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Cytokeratin Expression and Hyaluronic Acid Production in Cultures of Human Synovial Microvascular Endothelial Cells: Influence of Cytokines and Growth Factors
Author(s) -
Carley William W.,
Szczepanski Andrew,
Gerritsen Mary E.
Publication year - 1996
Publication title -
microcirculation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.793
H-Index - 83
eISSN - 1549-8719
pISSN - 1073-9688
DOI - 10.3109/10739689609148308
Subject(s) - microbiology and biotechnology , endothelial stem cell , biology , hyaluronic acid , cytokeratin , chemistry , immunology , in vitro , immunohistochemistry , biochemistry , genetics
Objectives: 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 europeaus 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 K4.62 (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 punctate cytoplasmic vesicular structures. Treatment of HSE cells with interleukin‐1α (IL‐1α) or acidic fibroblast growth factor (aFGF), but not tumor necrosis factor (TNFα), 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‐1α or aFGF. HA production was not significantly increased when HSE cells were incubated with TNFα, IL‐4 or interferon‐γ. 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.