Superoxide dismutase-dependent, catalase-sensitive peroxides in human endothelial cells infected by Rickettsia rickettsii

Abstract

The generation and intracellular accumulation of reactive oxygen species have been shown to be associated with the infection of human umbilical vein endothelial cells (HUVEC) by Rickettsia rickettsii. In response to the oxidant superoxide, the activity of the enzyme superoxide dismutase (SOD) increases following infection by this obligate intracellular bacterium. Other oxidants which are capable of oxidizing the fluorescent probe 2',7'-dichlorofluorescin (DCFH) also accumulate intracellularly within infected cells. In the study reported here, we show that (i) an inhibitor of SOD, diethyldithiocarbamic acid, reduces the observed rise in SOD activity in infected cells by 40 to 60% and at the same time reduces the degree of intracellular oxidation of DCFH; (ii) catalase-sensitive peroxides can be detected in supernatants of R. rickettsii-infected cells shortly after rickettsial exposure; and (iii) fluorescence-activated cell sorter analysis demonstrates significant intracellular oxidant activity in infected cells within 5 h after exposure to R. rickettsii. The results of these experiments indicate that hydrogen peroxide is a major oxidant associated with infection of HUVEC by R. rickettsii and that intracellular oxidant activity sensitive to SOD inhibition is detectable early and prior to significant rickettsial multiplication and much earlier than the ultrastructural manifestations of cell injury seen by electron microscopy.

FIG. 1

Relative percentage SOD activity in control (⊡) (uninfected, untreated) and infected, DDC-treated (◘, ◊) endothelial cells in comparison to untreated (⧫) cells infected with R. rickettsii. SOD concentrations found in untreated, infected cells were set at 100%. The data presented represent mean values ± the standard errors of at least three separate experiments at each time point.

FIG. 2

Representative FACScan fluorescence histograms constructed from HUVEC loaded with DCFH-DA and analyzed at 1 (a), 5 (b) and 24 (c) h after infection with R. rickettsii. Cells were incubated with 1 μM DCFH-DA at 37°C for 15 min and fixed in 1% paraformaldehyde before being analyzed on a FACScan flow cytometer. Excitation was 488 nm and emission was collected at 530/30 nm. Peak 1, unstained, uninfected endothelial cells; peak 2, uninfected endothelial cells stained with DCFH-DA; peak 3, R. rickettsii-infected endothelial cells stained with DCFH-DA. Each panel is a composite of separate runs, and the histogram represents the DCFH fluorescence of approximately 20,000 endothelial cells.

FIG. 3

Relative percentage of DCFH fluorescence in R. rickettsii-infected endothelial cells not treated with DDC (column 2), infected endothelial cells treated with 10⁻⁶ and 10⁻⁵ M DDC (columns 3 and 4, respectively), and uninfected, untreated endothelial cells (column 1) at 1, 5, and 24 h. DCFH fluorescence found in untreated, infected cells (column 2) was set at 100%. The data presented represent mean values ± standard errors of at least four separate experiments at each time point.

FIG. 4

Relative percentage of DCFH fluorescence in supernatant fluids of uninfected, untreated control (column a) and R. rickettsii-infected (column b) endothelial cells in comparison to catalase-treated infected cells (column c), glutathione peroxidase-treated infected cells (column d), and infected cells treated with both catalase and glutathione peroxidase (column e). DCFH fluorescence was normalized against the untreated infected cell mean, set at 100%. The data represent mean values ± standard error of the means of two separate experiments with three determinations at each condition. Statistically significant values compared with (column b), the untreated infected cell population: (columns a, c, and e, P < 0.01; column d, P < 0.05.