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.
Sporn
L A
LA
Department of Medicine, University of Rochester School of Medicine & Dentistry, New York 14642, USA.
Sahni
S K
SK
Lerner
N B
NB
Marder
V J
VJ
Silverman
D J
DJ
Turpin
L C
LC
Schwab
A L
AL
D002478:Cells, Cultured; D003572:Cytochalasins / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000382:microbiology*; D006801:Humans; D016328:NF-kappa B / Q000502:physiology*; D011401:Promoter Regions, Genetic; D012284:Rickettsia rickettsii / Q000502:physiology*; D014158:Transcription, Genetic
D003572:Cytochalasins; D016328:NF-kappa B