Quantitative analyses of variations in the injury of endothelial cells elicited by 11 isolates of Rickettsia rickettsii.

Eleven isolates of spotted fever group rickettsiae from the blood of patients or ixodid ticks from North and South America were characterized. All isolates were identified as Rickettsia rickettsii using restriction fragment length polymorphism analysis of a 532-bp rOmpA gene fragment obtained by PCR. The ability of the R. rickettsii isolates to elicit cytopathic effects and parameters of oxidative injury were examined in cultured human EA.hy 926 endothelial cells. Cytopathic effects were determined by direct observation of infected cultures, by measuring the release of cytoplasmic lactate dehydrogenase (LDH), and by determination of intracellular pools of peroxide and reduced glutathione. Four biotypes of R. rickettsii were defined. Group I included two highly cytopathic isolates from Montana, Bitterroot and Sheila Smith, and three isolates from Maryland, North Carolina, and Brazil. These isolates rapidly damaged cells, released large amounts of cytoplasmic LDH, caused accumulation of intracellular peroxide, and depleted intracellular pools of reduced glutathione. Group II contained three isolates, two from Montana, Hlp#2 and Lost Horse Canyon, and an isolate from Colombia, which were similar to group I but caused either lower responses in LDH release or smaller changes in intracellular peroxide levels. The group III isolates, Sawtooth from Montana and 84JG from North Carolina, caused lower cellular injury by all measures. Group IV isolate Price T from Montana was the least cytopathic and caused minimal alterations of all parameters measured. Understanding the molecular basis for the varied cellular injury caused by different isolates of R. rickettsii may contribute to improved treatment of Rocky Mountain spotted fever and to the rapid identification of those isolates which are more likely to cause fulminant disease.

Post-transcriptional regulation of endothelial cell plasminogen activator inhibitor-1 expression during R. rickettsii infection.

Intracellular infection of endothelial cells with Rickettsia rickettsii results in increased steady-state levels of plasminogen activator inhibitor-1 (PAI-1) mRNA. Control mechanisms governing such increased expression in response to this novel stimulus have not been defined. In this study, we compared the stability of PAI-1 mRNA in infected and uninfected endothelial cells (EC) and explored the requirement for de novo host cell protein synthesis in the infection-induced increase of steady-state levels. The half-life of PAI-1 mRNA, which is constitutively expressed in cultured EC, increased from 18 h in uninfected EC to greater than 30 h in EC infected for 24 h, a time point at which increases in steady-state PAI-1 mRNA levels are noted. There was no change in stability of gamma-actin due to infection. Nuclear run-on studies revealed no apparent increase in transcription rate at 4, 18 and 24 h. R. rickettsii -induced increase in PAI-1 mRNA was blocked by the eukaryotic protein synthesis inhibitor, cycloheximide, which suggests that this response requires de novo host cell protein synthesis. These results provide evidence that post-transcriptional control mechanisms are operative in the regulation of PAI-1 during R. rickettsii infection.

Western blotting analysis of heat shock proteins of Rickettsiales and other eubacteria.

Heat shock proteins (Hsp) of four Rickettsia species, three Bartonella species, two Ehrlichia species, Orientia tsutsugamushi and seventeen other eubacterial species were characterized by the enhanced chemiluminescence Western blotting (WB) technique with antibodies raised against recombinant Hsp from Escherichia coli and purified GroES from R. typhi. Although E. coli DnaK and GroEL have epitopes that are highly conserved among the homologous proteins found in Rickettsia, Ehrlichia, O. tsutsugamushi, Bartonella and other Proteobacteria, anti-E. coli DnaK and GroEL monoclonal antibodies (Dasch et al. (1990) Ann. N.Y. Acad. Sci. 590, 352-369) recognize less conserved epitopes. In contrast, epitopes on E. coli DnaJ, GrpE and GroES are much less conserved since anti-E. coli DnaJ, GrpE and GroES polyclonal antibodies did not recognize DnaJ, GrpE or GroES homologues in Rickettsia, Bartonella, Orientia, Ehrlichia and Legionella. Polyclonal antiserum prepared against GroES from R. typhi reacted strongly with purified 10 kDa GroES peptide from Rickettsia and Bartonella, and strongly bound to proteins of varying electrophoretic mobility from Wolbachia, Legionella, Proteus and Shigella flexneri and more weakly to other GroES homologues including that found in E. coli. Consequently, commercially available anti-DnaJ, anti-GrpE and anti-GroES polyclonal antibodies and anti-DnaK monoclonal antibody raised against their respective recombinant E. coli Hsp are not suitable for detection and identification of homologues of these proteins in a wide range of eubacteria.

NF-kappa B-dependent inhibition of apoptosis is essential for host cellsurvival during Rickettsia rickettsii infection.

