Expression of chemokine by human coronary-artery and umbilical-vein endothelial cells and its regulation by inflammatory cytokines.
BACKGROUND
Activation of the endothelium is a critical event in the process of inflammation and is associated with the production of chemokines.


OBJECTIVE
To evaluate the proinflammatory cytokine-induced chemokine repertoire of human coronary-artery endothelial cells (HCAEC) both at the messenger RNA (mRNA) level and at protein level in direct comparison with that of human umbilical-vein endothelial cells (HUVEC).


METHODS
Human coronary-artery and human umbilical-vein endothelial cells were obtained commercially and experimental data were derived from cell cultures between passage levels 3 through 6. Supernatant fluids from cytokine [tumor necrosis factor-alpha (TNF-alpha), interleukin-1-alpha, and anti-TNF R55] stimulated endothelial cell cultures were used to study chemokine release. Sandwiched ELISA assays, obtained commercially, were used to estimate cell culture supernatant fluid levels of the selected chemokines: monocytic chemotactic protein-1, regulated upon activated normal T cells expressed and secreted, interleukin-8, transforming growth factor-beta-2 (TGF-beta2), and gamma interferon protein-10. Expression of messenger RNA was determined using selected labeled riboprobes (32P UTP) in a ribonuclease protection assay using total cellular mRNA.


RESULTS
Upon in-vitro stimulation with TNF-alpha and interleukin-1-alpha, production of regulated-upon-activated-normal-T-cells expressed and secreted (RANTES) protein by HCAEC was significantly increased relative to that by HUVEC, the greatest effect being found with interleukin-1-alpha. The opposite effect, however, was noted for levels of monocytic-chemotactic-protein-1 protein, which were detected in HUVEC at significantly higher levels than they were in HCAEC challenged by those cytokines. Production of gamma interferon-inducible protein-10 (gammaIP-10) by HUVEC was induced by TNF-alpha and interleukin-1-alpha, whereas only a modest induction by interleukin-1-alpha was seen in HCAEC. TGF-beta-2 protein was constitutively expressed in HCAEC but not in HUVEC. Expression of mRNA was analyzed by the ribonuclease-protectionassay. RANTES mRNA was expressed in HCAEC from 3 h through 48 h after treatment with TNF-alpha, whereas only a modest induction of RANTES was expressed in HUVEC 24 h and 48 h after treatment with TNF-alpha. Monocytic-chemotactic-protein-1 mRNA was constitutively expressed by both types of cell, but the basal levels in HCAEC was significantly higher than in HUVEC. HCAEC constitutively expressed both TGF-beta-1 and TGF-beta-2 mRNA, whereas HUVEC constitutively expressed TGF-beta-1 only.


CONCLUSIONS
Our data indicate that HCAEC and HUVEC express chemokines differently, which could contribute to or influence site-specific recruitment of subsets of leukocytes.
Briones
M A
MA
Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia, USA.
Phillips
D J
DJ
Renshaw
M A
MA
Hooper
W C
WC
D002478:Cells, Cultured; D018925:Chemokines / Q000378:metabolism* / Q000502:physiology*; D003327:Coronary Disease / Q000378:metabolism* / Q000503:physiopathology*; D003331:Coronary Vessels / Q000378:metabolism* / Q000503:physiopathology*; D016207:Cytokines / Q000378:metabolism* / Q000502:physiology*; D004730:Endothelium, Vascular / Q000378:metabolism* / Q000503:physiopathology*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D066298:In Vitro Techniques; D012333:RNA, Messenger / Q000502:physiology*; D014471:Umbilical Veins / Q000378:metabolism* / Q000503:physiopathology*
D018925:Chemokines; D016207:Cytokines; D012333:RNA, Messenger