Induction of HHV-8 lytic cycle replication by inflammatory cytokines produced by HIV-1-infected T cells

Abstract

Human herpesvirus 8 (HHV-8) is a gamma2-herpesvirus consistently identified in Kaposi's sarcoma (KS), primary effusion lymphoma, and multicentric Castleman's disease. Although HHV-8 infection appears to be necessary, it may not be sufficient for development of KS without the involvement of other cofactors. One potentially important cofactor is HIV-1. HIV-1-infected cells produce HIV-1-related proteins and cytokines, both of which have been shown to promote growth of KS cells in vitro. Though HIV-1 is not absolutely necessary for KS development, KS is the most frequent neoplasm in AIDS patients, and AIDS-KS is recognized as a particularly aggressive form of the disease. To determine whether HIV-1 could participate in the pathogenesis of KS by modulating HHV-8 replication (rather than by inducing immunodeficiency), HIV-1-infected T cells were cocultured with the HHV-8-infected cell line, BCBL-1. The results demonstrate soluble factors produced by or in response to HIV-1-infected T cells induced HHV-8 replication, as determined by production of lytic phase mRNA transcripts, viral proteins, and detection of progeny virions. By focusing on cytokines produced in the coculture system, several cytokines known to be important in growth and proliferation of KS cells in vitro, particularly Oncostatin M, hepatocyte growth factor/scatter factor, and interferon-gamma, were found to induce HHV-8 lytic replication when added individually to BCBL-1 cells. These results suggest specific cytokines can play an important role in the initiation and progression of KS through reactivation of HHV-8. Thus, HIV-1 may participate more directly than previously recognized in KS by promoting HHV-8 replication and, hence, increasing local HHV-8 viral load.

Figure 1.

Expression of ORF26 mRNA is increased in BCBL-1 cells following coculture with CEM + HIV-1_IIIB. BCBL-1 cells were cultured in Transwell dishes with uninfected or HIV-1_IIIB-infected CEM cells as indicated. Lane 1, BCBL-1 cells cultured with CEM cells, 72 hours (2.7-fold increase compared to BCBL-1 cells alone); lane 2, BCBL-1 cells cultured with CEM + HIV-1_IIIB, 24 hours (1.8-fold increase); lane 3, BCBL-1 cells cultured with CEM + HIV-1_IIIB, 48 hours (4.8-fold increase); lane 4, BCBL-1 cells cultured with CEM + HIV-1_IIIB, 72 hours (5.5-fold increase); lane 5, BCBL-1 cells cultured with CEM + HIV-1_IIIB, 96 hours (7.0-fold increase); lane 6, BCBL-1 cells alone. A representative experiment is shown; seven independent experiments were run with similar results (increased ORF26 mRNA at 96 hours of coculture ranged from 6.0- to 12.9-fold increase).

Figure 2.

RT-PCR analysis for ORF26 mRNA expression in BC-1 cells cocultured with CEM + HIV-1_IIIB. Transcripts for ORF26 were not detected in unstimulated BC-1 cells, but were induced after coculture with CEM + HIV-1_IIIB or TPA stimulation (positive control). β-actin was readily detectable in all samples indicating the presence of amplifiable cDNA, and no bands were seen in control reactions performed in the absence of RT, indicating that the RNA template was not contaminated with DNA (data not shown).

Figure 3.

Northern blot analysis for ORF26 mRNA in BCBL-1 cells cultured for 24 to 72 hours in 50% conditioned media. ORF26 mRNA was increased following culture of BCBL-1 cells for 24 hours with conditioned media (10.8-fold increase compared with untreated BCBL-1 cells) which decreased at both the 48- and 72-hour time points (7.1-fold and 2.4-fold increase compared to untreated cells, respectively).

Figure 4.

