A three-dimensional model system to study the interactions between human leukocytes and endothelial cells.
Leukocyte adhesion to endothelial cells and migration into the subendothelial matrix was studied with a three-dimensional model system, consisting of human endothelial cells cultured on a loose collagen matrix. We developed a new method to separate the endothelial cell monolayer and adhering leukocytes, from the subendothelial matrix, allowing simultaneous analysis of leukocyte adhesion and transendothelial migration. Monocytes adhered more avidly to untreated endothelial cells than did neutrophils (2.5 +/- 0.3 vs. 1.0 +/- 0.2 leukocytes per endothelial cell). Only a small fraction (10%-20%) of these leukocytes migrated into the subendothelium. Pretreatment of endothelial cells with interleukin 1 (IL 1) enhanced adhesion (20%), but not migration of monocytes. In contrast, neutrophil adhesion was markedly and in a time-dependent manner increased by IL 1 treatment (i.e. 200% after 6 h and 110% after 24 h of IL 1 treatment). Moreover, IL 1 pretreatment enhanced neutrophil migration twofold. Activation of leukocytes with formyl-methionyl-leucyl-phenylalanine (fMLP) enhanced both monocyte and neutrophil adhesion, but did not affect leukocyte migration. Under all conditions, monocyte adhesion was only partly (30%-40%) inhibited by monoclonal antibodies (mAb) against the common beta subunit of the leukocyte-cell adhesion molecules (LeuCAM: CD18) and 25%-30% by mAb against the alpha subunit of LFA-1 (CD11a). In contrast, mAb against the alpha subunits of Mac-1 (CD11b) and p150.95 (CD11c) were hardly effective. fMLP-mediated neutrophil adhesion was reduced to below baseline levels by anti-LeuCAM (CD18) mAb, whereas the LeuCAM contribution in IL 1-mediated neutrophil adhesion was less pronounced and varied in time. IL 1-mediated neutrophil migration, however, was completely blocked by anti-LeuCAM mAb. fMLP-mediated neutrophil adhesion was inhibited by mAb against the alpha subunits of Mac, while mAb against the alpha subunits of LFA-1 and Mac-1 both reduced IL 1-mediated adherence. In summary, we describe a novel leukocyte adhesion/migration method and demonstrate that the contribution of the LeuCAM complex in leukocyte-endothelium interaction varies depending on cell type and stimulus used.
Hakkert
B C
BC
Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam.
Rentenaar
J M
JM
Van Aken
W G
WG
Roos
D
D
Van Mourik
J A
JA
D000911:Antibodies, Monoclonal; D015703:Antigens, CD / Q000502:physiology; D000943:Antigens, Differentiation / Q000502:physiology; D018845:CD11 Antigens; D018821:CD18 Antigens; D002448:Cell Adhesion; D002465:Cell Movement; D002478:Cells, Cultured; D003094:Collagen; D004730:Endothelium, Vascular / Q000166:cytology*; D005109:Extracellular Matrix / Q000502:physiology; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology; D007962:Leukocytes / Q000166:cytology*; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D016028:Receptors, Leukocyte-Adhesion / Q000502:physiology; D013997:Time Factors
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D000943:Antigens, Differentiation; D018845:CD11 Antigens; D018821:CD18 Antigens; D007375:Interleukin-1; D016028:Receptors, Leukocyte-Adhesion; D009240:N-Formylmethionine Leucyl-Phenylalanine; D003094:Collagen