Inflammatory cytokines inhibit Kaposi's sarcoma-associated herpesvirus lytic gene transcription in in vitro-infected endothelial cells

Abstract

The response of Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) to inflammatory cytokine treatment of experimentally infected endothelial cells was investigated. The cytokines inhibited spontaneous KSHV lytic gene expression but not the level of infection. The data suggest that if inflammatory cytokines present in KS lesions contribute to KSHV pathogenesis, they do so in part by promoting latent KSHV infection of the endothelial cells.

FIG. 1.

Immunohistochemical detection of IFN-γ-producing cells in a paraffin-embedded KS liver biopsy sample. A representative cell producing IFN-γ is indicated by the arrow. Similar data were found in analyses of a further 37 KS biopsy samples from a variety of tissue types (data not shown). (A) tissue stained with anti-IFN-γ primary antibody; (B) negative control with no primary antibody. IFN-γ antigen was retrieved by microwaving in citrate buffer (pH 6.0) and detected by staining with a mouse anti-IFN-γ monoclonal antibody that was detected with the Vectastain Elite and VIP kits (Vector Laboratories). Cells were counterstained with hematoxylin.

FIG. 2.

Electron microscopy of cell-free KSHV(JSC-1). (A) capsids; (B) tegumented virions. Virus was concentrated from sodium butyrate-treated cells before negative staining and visualization by transmission electron microscopy. Scale bar = 150 nm.

FIG. 3.

Immunofluorescence detection of KSHV infection of tDMVEC. (A) low-power magnification showing approximately 68% of cells infected; (B) high-power magnification of nuclei of tDMVEC from an infection separate from that shown in panel A (approximately 50% of the cells are infected). tDMVEC were infected with concentrated KSHV(JSC-1) for 2 days before staining with a rat monoclonal antibody against human herpesvirus 8 LANA-1. Nuclei were revealed by costaining with propidium iodide. Images were collected by confocal microscopy.

FIG. 4.

Infection of tDMVEC with KSHV(JSC-1). (A) uninfected tDMVEC (magnification, ×400); (B) tDMVEC at 7 days postinfection (magnification, ×400). Cells were infected as described in the text, and infection was confirmed by anti-LANA-1 IFA.

FIG. 5.

Modulation of KSHV gene expression by inflammatory cytokines. (A) tDMVEC cultures were infected with KSHV(JSC-1) and then expanded into replicate culture dishes that were treated with either phorbol ester (PMA at 20 ng/ml) or each cytokine individually for 48 h or with all four cytokines in combination for either 17 or 48 h before the effect on ORF50 and ORF29 transcript levels was measured in duplicate samples by real-time RT-PCR. Induction of gene expression was calculated relative to that in parallel cultures of untreated, infected tDMVEC. The data represent the mean values obtained from at least three independent experiments, except the ORF29 transcript measurements on cells treated with each cytokine individually, which were performed in duplicate. Recombinant cytokine concentrations were as follows: TNF-α, 2 ng/ml; IL-1β, 5 ng/ml; IFN-γ, 500 U/ml; IL-6, 100 U/ml. (B) Effect of inflammatory cytokines on the basal transcription activity of the region encompassing 250 bp upstream of the ORF50 translation initiation codon (cloned into a luciferase promoter reporter vector as pORF50/250-luc). Recombinant cytokine concentrations were as follows: TNF-α, 2 ng/ml; IL-1β, 5 ng/ml; IFN-γ, 500 U/ml; IL-6, 100 U/ml. (C) Effects of inflammatory cytokines on the transcription activity of an IFN-γ-responsive promoter in plasmid pGAS-luc. In panels B and C, modulation of firefly luciferase activity by cytokine treatment was calculated relative to the firefly luciferase activity of transfected, untreated cells cultured in parallel, which was assigned an activity of 100%. Each experiment was performed in duplicate and repeated at least three times, and all firefly luciferase data were normalized to Renilla luciferase activity expressed from a separate, constitutively active plasmid that was cotransfected with the firefly luciferase reporter plasmid. This normalization compensates for differences in transfection efficiency between replicate cultures. Recombinant cytokine concentrations: TNF-α, 2 ng/ml; IL-1β, 5 ng/ml; IFN-γ, 500 U/ml; IL-6, 100 U/ml.