Rta of the human herpesvirus 8/Kaposi sarcoma–associated herpesvirus up-regulates human interleukin-6 gene expression

Human interleukin-6 (hIL-6) is a multifunctional cytokine, and dysregulation of hIL-6 is implicated in the pathogenesis of several malignancies such as Kaposi sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman disease (MCD). hIL-6 serves as an autocrine growth factor for cultured AIDS-KS cells and may induce endothelial cell proliferation in KS through a paracrine pathway.1,2 Supernatants from PEL-derived cell lines and PEL effusions contain large quantities of hIL-6.3,4Anti–hIL-6 neutralizing antibodies delayed PEL tumor progression in SCID mice.5 Overproduction of IL-6 also reproduced some manifestations of MCD in a mouse model.6 Furthermore, anti–hIL-6 or anti–hIL-6 receptor antibodies exerted a therapeutic effect on MCD patients.7,8 Taken together, these data strongly support the involvement of hIL-6 in the pathogenesis of these malignancies.

Another common feature of KS, PEL, and MCD is their association with human herpesvirus 8 (HHV-8)/Kaposi sarcoma–associated herpesvirus (KSHV).9-11 HHV-8/KSHV encodes a potent transcriptional activator, Rta, which is necessary and sufficient for initiating viral lytic replication.12,13 Among the lytic genes expressed are homologues of cytokines and chemokines, including viral IL-6 (vIL-6) and viral macrophage inflammatory proteins.14,15In particular, vIL-6 has been detected in tumor lesions and sera from KS, PEL, and MCD patients and is thought to play an important role in viral pathogenesis.16-18 In addition to pirating cellular genes, it is likely that HHV-8/KSHV has developed strategies to enhance its replication by modulating the regulation of cellular factors. We are investigating the effect of Rta on cellular genes and report here that hIL-6 expression is up-regulated by Rta.

The 1.2-kb hIL-6 promoter region was amplified from total cellular DNA using primers F (5′-GGAAGATCTCTCCTGCAAGAGACACCATCCTGA-3′) and R (5′-CGGGAATTCAGGGCAGAATGAGCCTCAGAGACAT3-3′); the underlined nucleotides represent BglII and EcoRI sites, respectively. The PCR fragment was cloned into pSEAP2-basic (Clontech, Palo Alto, CA) to produce phIL6-1200/SEAP.

Transfections were performed in 12-well plates using a standard calcium phosphate method for the human embryonic kidney cell line 293T or LipofectAmine PLUS (Invitrogen, Carlsbad, CA) for the immortalized bone marrow stromal cell line R1T.19 At 48 hours after transfection, supernatants and cells were harvested. Supernatants were assayed for secreted alkaline phosphatase (SEAP) activities, using the Great EscAPe SEAP Chemiluminescence Detection Kit (Clontech). Cells were lysed in 1× passive lysis buffer and assayed forRenilla luciferase activities using the Luciferase Reporter Assay System (Promega, Madison, WI).

pcDNA3/Rta12 or pcDNA3 was transfected into 293T or R1T cells in 6-well plates using LipofectAmine PLUS. pcDNA3/Rta contained a 3.1-kb genomic sequence encoding Rta, whose expression was driven by the cytomegalovirus immediate-early promoter/enhancer in the vector. Supernatants from transfected cells were collected at 24, 48, and 72 hours after transfection and were assayed for hIL-6 protein levels using an hIL-6 enzyme-linked immunosorbent assay (ELISA) kit (Biosource International).

To investigate the role Rta may play in regulatinghIL-6 gene expression, we first examined whether Rta can activate the hIL-6 promoter in a reporter system. A 1200-bp promoter region upstream of the first hIL-6 exon was cloned into the pSEAP2-basic vector to produce phIL6-1200/SEAP. This reporter plasmid was cotransfected into 293T cells with either pcDNA3/Rta (an Rta expression plasmid) or vector alone. To control for transfection efficiency and other experimental variations, pRL-CMV, which constitutively expresses the Renilla luciferase, was included in each transfection. As shown in Figure1A, phIL6-1200/SEAP was potently activated (164-fold) by Rta. To confirm that activation of the hIL-6 promoter was mediated by the Rta protein, we examined the dose dependence of Rta activation. A fixed amount of the reporter plasmid phIL6-1200/SEAP was cotransfected with increasing amounts of pcDNA3/Rta into 293T cells. As the amount of pcDNA3/Rta in each transfection increased, so did the normalized SEAP activity (Figure 1B), indicating that activation of the hIL-6 promoter by Rta is specific.

