Cytokeratin expression and hyaluronic acid production in cultures of human synovial microvascular endothelial cells: influence of cytokines and growth factors.
OBJECTIVE
To isolate and characterize human synovial endothelial cells and to determine the effects of cytokines and fibroblast growth factor on human synovial endothelial (HSE) cell hyaluronic acid production.


METHODS
Endothelial cells were isolated from primary cultures of human synovial cells by fluorescent activated cell sorting based on the incorporation of a fluorescent derivative of acetylated low-density lipoprotein (DiI-Ac-LDL). Identity of endothelial cells was confirmed by positive immunostaining for von Willebrand factor (vWf), cytokeratins, endoglin, and reactivity with the lectin ulex europeans agglutinin (UEA). Hyaluronic acid production was measured by a radioligand-binding assay.


RESULTS
HSE cells were isolated and maintained in long-term culture. The identity of the cultured cells as endothelial was based on uniform uptake of a (DiI-Ac-LDL), immunoreactivity for vWf, and endoglin and the binding of the lectin UEA. In addition, small blood vessels in the synovium were stained selectively with anticytokeratin antibodies K462 (cytokeratin 19 specific) and K8.13 (reactive for cytokines 1, 5, 6, 7, 8, 10, 11, and 18). Isolated HSE cells also demonstrated immunoreactivity with these cytokeratin antibodies. The cytokeratins identified by the monoclonal antibody clone K8.13 demonstrated a diffuse, fibrillar staining pattern. The cytokeratin distribution revealed with monoclonal antibody K4.62 (cytokeratin 19) was also fibrillar; however, the majority of cells also demonstrated numerous punctuate cytoplasmic vesicular structures. Treatment of HSE cells with interleukin-1 alpha (IL-1 alpha) or acidic fibroblasts growth factor (aFGF), but not tumor necrosis factor (TNF alpha), dramatically reduced the vesicular structures staining with the K4.62 antibody. HSE cells produced hyaluronic acid (HA) at a constitutive rate of 200-800 ng/10(5) cells/24 h, which could be upregulated when the cells were incubated with either IL-1 alpha or aFGF. HA production was not significantly increased when HSE cells were incubated with TNF alpha, IL-4 or interferon-gamma.


CONCLUSIONS
Synovial microvascular endothelial cells produce and secrete HA and endothelial HA secretion is upregulated by IL-1 and aFGF. IL-1 and aFGF also reduce the number of vesicular-like structures immunoreactive with a monoclonal antibody to cytokeratin 19. These studies suggest that cytokine stimulation of local endothelial secretion and/or accumulation of HA may influence leukocyte adhesion to the synovial endothelium.
Carley
W W
WW
Institute for Bone and Joint Disease and Cancer, Bayer Corporation, West Haven, Connecticut 06516, USA.
Szczepanski
A
A
Gerritsen
M E
ME
D000328:Adult; D000911:Antibodies, Monoclonal / Q000276:immunology; D015703:Antigens, CD; D015415:Biomarkers; D002478:Cells, Cultured; D016207:Cytokines / Q000494:pharmacology*; D000071063:Endoglin; D004730:Endothelium, Vascular / Q000378:metabolism*; D005260:Female; D016220:Fibroblast Growth Factor 1 / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D006133:Growth Substances / Q000494:pharmacology*; D006801:Humans; D006820:Hyaluronic Acid / Q000096:biosynthesis*; D007375:Interleukin-1 / Q000494:pharmacology; D007633:Keratins / Q000096:biosynthesis*; D008077:Lipoproteins, LDL / Q000378:metabolism; D008833:Microcirculation; D008875:Middle Aged; D011956:Receptors, Cell Surface; D013583:Synovial Membrane / Q000098:blood supply*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D019010:Vascular Cell Adhesion Molecule-1 / Q000032:analysis; D014841:von Willebrand Factor / Q000032:analysis
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D015415:Biomarkers; D016207:Cytokines; C064206:ENG protein, human; D000071063:Endoglin; D005456:Fluorescent Dyes; D006133:Growth Substances; D007375:Interleukin-1; D008077:Lipoproteins, LDL; D011956:Receptors, Cell Surface; D014409:Tumor Necrosis Factor-alpha; D019010:Vascular Cell Adhesion Molecule-1; D014841:von Willebrand Factor; D016220:Fibroblast Growth Factor 1; D007633:Keratins; D006820:Hyaluronic Acid