-integrins. Migration from the subendothelial compartment was supported by β1- and β(2)-integrins for all cultures, but blockade of β(3)-integrin only inhibited migration effectively for 20-day cultures. Flow cytometry indicated that there was no net increase in expression of β1- or β3-integrins during neutrophil migration, and that their specific subendothelial function was likely dependent on turnover of integrins during migration. These studies show that BM is a distinct barrier to migration of human neutrophils, and that β(3)-integrins are particularly important in crossing this barrier. The lesser effect of BM on lymphocytes and monocytes supports the concept that crossing the BM is a separate, leukocyte-specific, regulated step in migration.
Centre for Cardiovascular Sciences and MRC Centre for Immune Regulation, College of Medical and Dental Sciences, The University of Birmingham, Birmingham B15 2TT, UK.
Victoria J
VJ
Burton
Lynn M
LM
Butler
Helen M
HM
McGettrick
Phil C
PC
Stone
Hannah C
HC
Jeffery
Caroline O
CO
Savage
G Ed
GE
Rainger
Gerard B
GB
Nash
D001485:Basement Membrane / Q000378:metabolism*; D018821:CD18 Antigens / Q000032:analysis; D018929:Cell Culture Techniques; D002634:Chemotaxis, Leukocyte; D042783:Endothelial Cells / Q000166:cytology* / Q000648:ultrastructure; D006801:Humans; D019012:Integrin beta1 / Q000032:analysis; D039661:Integrin beta3 / Q000032:analysis; D007962:Leukocytes / Q000166:cytology* / Q000502:physiology; D008214:Lymphocytes / Q000166:cytology; D009000:Monocytes / Q000166:cytology; D009504:Neutrophils / Q000166:cytology / Q000502:physiology; D013995:Time
D018821:CD18 Antigens; D019012:Integrin beta1; D039661:Integrin beta3