Interferon-γ Stimulates the Expression of CX3CL1/Fractalkine in Cultured Human Endothelial Cells

Abstract

CX3CL1/Fractalkine, a CX3C chemokine, is a potent agonist for the chemotaxis and adhesion of monocytes and lymphocytes. It was first identified as a membrane protein in endothelial cells activated with IL-1 or TNF-alpha. We have found the enhanced expression of fractalkine in human umbilical vein endothelial cells stimulated with interferon-gamma (IFN-gamma). Pretreatment of the cells with cycloheximide did not inhibit the expression of fractalkine mRNA. The majority of fractalkine protein was found in the cell lysate, and an antibody-blocking experiment disclosed that fractalkine contributes to the adhesion of mononuclear cells to endothelial monolayers stimulated with IFN-gamma. Vascular endothelial cells produce fractalkine in response to IFN-gamma, and this may play an important role in immune responses by eliciting a traffic of mononuclear cells through the vascular wall.

15‐Deoxy‐Δ12,14‐prostaglandin J2 inhibitsCX3CL1/fractalkine expression in human endothelial cells

“…WY14643 and ciglitazone were from Calbiochem (San Diego, CA). Primer oligo(dT) [12][13][14][15][16][17][18] and MMulv reverse transcriptase were from Gibco-BRL (Gainthersburg, MD). RNeasy total RNA isolation kit and Taq DNA polymerase were purchased from Qiagen (Hilden, Germany).…”

“…15,[23][24][25][26] Culture medium was Humedia EB-2 supplemented with 2% fetal bovine serum (FBS), 10 ng/mL recombinant human (r(h)) epidermal growth factor, 1 µ g/mL hydrocortisone, 5 ng/mL r(h) basic fibroblast growth factor and 10 µ g/mL heparin. When the cultures reached about 80% confluence, the medium containing growth factors was removed and the cells were cultured in Humedia EB-2 supplemented with 20% human serum (Humedia-HS).…”

“…The cells were stimulated for 16 h with IFN-γ and 6 h with IL-1 β according to the maximal expression of fractalkine observed in our previous study. 15 In the experiments using cycloheximide (CHX), the cells were pretreated with 500 ng/mL CHX for 1 h prior to the stimulation. The effect of actinomycin D was also examined.…”

“…Single-strand cDNA for a PCR template was synthesized from 1 µ g of total RNA using a primer oligo(dT) [12][13][14][15][16][17][18] and M-Mulv reverse transcriptase under the conditions indicated by the manufacturer. Specific primers were designed from cDNA sequences for fractalkine, ICAM-1, VCAM-1, PPAR-α , PPAR-γ and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).…”

“…13 Interferon-γ is also found to stimulate endothelial fractalkine expression. 14,15 Interferon-γ stimulates fractalkine expression in other types of cells such as bronchial epithelial cells 16 and brain astrocytes. 17 Fractalkine is incorporated into the cell surface membrane of activated endothelial cells and promotes strong adhesion of T lymphocytes and monocytes.…”