The vascular endothelial cell (EC) is a primary target of infection with Rickettsia rickettsii, the etiologic agent of Rocky Mountain spotted fever. Changes in gene transcription elicited by intracellular infection, including EC expression of the coagulation pathway initiator known as tissue factor (TF), may contribute to the vascular pathology observed during disease. Nuclear run-on analysis of uninfected and infected, cultured human endothelial cells revealed that the rate of TF mRNA transcription is enhanced more than twofold at 3 h following infection, thus coinciding with increased steady-state levels of TF mRNA. TF mRNA remained relatively unstable during infection, with a half-life of 1.6 h. The eukaryotic protein synthesis inhibitor cycloheximide did not block R. rickettsii-induced increase in TF mRNA levels and actually resulted in its superinduction, thus revealing that de novo synthesis of host cell protein was not prerequisite to this transcriptional response. Involvement of the transcription factor NF-κB in R. rickettsii-induced TF expression was demonstrated by using two unrelated inhibitors of NF-κB activation. The antioxidant pyrrolidinedithiocarbamate and the proteasome inhibitorN-tosyl-l-phenylalanine chloromethyl ketone blocked expression of TF mRNA and activity during infection. This study demonstrates that R. rickettsii infection results in transcriptional activation of the TF gene and that this response involves activation of the transcription factor NF-κB.

SummaryChanges in PAI-1 expression in human umbilical vein endothelial cells (HUVEC) were studied following in vitro infection with Rickettsia rickettsii. A 1.8-fold increase in secreted PAI-1 activity occurred in infected versus control cultures (p = 0.03) at 24 h but not at earlier timepoints. A similar increase (1.4-fold) in secreted PAI-1 antigen (p <0.005) was measured by ELISA. To determine whether this increase was due to increased synthesis of PAI-1, HUVEC were metabolically labeled with 35S-methionine concurrent with R. rickettsii infection. Such infection resulted in a 1.9-fold increase in labeled PAI-1 in the medium at 24 h (p = 0.036). Increase in steady-state levels of PAI-1 mRNA were detected as early as 18 h by Northern blot analysis, peaking (5.5-fold) at approximately 24 h. These results indicate that PAI-1 production is increased in RR-infected endothelial cells, an effect that may contribute to the vascular occlusions noted in Rocky Mountain spotted fever.

Microvascular thrombi underlie many of the clinical manifestations of Rocky Mountain spotted fever (RMSF), a disease characterized by Rickettsia rickettsii infection of vascular endothelial cells. Studies were designed to determine whether R rickettsii-infection of cultured human umbilical vein endothelial cells results in tissue factor (TF) induction, a process that could directly activate coagulation in infected vessels. Whereas uninfected endothelial cell cultures showed essentially undetectable TF mRNA and activity, both TF mRNA and activity were present after R rickettsii infection. TF mRNA levels were transient, peaking at 4 hours after the initiation of infection, whereas the peak of TF activity occurred at 8 hours. Induction of the TF response requires the intracellular presence of R rickettsii organisms, because uninfected rickettsia were ineffective and the response was blocked by inhibiting rickettsial entry using cytochalasin B. TF induction was not mediated by endothelial cell release of soluble factor, because no response was induced using culture medium conditioned by R rickettsii-infected cells. Furthermore, preadsorption of suspensions of R rickettsii with polymyxin B to remove contaminating lipopolysaccharide did not eliminate the TF response. Induction of TF in vital endothelial cells during R rickettsii infection could be the trigger for vascular thrombus formation of RMSF.

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The clinical manifestations of Rocky Mountain spotted fever (RMSF) result from Rickettsia rickettsii (R rickettsii) infection of endothelial cells and are mediated by pathologic changes localized to the vessel, including in situ thrombosis and tissue ischemia. This study uses in vitro infection of cultured human umbilical vein endothelial cells with R rickettsii to test the hypothesis that such infection induces von Willebrand factor (vWF) release from Weibel- Palade bodies, a process that could contribute to thrombotic changes. At 24 hours postinfection, there was an increase in metabolically prelabeled large multimers of vWF in the culture medium, with a concomitant decrease of these forms in the cell lysate samples. This release reaction was specific for the large multimer pool of vWF, localized to Weibel-Palade bodies, because no change in the distribution of dimeric forms between cells and culture medium was detected. Double-label immunofluorescence staining showed an inverse correlation between the number of R rickettsii and the number of Weibel- Palade bodies in infected cells. Cell lysis was minimal at 24 hours postinfection, as no detectable intracellular precursor forms (molecular weight 260,000) of vWF were released into the culture medium, there was no decrease in cell viability as measured by trypan blue exclusion, and no increase in 51Cr-release into the culture medium was observed when compared with uninfected controls. Release was likely a direct effect of the intracellular presence of the organism, rather than due to a noxious soluble factor such as endotoxin, because culture medium conditioned by infected endothelial cells was ineffective at inducing release in uninfected endothelial cell cultures. In summary, in vitro infection of endothelial cells by R rickettsii induces release of Weibel-Palade body contents, a process that may contribute to the pathogenesis of RMSF.