CYTOKINE-INDUCED, NITRIC OXIDE-DEPENDENT, INTRACELLULAR ANTIRICKETTSIAL ACTIVITY OF MOUSE ENDOTHELIAL-CELLS

In a murine model of rickettsial disease in which, as in human rickett sioses, endothelial cells are the major target of infection, depletion of IFN-gamma or TNF-alpha converts a sublethal infection into a unifo rmly fatal disease with overwhelming rickettsial growth and decreased nitric oxide (NO) synthesis. The kinetics of NO production and rickett sial survival and growth were examined on Days 1, 2, and 3 after inocu lation of endothelial cells with Rickettsia conorii under four differe nt experimental conditions: (a) no cytokine treatment, (b) treatment w ith IFN-gamma and TNF-alpha, (c) treatment with cytokines and N-G mono methyl-L-arginine, a competitive inhibitor of NO synthesis, and (d) tr eatment with sodium nitroprusside, a source of NO. Endothelial cells w ere examined for the presence of inducible nitric oxide synthase mRNA by specific reverse transcriptase-PCR after stimulation with lFN-gamma and TNF-cv. Cytokine-stimulated and unstimulated rickettsiae-infected endothelial cells were examined by electron microscopy to observe the cellular and rickettsial events. Transformed and diploid mouse endoth elial cells stimulated by the combination of recombinant murine IFN-ga mma and TNF-alpha killed intracellular Ricketssia conorii by a mechani sm that required the synthesis of NO. The antirickettsial effect and N O synthesis were inhibited by treatment of endothelial cells with N-G monomethyl-L-arginine. Addition of nitroprusside, which released NO, a lso exerted a strong antirickettsial effect in the absence of IFN-gamm a and TNF-alpha. Endothelial inducible nitric oxide synthase mRNA was detected 4 hours after cytokine stimulation, increased substantially a t 8 hours, and decreased to low levels by 72 hours. Ultrastructural ev aluation revealed that endothelial cells effected rickettsial killing in association with autophagy. Double membranes of endothelial cell gr anular endoplasmic reticulum surrounded rickettsiae, which were also o bserved being destroyed within phagolysosomes. This study demonstrated for the first time that endothelial cells are capable of killing rick ettsiae. When stimulated by the combination of IFN-gamma and TNF-alpha , mouse endothelial cells kill Rickettsia conorii by an NO-dependent m echanism. Within the endothelium, NO exerts a rickettsicidal effect.