Immunohistochemical staining of BCBL-1 cells cocultured in Transwell dishes with CEM or CEM + HIV-1_IIIB (original magnification, ×60). Top row (ORF59 mAb): CEM cells (0% positive), untreated BCBL-1 cells (1.3% positive), BCBL-1 cells cultured with uninfected CEM cells for 8 days (16.4% positive), BCBL-1 cells cultured with CEM + HIV-1_IIIB for 8 days (24.8% positive). Inset figures are enlarged to show nuclear staining in the indicated samples with ORF59 mAb. Middle row (K8.1 mAb): CEM cells (0% positive), untreated BCBL-1 cells (1.1% positive), BCBL-1 cells cocultured with CEM cells for 10 days (4.6% positive), BCBL-1 cells cocultured with CEM + HIV-1_IIIB for 10 days (7.9% positive). Bottom row (CD4 mAb): CEM cells (88.6% positive), untreated BCBL-1 cells (0.1% positive), BCBL-1 cells cocultured with CEM cells cultured for 10 days (0% positive), BCBL-1 cells cocultured with CEM + HIV-1_IIIB for 10 days (0.4% positive). Note the low level of nonspecific mAb binding using the isotype-matched control mAb (CD4) in the coculture conditions. **Analysis of variance demonstrates statistically significant increases in K8.1 and/or ORF59 expression compared to untreated BCBL-1 cells.

Figure 5.

Northern blot analysis of ORF26 expression in BCBL-1 cells cocultured with normal human PBMCs infected with HIV-1_IIIB or HIV-1_SF2. Lane 1: BCBL-1 alone, 72 hours; lane 2, BCBL with PBMC + HIV-1_IIIB, 24 hours (1.6-fold increase); lane 3, BCBL with PBMC + HIV-1_IIIB, 48 hours (4.2-fold increase); lane 4, BCBL with PBMC + HIV-1_IIIB, 72 hours (8.2-fold increase); lane 5, BCBL with PBMC + HIV-1_IIIB, 96 hours (8.4-fold increase); lane 6, BCBL with PBMC + HIV-1_SF2, 24 hours (1.1-fold increase); lane 7, BCBL with PBMC + HIV-1_SF2, 48 hours (4.5-fold increase); lane 8, BCBL with PBMC + HIV-1_SF2, 72 hours (6.4-fold increase); lane 9, BCBL with PBMC + HIV-1_SF2, 96 hours (12.8-fold increase).

Figure 6.

Northern blot analysis of ORF26 mRNA expression after stimulation of BCBL-1 cells with recombinant cytokines. A: RNA isolated 3 days after initiation of culture. Lane 1, untreated BCBL-1 cells; lane 2, OSM (2.7-fold increase); lane 3, HGF/SF (3.0-fold increase); lane 4, IFN-γ (4.4-fold increase); lane 5, IL-6 (0.8-fold increase); lane 6, TNF-α (0.5-fold increase); lane 7, IL-2 (no change). B: RNA isolated 7 days after initiation of culture. Lane 1, untreated; lane 2, OSM (3.5-fold increase); lane 3, HGF/SF (4.6-fold increase); lane 4, IFN-γ (4.2-fold increase); lane 5, TNF-α (1.0-fold increase); lane 6, IL-2 (1.7-fold increase); lane 7, Combination of cytokines (4.3-fold increase). Three independent experiments were run with similar results.

Figure 7.

Immunohistochemical staining of cytokine-stimulated BCBL-1 cells (original magnification, ×60). BCBL-1 cells were stimulated for 7 days with recombinant cytokines, cytospin preparations were made and stained for expression of HHV-8 ORF K8.1. BCBL-1 cells alone: 0.4% positive; BCBL-1 cells stimulated with 0.3 mmol/L n-butyrate for 96 hours: 10.5-fold increase in K8.1 expression compared to BCBL-1 alone; BCBL-1 cells stimulated with IFN-γ: 3.6-fold increase (P < 0.05); BCBL-1 cells stimulated with OSM: 4.2-fold increase (P < 0.05); BCBL-1 cells stimulated with HGF/SF: 5.1-fold increase (P < 0.05). Stimulation with IL-2 (1.3-fold increase), TNF-α (1.2-fold increase) or IL-6 (1.7-fold increase) did not result in significant induction of HHV-8 K8.1 (data not shown). Electron microscopy (original magnification, ×20,000) from the BCBL-1 cells stimulated with HGF/SF for 7 days demonstrates the presence of both intranuclear nucleocapsids (arrows) and extracellular virions (data not shown).