The possibility that bacteria may have evolved strategies to overcome host cell apoptosis was explored by using Rickettsia rickettsii, an obligate intracellular Gram-negative bacteria that is the etiologic agent of Rocky Mountain spotted fever. The vascular endothelial cell, the primary target cell during in vivo infection, exhibits no evidence of apoptosis during natural infection and is maintained for a sufficient time to allow replication and cell-to-cell spread prior to eventual death due to necrotic damage. Prior work in our laboratory demonstrated that R. rickettsii infection activates the transcription factor NF-kappa B and alters expression of several genes under its control. However, when R. rickettsii-induced activation of NF-kappa B was inhibited, apoptosis of infected but not uninfected endothelial cells rapidly ensued. In addition, human embryonic fibroblasts stably transfected with a superrepressor mutant inhibitory subunit Ikappa B that rendered NF-kappa B inactivatable also underwent apoptosis when infected, whereas infected wild-type human embryonic fibroblasts survived. R. rickettsii, therefore, appeared to inhibit host cell apoptosis via a mechanism dependent on NF-kappa B activation. Apoptotic nuclear changes correlated with presence of intracellular organisms and thus this previously unrecognized proapoptotic signal, masked by concomitant NF-kappa B activation, likely required intracellular infection. Our studies demonstrate that a bacterial organism can exert an antiapoptotic effect, thus modulating the host cell's apoptotic response to its own advantage by potentially allowing the host cell to remain as a site of infection.

Effects of the antioxidant alpha-lipoic acid on human umbilical vein endothelial cells infected with Rickettsia rickettsii.

Rickettsia rickettsii infection of endothelial cells is manifested in very distinctive changes in cell morphology, consisting of extensive dilatation of the membranes of the endoplasmic reticulum and outer nuclear envelope and blebbing of the plasma membrane, as seen by transmission electron microscopy (D. J. Silverman, Infect. Immun. 44:545-553, 1984). These changes in cellular architecture are thought to be due to oxidant-mediated cell injury, since their occurrence correlates with dramatic alterations in cellular metabolism, particularly with regard to antioxidant systems. In this study, it was shown that R. rickettsii infection of human umbilical vein endothelial cells resulted in a significant depletion of intracellular reduced glutathione (thiol) content at 72 and 96 h and decreased glutathione peroxidase activity at 72 h postinfection. Infected cells displayed a dramatic increase in the concentration of intracellular peroxides by 72 h. Supplementation of the cell culture medium with 100, 200, or 500 microM alpha-lipoic acid, a metabolic antioxidant, after inoculation with R. rickettsii restored the intracellular levels of thiols and glutathione peroxidase and reduced the intracellular peroxide levels in infected cells. These effects were dose dependent. Treated infected monolayers maintained better viability at 96 h after inoculation with R. rickettsii than did untreated infected cells. Moreover, supplementation of the cell culture medium with 100 microM alpha-lipoic acid for 72 h after infection prevented the occurrence of morphological changes in the infected cells. The presence of 100 or 200 microM alpha-lipoic acid did not influence rickettsial growth in endothelial cells, nor did it affect the ability of R. rickettsii to form lytic plaques in Vero cells. Treatment with 500 microM alpha-lipoic acid decreased by 50% both the number and size of lytic plaques in Vero cells, and it also decreased the recovery of viable rickettsiae from endothelial cells. However, under all treatment conditions, a significant number of rickettsiae could be detected microscopically. Furthermore, the rickettsiae apparently retained their capacity for intracellular movement, since they possessed long polymerized actin tails after 72 and 96 h of treatment regardless of the concentration of alpha-lipoic acid used. Since alpha-lipoic acid does not seem to exhibit direct antirickettsial activity except with long-term exposure at very high concentrations, the mechanism of its protective activity for endothelial cells infected with rickettsiae may involve complex changes in cellular metabolism that only indirectly affect rickettsiae.

Proteasome-independent activation of nuclear factor kappaB in cytoplasmic extracts from human endothelial cells by Rickettsia rickettsii.

