Interaction Between Toxoplasma gondii Tachyzoites and Retinal Microvascular Endothelium

Abstract

Abstract: : Purpose: Retinochoroiditis secondary to infection with the protozoan parasite, Toxoplasma gondii, is a prevalent form of posterior uveitis worldwide. Previous work from our group suggests a relative susceptibility of the human retinal microvascular endothelium to infection with T. gondii tachyzoites. We have developed a novel binding assay to investigate the possibility that susceptibility is related to preferential binding between tachyzoite and endothelial cell. Methods:Recombinant yellow fluorescent protein–expressing RH strain T. gondii tachyzoites (gifted by Dr. Boris Streipen, University of Georgia) were grown in confluent monolayers of neonatal human dermal fibroblasts. Tachyzoites were suspended in DMEM with 1% FBS containing Alexa Fluor 594 (Molecular Probes)–conjugated rabbit anti–human von Willebrand factor (vWF) antibody (0.02 µg/ml) (Dako) at a concentration of 2.5 x 10⁷. 100 µL of this suspension was placed on glass slide–mounted 6 micron sections of unfixed human retina (2 donors) and rotated 30 minutes at 37 °C. Following fixation in 10% buffered formalin, separate images of the retina were obtained using two filter sets (590nmEX/660nmEM, 550nmEX/550EM) and under darkfield. A masked observer quantified the numbers of tachyzoites overlying vWF–positive endothelium versus vWF–negative non–endothelial retina. Areas of endothelium and non–endothelial retina were determined using Image Pro 5.0 (Media Cybernetics). Results:For one donor retina, significantly more tachyzoites bound to vWF–positive endothelium (median = 961 tachyzoites/mm², range = 0–6611, n = 108 vessels) than vWF–negative non–endothelial retina (median = 230 tachyzoites/mm², range = 0–5511), p < 0.0001 (Mann–Whitney test). For the other donor, there was no significant difference in binding between endothelium (median = 237 tachyzoites/mm², range = 0–5895, n = 165 vessels) and non–endothelial retina (median = 230 tachyzoites/mm², range = 0–4287), p = 0.15. Autofluorescence of retina complicated quantification of this assay. Conclusions:The ability of T.gondii tachyzoites to bind to retinal microvascular endothelium may explain in part why retinal endothelial cells are highly susceptible to infection with T. gondii. Our data suggest some persons may be more prone to T. gondii infection than others if binding of T. gondii to retinal endothelium is critical for disease propagation.

Keywords: toxoplasmosis • retinochoroiditis • vascular cells