Rickettsia rickettsii infection of cultured human endothelial cells induces NF-kappa B 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 K. rickettsii-induced activation of the nuclear factor-kappa B/ReI (NF-kappa B) family of transcription factors involved in early transcriptional responses to injurious stimuli, Two NF-kappa B species were activated by infection and reacted with a doable-stranded oligonucleotide probe corresponding to the kappa B 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-slate levels of mRNA coding for the inhibitory subunit of NF-kappa B (I kappa B alpha, transcription of which is enhanced bg the binding of NF-kappa B within the I kappa B alpha promoter region. NF-kappa B 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 13 h, and even higher levels of activation at 24 h. It is likely that NF-kappa B 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, B, rickettsii-induced activation of NF-kappa B may be an important controlling factor in the transcriptional responses of EC to infection with this obligate intracellular organism.