Rickettsia rickettsii infection of human macrovascular and microvascular endothelial cells reveals activation of both common and cell type-specific host response mechanisms

Abstract

Although inflammation and altered barrier functions of the vasculature, due predominantly to the infection of endothelial cell lining of small and medium-sized blood vessels, represent salient pathological features of human rickettsioses, the interactions between pathogenic rickettsiae and microvascular endothelial cells remain poorly understood. We have investigated the activation of nuclear transcription factor-kappa B (NF-kappaB) and p38 mitogen-activated protein (MAP) kinase, expression of heme oxygenase 1 (HO-1) and cyclooxygenase 2 (COX-2), and secretion of chemokines and prostaglandins after Rickettsia rickettsii infection of human cerebral, dermal, and pulmonary microvascular endothelial cells in comparison with pulmonary artery cells of macrovascular origin. NF-kappaB and p38 kinase activation and increased HO-1 mRNA expression were clearly evident in all cell types, along with relatively similar susceptibility to R. rickettsii infection in vitro but considerable variations in the intensities/kinetics of the aforementioned host responses. As expected, the overall activation profiles of macrovascular endothelial cells derived from human pulmonary artery and umbilical vein were nearly identical. Interestingly, cerebral endothelial cells displayed a marked refractoriness in chemokine production and secretion, while all other cell types secreted various levels of interleukin-8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) in response to infection. A unique feature of all microvascular endothelial cells was the lack of induced COX-2 expression and resultant inability to secrete prostaglandin E(2) after R. rickettsii infection. Comparative evaluation thus yields the first experimental evidence for the activation of both common and unique cell type-specific host response mechanisms in macrovascular and microvascular endothelial cells infected with R. rickettsii, a prototypical species known to cause Rocky Mountain spotted fever in humans.

FIG. 1.

Effect of R. rickettsii infection on NF-κB activation in micro- and macrovascular human endothelial cells. ECs were either infected with R. rickettsii or incubated with the antibiotic-free culture medium alone (Control). Gel-shift analysis was carried out to measure the DNA-binding activity using nuclear extracts (2 μg protein) and a consensus NF-κB sequence. Specificity of DNA-protein binding was ascertained by inclusion of approximately 50-fold molar excess of unlabeled probe in the assay (+cold). The autoradiographic exposures from a typical experiment with human microvascular cerebral ECs (SV-HCECs [A]) and pulmonary artery ECs (HPAECs [B]) are shown. The arrows indicate relative positions of the NF-κB complex, a nonspecific band (NS), and unbound radioactivity in the reaction mixture (Free probe) on the gel. Panel C represents quantitative densitometric analysis of NF-κB activation in organ-specific ECs after R. rickettsii infection in relation to the uninfected controls (0 h), which were assigned a value of 1. The data are presented as the mean ± standard error of the mean (SEM) from a minimum of three independent experiments (n ≥ 3). The asterisks indicate statistically significant differences (P ≤ 0.05) in comparison to the corresponding uninfected controls.

FIG. 2.

Activation of p38 MAP kinase in different types of ECs following R. rickettsii infection for various lengths of time. Total protein lysates from ECs infected with R. rickettsii for 1.5 to 24 h or incubated with the culture medium alone (Control) were subjected to Western blot analysis to determine the steady-state levels of phosphorylated p38 kinase. Blots were probed with a phosphorylation-state-specific (Thr180/Tyr182) p38 antibody, followed by an anti-α-tubulin antibody to normalize for any variations in protein loading among different lanes. Shown are the changes in phospho-p38 levels in different types of ECs infected with R. rickettsii, as determined by quantitative densitometric analysis. For comparison, the basal level of p38 phosphorylation in each cell type was assigned a value of 1. The data are presented as the mean ± standard error from a minimum of three independent observations (n ≥ 3). The asterisks indicate statistically significant increase in cellular phospho-p38 levels in comparison to the corresponding uninfected controls.

FIG. 3.

IL-8 and MCP-1 production by R. rickettsii-infected ECs of different origin. Culture supernatants from cells infected with R. rickettsii for 3, 7, 14, and 21 h and corresponding uninfected controls were collected and assessed for IL-8 and MCP-1 levels by ELISA. At least two dilutions of each sample were assayed in duplicate, and OD values corresponding to the linear range of the standard curve were used for the determination of chemokine concentrations. Results are expressed as mean fold induction of IL-8 (A) and MCP-1 (B) over the basal levels of secretion by uninfected cells (Control), which were assigned a value of 1 for the ease of comparison. The data sets are presented as the mean ± standard error (SE) of at least three independent experiments (n ≥ 3). Statistically significant differences in comparison to the basal IL-8/MCP-1 secretion by uninfected cells are indicated by the asterisks.

FIG. 4.

Dynamics of HO-1 mRNA expression in organ-specific micro- and macrovascular ECs infected with R. rickettsii. Total RNA (8 μg) from uninfected ECs (Control) or those infected with R. rickettsii for 3 to 21 h were processed for Northern blot analysis to determine the level of HO-1 transcriptional activation. Blots were first probed with a cDNA probe specific for human HO-1, followed by a GAPDH probe to normalize for potential variations in RNA loading among different samples. Results of a representative blot obtained from infected HPMECs are shown in panel A. The detailed densitometric analysis of changes in steady-state levels of HO-1 transcript due to R. rickettsii infection of different types of ECs is presented as the mean fold induction over the basal level of 1 (B). The data are presented as the mean ± standard error of three independent experiments (n ≥ 3). The asterisks indicate statistically significant differences in HO-1 mRNA expression relative to the corresponding uninfected controls.

FIG. 5.

Increased expression of COX-2 mRNA and protein synthesis in macrovascular ECs infected with R. rickettsii. Panel A depicts densitometric analysis of changes in steady-state levels of COX-2 transcriptional activation in HPAECs and HUVECs after R. rickettsii infection. The results are presented as the mean fold induction over the basal level ± SE (n ≥ 3). The asterisks indicate statistically significant differences in COX-2 mRNA expression relative to the baseline in uninfected controls. Panel B shows the results of a typical Western blot with uninfected (Control) and R. rickettsii-infected HPAECs. Total protein lysates of HPAECs infected for 1.5 to 24 h or incubated with medium alone (Control) were analyzed by Western blotting to determine the steady-state levels of cellular COX-2 protein. Blots were probed in succession with anti-human COX-2-specific antibody and an anti-α-tubulin antibody. Densitometric analysis of changes in COX-2 protein levels in HPAECs and HUVECs infected with R. rickettsii was then performed (C). The data are presented as the mean fold induction ± standard error over the baseline value of 1 (n ≥ 3). Panel D shows a representative Northern blot for the detection of basal and R. rickettsii-induced COX-2 expression in HPMECs infected for different lengths of time. HUVECs infected with R. rickettsii for 7 h were included as a positive control in this experiment.