Interaction of many infectious agents with eukaryotic host cells is known to cause activation of the ubiquitous transcription factor nuclear factor kappaB (NF-kappaB) (U. Siebenlist, G. Franzoso, and K. Brown, Annu. Rev. Cell Biol. 10:405-455, 1994). Recently, we reported a biphasic pattern of NF-kappaB activation in cultured human umbilical vein endothelial cells consequent to infection with Rickettsia rickettsii, an obligate intracellular gram-negative bacterium and the etiologic agent of Rocky Mountain spotted fever (L. A. Sporn, S. K. Sahni, N. B. Lerner, V. J. Marder, D. J. Silverman, L. C. Turpin, and A. L. Schwab, Infect. Immun. 65:2786-2791, 1997). In the present study, we describe activation of NF-kappaB in a cell-free system, accomplished by addition of partially purified R. rickettsii to endothelial cell cytoplasmic extracts. This activation was rapid, reaching maximal levels at 60 min, and was dependent on the number of R. rickettsii organisms added. Antibody supershift assays using monospecific antisera against NF-kappaB subunits (p50 and p65) confirmed the authenticity of the gel-shifted complexes and identified both p50-p50 homodimers and p50-p65 heterodimers as constituents of the activated NF-kappaB pool. Activation occurred independently of the presence of endothelial cell membranes and was not inhibited by removal of the endothelial cell proteasome. Lack of involvement of the proteasome was further confirmed in assays using the peptide-aldehyde proteasome inhibitor MG 132. Activation was not ATP dependent since no change in activation resulted from addition of an excess of the unhydrolyzable ATP analog ATPgammaS, supplementation with exogenous ATP, or hydrolysis of endogenous ATP with ATPase. Furthermore, Western blot analysis before and after in vitro activation failed to demonstrate phosphorylation of serine 32 or degradation of the cytoplasmic pool of IkappaB alpha. This lack of IkappaB alpha involvement was supported by the finding that R. rickettsii can induce NF-kappaB activation in cytoplasmic extracts prepared from T24 bladder carcinoma cells and human embryo fibroblasts stably transfected with a superrepressor phosphorylation mutant of IkappaB alpha, rendering NF-kappaB inactivatable by many known signals. Thus, evidence is provided for a potentially novel NF-kappaB activation pathway wherein R. rickettsii may interact with and activate host cell transcriptional machinery independently of the involvement of the proteasome or known signal transduction pathways.

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

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.

Transcriptional regulation of endothelial cell tissue factor expression during Rickettsia rickettsii infection: involvement of the transcription factor NF-kappaB.

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-kappaB in R. rickettsii-induced TF expression was demonstrated by using two unrelated inhibitors of NF-kappaB activation. The antioxidant pyrrolidinedithiocarbamate and the proteasome inhibitor N-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-kappaB.

Rickettsia rickettsii infection of cultured human endothelial cells induces NF-kappaB activation.

Rickettsia rickettsii, the etiologic agent of Rocky Mountain spotted fever, is an obligate intracellular bacterial organism that infects primarily the vascular endothelial cells (EC). A component of the EC response to infection is transcriptional activation, which may contribute to the thrombotic and inflammatory consequences of disease. In this study, we explore R. rickettsii-induced activation of the nuclear factor-kappaB/Rel (NF-kappaB) family of transcription factors involved in early transcriptional responses to injurious stimuli. Two NF-kappaB species were activated by infection and reacted with a double-stranded oligonucleotide probe corresponding to the kappaB binding domain of the murine kappa light-chain gene enhancer. Gel supershift analysis demonstrated the reactivity of these complexes with antibodies against p65 and p50, and the induced species were tentatively identified as p50-p50 homodimers and p50-p65 heterodimers. Semiquantitative reverse transcription-PCR analysis revealed dramatic increases in the steady-state levels of mRNA coding for the inhibitory subunit of NF-kappaB (IkappaB alpha), transcription of which is enhanced by the binding of NF-kappaB within the IkappaB alpha promoter region. NF-kappaB activation was first detected 1.5 h following infection and was biphasic, with an early peak of activation at approximately 3 h, a return to baseline levels at 14 h, and even higher levels of activation at 24 h. It is likely that NF-kappaB activation requires cellular uptake of R. rickettsii, since treatment of EC with cytochalasin B during infection to block entry inhibited activation by only 70% at 3 h. R. rickettsii-induced activation of NF-kappaB may be an important controlling factor in the transcriptional responses of EC to infection with this obligate intracellular organism.

Increased expression of plasminogen activator inhibitor-1 in R. rickettsii-infected endothelial cells.

Changes 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 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.

Rickettsia rickettsii has proteins with cross-reacting epitopes to eukaryotic phospholipase A2 and phospholipase C.

The entry, and possibly the exit, of rickettsiae from eukaryotic cells, as well as erythrocyte lysis by some members of this group of organisms, is thought to be mediated by a phospholipase A activity even though the enzyme has not been isolated from these organisms. Evidence for phospholipase C, on the other hand, has not been reported for the genus Rickettsia. In this study, in a preliminary attempt to demonstrate the presence of phospholipase A2 and phospholipase C in the virulent Sheila Smith strain of Rickettsia rickettsii, we performed immunoblotting and immuno-gold electron microscopy using anti-phospholipase A2 and anti-phospholipase C IgG antibodies (raised against mammalian enzymes). We provide evidence for cross-reactivity of the antibodies with proteins present in R. rickettsii. Western blots showed a higher staining intensity with anti-phospholipase C antibody than with anti-phospholipase A2. According to the results obtained with the immuno-gold labeling of phospholipase A2 and phospholipase C reactive epitopes, most of the phospholipase A2 cross-reactive material appears to be associated with the membrane of the organism while the phospholipase C cross-reactive material appears to be randomly distributed throughout the cell.

