Rickettsia rickettsii infection of cultured human endothelial cells induces heme oxygenase 1 expression

Rickettsiae are obligate, intracellular parasites and causative agents of severe bacterial diseases of humans, including epidemic typhus and Rocky Mountain spotted fever, caused, respectively, by Rickettsia prowazekii and R. rickettsii. These bacteria are transmitted to their mammalian hosts by arthropod vectors such as ticks, fleas, lice, and mites and grow within the cytoplasm (and occasionally the nucleus) of eukaryotic cells (37, 62). Rickettsiae exhibit a tropism for the endothelium and invade vascular endothelial cells as a major target. The proliferation of organisms in capillary endothelium and associated cytopathic effects of infection contribute to increased vascular permeability (57). Studies have suggested that the host endothelial cells actively respond to intracellular infection by altering the expression of several proteins, including tissue factor (50, 56), plasminogen activator inhibitor 1 (13, 44), E-selectin (12, 49), interleukin-1 (IL-1), IL-6, and IL-8 (22, 51). Ultrastructural studies have shown that infected endothelial cells sustain severe damage at late stages of infection, as indicated by dilatation of the rough endoplasmic reticulum and outer nuclear envelope, loss of osmoregulatory control, and cell lysis (45, 46). The mechanisms of cell injury induced by R. rickettsii remain largely unknown. Based on the striking architectural changes in the cytoskeleton of infected cells, it was speculated that reactive oxygen species may be one of the major causes of cell injury by R. rickettsii. Subsequent biochemical analysis confirmed the accumulation of intracellular peroxides and superoxide radicals (42, 47), detection of higher amounts of extracellular hydrogen peroxide in the culture medium of infected cells (21), and reduction in levels of intracellular thiols (48). Further, the activities of three important enzymes of the cellular antioxidant system, namely, glucose-6-phosphate dehydrogenase, catalase, and glutathione peroxidase, were downregulated, while that of superoxide dismutase was increased in endothelial cells exposed to R. rickettsii (11, 42). α-Lipoic acid, a lipoamide compound with potent antioxidant properties, exhibited a protective effect against oxidative changes by decreasing the levels of peroxides and elevating reduced glutathione and glutathione peroxidase activity (15). These observations with human umbilical vein endothelial cells and similar studies utilizing a permanent endothelial cell line, EA.hy 926 (16), provide evidence for oxidative stress during R. rickettsii infection, which may be responsible for cell injury. The cellular response to oxidative stress-inducing agents such as heavy metals, UV irradiation, heme, hemoglobin, and hydrogen peroxide involves the production of a number of cellular mediators, including acute-phase proteins, eicosanoids, and cytokines. Heme oxygenase (HO; EC 1.14.99.3), the initial and rate-limiting enzyme in the pathway for heme catabolism, plays a vital role in diverse biological processes, including cell respiration, energy generation, oxidative biotransformation, and cell growth and differentiation (2, 7). HO is present in most mammalian tissues and catalyzes the degradation of heme to biliverdin, releasing equimolar amounts of biliverdin IXa, iron, and carbon monoxide (CO) (28). Biliverdin is subsequently converted into bilirubin by the enzyme biliverdin reductase. The HO system consists of at least three isozymes. Two of them, HO-1 and HO-2, are products of distinct genes; differ in their tissue distribution and regulation and have been characterized in detail. Of these, HO-1 is inducible and designated a stress response protein, whereas HO-2 is predominantly constitutive (28). A third isozyme, HO-3, which is closely related to HO-2, has been described recently (29). Although heme is purported to be the typical HO-1 inducer, the inflammatory cytokines IL-1α and tumor necrosis factor-α (TNF-α) have been shown to be effective inducers of HO-1 in cultured human EC (54). The expression of HO-1 is sensitive to induction by oxidants, and recent work has demonstrated that HO-1 provides cytoprotective effects in models of oxidant-induced cellular and tissue injury (34). Increased HO-1 activity enhances the survival of EC exposed to heme iron (1), and bilirubin, a potent antioxidant, also protects against hydrogen peroxide-induced toxicity in an aortic endothelial cell line (32). A recent study has demonstrated that CO generated through the action of HO-1 acts as an antiapoptotic molecule, and induction of HO-1 is able to prevent a series of inflammatory reactions associated with apoptosis (4). Since HO activity is known to protect EC in response to oxidative stress induced by various stimuli, the present study was undertaken to investigate the regulation of endothelial HO-1 and HO-2 during R. rickettsii infection. (A portion of this work was presented at the 101st General Meeting of the American Society for Microbiology, in Orlando, Fla., 20 to 24 May 2001.)