Up-regulation of the vascular cell adhesion molecule-1 (VCAM-1) induced by Theiler's murine encephalomyelitis virus infection of murine brain astrocytes.

Abstract

The present article reports the up-regulation of the expression of the vascular cell adhesion molecule-1 (VCAM-1) by SJL/J mouse brain astrocytes infected with Theiler's murine encephalomyelitis virus (TMEV). Complementary RNA (cRNA) from mock- and TMEV-infected cells was hybridized to the Affymetrix whole murine genome U74v2 DNA microarray. Hybridization data analysis revealed background expression in untreated cells and the up-regulation of three sequences coding for VCAM-1, as described by the SCOP (Structural Classification Of Proteins) database. The authors further studied its regulation, confirming and validating their mRNA increase by reverse transcriptase-polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR. The presence of the 100-kDa VCAM-1 protein in mock- and TMEV-infected cells was demonstrated in the cell membrane by a specific cell-based enzyme-linked immunosorbent assay (ELISA), in addition to flow cytometry and confocal immunohistochemistry. Further, Western blots were used to quantify the amount of VCAM-1 molecules in cell extracts. All these data demonstrated a mean 75% increase in the expression of VCAM-1 on the surface of TMEV-infected cells. Three inflammatory cytokines, interleukin-1alpha (IL-1alpha), interferon gamma (IFNgumma), and specially tumor necrosis factor alpha (TNF-α), some of which are also induced by TMEV in astrocytes (IL-1alpha and TNF-alpha), were potent inducers of VCAM-1 expression. To demonstrate whether the VCAM-1 molecules were biologically active, mediating adhesion to other cells as the integrin alpha4-expressing CD4+ T lymphocytes, the authors used a cell adhesion test. It was also demonstrated by immunohistochemistry that in vivo VCAM-1 expression is enhanced after TMEV intracraneal infection. The present data show a small but statistically significant overexpression of VCAM-1 after astrocyte infection with TMEV that could play a significant role in vivo.