Analysis of chemokine expression of rat retinal vascular endothelial cells infected with Toxoplasma gondii

Abstract

Abstract: : Purpose:To investigate changes in chemokine expression following T.gondii infection of retinal vascular endothelial (RVE). Methods:Rat RVE cell line JG2/1(a kind gift of Prof J. Greenwood) were infected or mock infected with the RH strain of T.gondii. RNA and supernatants were collected from infected and uninfected cells at two and twenty four hours. In order to elucidate changes in gene expression, RNA from infected and uninfected cells from the two time points were compared using the Clontech Atlas^TM Plastic array system. RT–PCR and/or ELISA were used to confirm these changes. Results:After two hours exposure to T.gondii there was an overall change of expression in ∼6% of genes on the array which include those involved in cell structure, protein and vesicle trafficking, cell–cycle regulation, transcriptional and translational machinery, and apoptosis. We focused specifically on the chemokine genes and found that MCP–1, GROα, MIP–1ß, and fractalkine were upregulated greater than two fold over background. Thus far ELISA has confirmed GROα and MCP–1 expression. At 24 hours GROα levels in the supernatant of the control sample was 1 pg/ml per million cells and in the supernatant of the infected sample it was 13.3 ng/ml per million cells. At 24 hours MCP–1 levels in the supernatant of the control sample was 14.9 pg/ml per million cells and in the supernatant of the infected sample it was 798 pg/ml per million cells. Conclusion:Although we saw upregulation of MCP–1 and GROα, which has been seen in previous microarray studies on fibroblasts (Blader IJ et al. 2001), this is the first time upregulation of MIP–1ß and fracktalkine by T.gondii infection of endothelial cells has been observed. Endothelial cells normally secrete GROα and MCP–1 in response to injury to facilitate recruitment of neutrophils and macrophages. The production of fractalkine by endothelial cells is an important step in the adaptive response by attracting T–cells and NK cells. The production of chemokines secondary to infection by T.gondii by the cells of the inner blood retinal barrier is important in the initiation of the immune response to clear infection. Acknowledgments We would like to thank the Iris Fund for the Prevention of Blindness for their financial support of this study.

Keywords: cytokines/chemokines • gene/expression • gene microarray