S100A8, S100A9 and the S100A8/A9 heterodimer complex specifically bind to human endothelial cells: identification and characterization of ligands for the myeloid-related proteins S100A9 and S100A8/A9 on human dermal microvascular endothelial cell line-1 cells.
The natural ligands of the S100 EF hand proteins S100A8 and A9 [myeloid-related proteins 8 and 14] have long been searched for in order to further the understanding of the role of the S100A8/A9-expressing monocyte subpopulation in progressing inflammatory processes. We demonstrate that S100A8, S100A9 and the S100A8/A9 heterodimeric complex bind to human dermal microvascular endothelial cell line (HMEC)-1 with an increasing binding capacity progressing from S100A8 < or = S100A9 < or = S100A8/A9. Similar results were obtained in the apolipoprotein E knockout mouse model, where preferably recombinant S100A9 but no S100A8 bound to the endothelium of the aorta ascendens. The binding of the S100A8/A9 heterodimer complex to activated HMEC-1 is specific as demonstrated by a dose-responding and satiable binding curve and the competition of FITC-labeled versus unlabeled protein. The protein character of the binding site was proven by treatment with trypsin. S100A8/A9 binding to HMEC-1 is inducible by lipopolysaccharide and tumor necrosis factor-alpha, and in the presence of calcium. A 163-kDa protein was isolated from a cell lysate of activated HMEC-1 cells using an affinity-chromatography protocol. The endothelial cell-associated ligand proteins isolated by the use of the S100A9 monomer and the S100A8/A9 dimer were subjected to mass spectrometry for protein identification. Clearly, alpha(2)-macroglobulin was identified as a binding partner for the S100A9 monomer, whereas no protein could be identified from the database for the ligand of the S100A8/A9 dimer.
Eue
Ines
I
PAN Clinic, Zeppelinstrasse 1, 50667 Köln, Germany. Ines.Eue@aol.com
König
Simone
S
Pior
Jolanthe
J
Sorg
Clemens
C
D000818:Animals; D000943:Antigens, Differentiation / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D040501:Calgranulin A; D040502:Calgranulin B; D002196:Capillaries / Q000166:cytology; D002460:Cell Line; D002478:Cells, Cultured; D020405:Dermis / Q000098:blood supply* / Q000166:cytology; D019281:Dimerization; D004730:Endothelium, Vascular / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D046911:Macromolecular Substances; D051379:Mice; D008815:Mice, Inbred Strains; D022423:Myeloid Cells / Q000276:immunology; D009418:S100 Proteins / Q000378:metabolism*
D000943:Antigens, Differentiation; D002135:Calcium-Binding Proteins; D040501:Calgranulin A; D040502:Calgranulin B; D008024:Ligands; D046911:Macromolecular Substances; D009418:S100 Proteins