These results from the reporter system indicate that Rta activates the hIL-6 promoter in the absence of chromatin structure. We next examined whether Rta also activates the endogenous hIL-6 gene. pcDNA3/Rta or pcDNA3 was transfected into 293T cells, and supernatants were harvested at different time points after transfection. The hIL-6 protein levels in these samples were then assayed by ELISA. Consistent with the lack of endogenous hIL-6 expression in 293T cells, the hIL-6 protein levels were low (less than 7.8 pg/mL, the detection limit of the kit) in pcDNA3-transfected cells (Figure2A). However, the expression of Rta in 293T cells stimulated hIL-6 expression and resulted in progressively higher amounts of hIL-6 protein accumulating in the supernatant at 48 and 72 hours after transfection (54.0 and 84.5 pg/mL, respectively).

To further establish the ability of Rta to activate the hIL-6 promoter and to induce hIL-6 protein expression, we performed similar experiments in R1T cells. R1T cells manifest a significant level of basal hIL-6 expression19 and thus complement the use of 293T cells. The reporter plasmid phIL6-1200/SEAP was activated 27-fold by Rta in transient transfection reporter assays in R1T cells (Figure1A). The fold activation in R1T cells was lower than that in 293T cells because of the higher basal level of the reporter plasmid. Moreover, transfection of pcDNA3/Rta stimulated the expression of endogenous hIL-6 in R1T cells, when compared to transfection of pcDNA3, and resulted in hIL-6 levels of 1209, 5762, and 21 447 pg/mL at 24, 48, and 72 hours after transfection, respectively (Figure 2B).

Up-regulation of the hIL-6 gene has emerged as a common theme among herpesvirus infections, and multiple mechanisms may be involved.20-22 In the case of HHV-8/KSHV, latently infected B-cell lines (eg, BC-1 and KS-1) express hIL-6 at high levels.3,4 This is attributed in part to the responsiveness of the hIL-6 promoter to an HHV-8/KSHV-latent gene product, the latency-associated nuclear antigen.19 Because HHV-8/KSHV exists predominantly in a latent state in KS and PEL lesions, the induction of hIL-6 expression by the latency-associated nuclear antigen may play a critical role in the development of these malignancies. Here we have demonstrated that HHV-8/KSHV also stimulates hIL-6 expression through its lytic transcriptional activator, Rta. We hypothesize that activation of hIL-6 by Rta plays an important role in lytic infections. This is especially relevant in patients with HHV-8/KSHV-associated MCD. Our results are consistent with the high plasma hIL-6 levels observed in MCD patients and with the fact that most HHV-8/KSHV–infected cells in MCD lesions express the viral lytic gene expression program driven by Rta.16,17 

Interestingly, in a separate study, we demonstrated that Rta also strongly activates the HHV-8/KSHV vIL-6 gene.23Like hIL-6, vIL-6 promotes the growth of IL-6–dependent B cells and activates signal transduction pathways. However, vIL-6 may stimulate a broader spectrum of target cells because it requires only the ubiquitously expressed gp130 receptor, whereas hIL-6 requires both gp130 and IL-6Rα for signal transduction.14,15,24 On the other hand, the amount of vIL-6 required to stimulate the growth of IL-6–dependent B cells was greater than that of hIL-6, and the binding affinity of vIL-6 for soluble gp130 was determined to be 1000-fold lower than that of hIL-6/soluble IL-6R complex for gp130.25 Therefore, hIL-6 and vIL-6 may both be important in HHV-8/KSHV replication and pathogenesis, but they may play overlapping yet different roles.

We thank Mike Johnson and Jiabin An for excellent technical assistance, Dr Tonia Symensma for critical reading of the manuscript, and members of the Sun and Martinez-Maza laboratories for discussion.