Infection of human endothelial cells by Rickettsia rickettsii causes a significant reduction in the levels of key enzymes involved in protection against oxidative injury.

The activities of glucose-6-phosphate dehydrogenase, catalase, and glutathione peroxidase were significantly decreased in human endothelial cells infected with the obligate intracellular bacterium Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever. This observation lends additional support to our hypothesis implicating oxidative damage in endothelial cell injury caused by this microorganism.

Rickettsia rickettsii infection of cultured human endothelial cells induces tissue factor expression.

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.

E-selectin-dependent neutrophil adhesion to Rickettsia rickettsii-infected endothelial cells.

Increased neutrophil or HL60 cell adhesion to Rickettsia rickettsii-infected endothelial cells (ECs) was observed at 6 to 8 hours after the initiation of infection, diminishing by 24 hours. Similar increases were observed using formaldehyde-fixed neutrophils. Cellular association and likely the intracellular presence of rickettsiae was required for enhanced neutrophil adhesion, because culture medium conditioned by infected cells or rickettsiae rendered noninfective by pretreatment with tetracycline were ineffective at inducing neutrophil adhesion. Increases in neutrophil adhesion caused by infection were blocked by pretreatment of ECs with cycloheximide, suggesting the involvement of new protein synthesis in the cells' response. Flow cytometric analysis of infected cells showed increases in cell surface expression of E-selectin compared with uninfected control cells. Furthermore, incubation of 6- to 8-hour infected cells with a blocking monoclonal antibody against E-selectin (BB11) inhibited neutrophil adhesion an average of 61%. These results suggest the involvement of E-selectin in neutrophil adhesion to infected ECs occurring early in the course of the infection process. EC-initiated recruitment of neutrophil adhesion during rickettsiae infection could contribute to the pathologic changes associated with Rocky Mountain Spotted Fever.

Rhythmic and patterned neuronal firing in visual cortex.

We present evidence from multi-electrode recordings that the stimulus driven activity in a region of monkey visual cortex displays synchronous, oscillatory behaviour with temporally changing patterns of firing. The spatial-temporal patterns are dependent on cortical layer and on visual stimulus, 'build up and die down' with a period of 70-100 msec, and cannot be factorized into separate spatial and temporal components. This finding supports neural network models which explicitly incorporate spatial-temporal structure, ruling against a purely spatial code for information processing. The new type of analysis presented here is also suitable for direct on-line interactive analysis during multi-electrode recording experiments. Further, it could be modified for use in human EEG or evoked response recordings.

Rickettsia rickettsii induces superoxide radical and superoxide dismutase in human endothelial cells.

Human endothelial cells infected with Rickettsia rickettsii, the etiological agent of Rocky Mountain spotted fever, undergo striking morphological changes to the endoplasmic reticulum-outer nuclear envelope complex. These changes are accompanied by concurrent accumulation of intracellular peroxides. Both of these findings are consistent with the notion that cells undergo some form of oxidative stress. Since oxidant injury is often initiated or mediated through oxygen radicals, we examined superoxide radical generation when endothelial cells were exposed to R. rickettsii. We also examined the levels of superoxide dismutase, an enzyme induced in response to increased superoxide formation. The levels of both superoxide and superoxide dismutase increased when endothelial cells were exposed to R. rickettsii. These results, together with our previous findings, support our hypothesis that cells infected by this intracellular bacterium experience oxidant-mediated injury that may eventually contribute to cell death.

Penetration of host cells by Rickettsia rickettsii appears to be mediated by a phospholipase of rickettsial origin.

Internalization of obligate intracellular bacteria belonging to the genus Rickettsia by eukaryotic cells requires participation of both the parasitized host and the microorganism. The term "induced phagocytosis" has been used specifically to describe the entry of Rickettsia prowazekii, although a similar mechanism is likely for R. rickettsii. A role for a phospholipase in the internalization process has been proposed for both of these organisms, with the strongest supporting evidence provided for R. prowazekii. Despite general acceptance of the notion that phospholipase activity is involved in the internalization process of these bacteria, the origin of the enzyme is not known. The results of the study presented here, which used R. rickettsii and Vero cells, suggest that a rickettsial phospholipase, rather than a host cell phospholipase, mediates internalization of the organism. This conclusion is based upon results which show that pretreatment of R. rickettsii, but not of host cells, with a specific chemical inhibitor of phospholipase, and also antiserum to this enzyme, significantly reduces uptake of the organism and its ability to cause plaque formation.