RICKETTSIA-RICKETTSII INFECTION OF THE EA.HY-926 ENDOTHELIAL-CELL LINE - MORPHOLOGICAL RESPONSE TO INFECTION AND EVIDENCE FOR OXIDATIVE INJURY

EA.hy 926 is a permanent human cell line that expresses highly differe ntiated functions characteristic of human vascular endothelium. Ricket tsia rickettsii can efficiently infect and cause a cytopathic effect i n EA.hy 926 cells. R. rickettsii produced visible lytic plaques in EA. hy 926 cells at 10 d postinfection (p.i.) following application of a s econdary agarose overlay containing 2 mu g emetine ml(-1) and 40 mu g NaF ml(-1) on day 2. Rickettsial growth in EA.hy 926 cells had a simil ar profile to that occurring in human umbilical vein endothelial cells (HUVEC) and rickettsiae catalysed polymerization of actin tails. Intr acellular multiplication of R. rickettsii resulted in significant chan ges in the internal morphology of EA.hy 926 cells, most notably extens ive dilatation of the membranes of the endoplasmic reticulum and outer nuclear envelope by 72 h p.i, These events correlated with significan t alterations in the host-cell antioxidant system, including decreased levels of intracellular reduced glutathione and glutathione peroxidas e 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 m echanisms 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 infecti on. Overnight pretreatment with 1-500 mu M alpha-lipoic acid did not p revent cells from being destroyed following infection with rickettsiae . Supplementation of the culture medium with 1 and 10 mu M alpha-lipoi c acid 2 h after rickettsial inoculation also did not provide any prot ective effect. However, 100, 200 and 500 mu M alpha-lipoic acid increa sed the viability of infected cells at 96 h to 45, 51 and 70%, respect ively compared with 26% for untreated, infected samples. Thiol levels and glutathione peroxidase activity in treated, infected cells increas ed and peroxide content decreased proportionally to increasing alpha-l ipoic acid concentrations. Furthermore, treatment with 500 mu M 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 ident ical in all aspects to that demonstrated previously in HUVEC, the incr eased reproducibility and convenience of EA.hy 926 cells make them sui table for biochemical and morphological studies.