Rickettsia Rickettsii Infection of the Ea.Hy 926 Endothelial Cell Line: Morphological Response To Infection and Evidence for Oxidative Injury

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Abstract
EA.hy 926 is a permanent human cell line that expresses highly differentiated functions characteristic of human vascular endothelium. Rickettsia rickettsii can efficiently infect and cause a cytopathic effect in EA.hy 926 cells. R. rickettsii produced visible lytic plaques in EA.hy 926 cells at 10 d post-infection (p.i.) following application of a secondary agarose overlay containing 2 micrograms emetine ml-1 and 40 micrograms NaF ml-1 on day 2. Rickettsial growth in EA.hy 926 cells had a similar profile to that occurring in human umbilical vein endothelial cells (HUVEC) and rickettsiae catalysed polymerization of actin tails. Intracellular multiplication of R. rickettsii resulted in significant changes in the internal morphology of EA.hy 926 cells, most notably extensive dilatation of the membranes of the endoplasmic reticulum and outer nuclear envelope by 72 h p.i. These events correlated with significant alterations in the host-cell antioxidant system, including decreased levels of intracellular reduced glutathione and glutathione peroxidase activity and increased amounts of intracellular peroxide through to 96 h of infection. These findings are similar to the changes described previously for R. rickettsii-infected HUVEC and suggest that common mechanisms associated with rickettsia-induced oxidative injury occur in the two cell lines. EA.hy 926 cells were also used to investigate the influence of the antioxidant alpha-lipoic acid on rickettsial infection. Overnight pretreatment with 1-500 microM alpha-lipoic acid did not prevent cells from being destroyed following infection with rickettsiae. Supplementation of the culture medium with 1 and 10 microM alpha-lipoic acid 2 h after rickettsial inoculation also did not provide any protective effect. However, 100, 200 and 500 microM alpha-lipoic acid increased the viability of infected cells at 96 h to 45, 51 and 70%, respectively compared with 26% for untreated, infected samples. Thiol levels and glutathione peroxidase activity in treated, infected cells increased and peroxide content decreased proportionally to increasing alpha-lipoic acid concentrations. Furthermore, treatment with 500 microM alpha-lipoic acid for 72 h p.i. completely prevented ultrastructural changes in infected cells. In conclusion, the permanent endothelial cell line EA.hy 926 is susceptible to injury induced by R. rickettsii infection. Although the cellular changes resulting from infection are not identical in all aspects to that demonstrated previously in HUVEC, the increased reproducibility and convenience of EA.hy 926 cells make them suitable for biochemical and morphological studies.