Latent Membrane Protein 1 of EBV Activates Phosphatidylinositol 3-Kinase to Induce Production of IL-10

Abstract

EBV is a B lymphotrophic gamma-herpesvirus that is associated with multiple human malignancies, including posttransplant lymphoproliferative disorder. The EBV-encoded protein, latent membrane protein 1 (LMP1), is required for oncogenic transformation of human B cells by EBV. An important consequence of LMP1 expression in EBV-infected B cells is the induction of cellular IL-10, which acts as an autocrine growth factor for B cell lymphomas. However, the mechanisms by which LMP1 induces IL-10 are incompletely understood. We previously showed that rapamycin, a clinically relevant immunosuppressant and mammalian target of rapamycin inhibitor, could suppress IL-10 production by EBV-infected B cell lines. To test the hypothesis that PI3K, which acts upstream of mammalian target of rapamycin, might also be involved in LMP1-dependent IL-10 production, we generated B cell lines expressing signaling-inducible chimeric LMP1. Our results show that induced LMP1 signaling elicits both p38- and PI3K-dependent IL-10 production in EBV- B cells. Moreover, distinct regions of the LMP1 signaling tail are associated with p38- vs PI3K-dependent IL-10 induction. We also demonstrate that the LMP1-dependent p38 and PI3K activation regulates IL-10 induction through discrete mechanisms. Whereas p38 activation is critical for the phosphorylation of the transcription factor CREB, PI3K activation is required for the inactivation of glycogen synthase kinase 3beta (GSK3beta), an inhibitory kinase that can regulate CREB function. We find that GSK3beta regulates LMP1-dependent IL-10 induction, with GSK3beta inhibition by pharmacologic or small interfering RNA strategies enhancing LMP1-induced IL-10 induction. These findings demonstrate that LMP1 uses both p38 and PI3K activation for maximal up-regulation of IL-10.

Epstein-Barr Virus Zta-Induced Immunomodulators from Nasopharyngeal Carcinoma Cells Upregulate Interleukin-10 Production from Monocytes

“…BCRF1 is the EBV homolog of IL-10 and exerts immunosuppressive functions at the late stage of lytic infection (53,60). Furthermore, in B cells, expression of human IL-10 can be induced by some EBV gene products, including the immediate-early lytic protein Zta and two latent products, latent membrane protein 1 (LMP1) and EBV-encoded small RNA (EBER) (35,42,54). On the other hand, how EBV regulates IL-10 expression in the context of epithelial cells is unclear.…”

Syk Activation of Phosphatidylinositol 3-Kinase/Akt Prevents HtrA2-dependent Loss of X-linked Inhibitor of Apoptosis Protein (XIAP) to Promote Survival of Epstein-Barr Virus+ (EBV+) B Cell Lymphomas

“…Data are representative of n Ն 3 independent experiments in which Syk knockdown was Ն 90%. (44) and is required for the autonomous proliferation of PTLD lines (48). Whereas the endogenously high secretion of the autocrine growth factor IL-10 was markedly decreased after Syk inhibition in our R406-sensitive, EBVϩ PTLD-derived B cell lines (Fig.…”

“…After 48 h, viable cells were counted by trypan blue exclusion and supernatant. IL-10 production was determined as previously described (44). Briefly, supernatants were added to coated (anti-IL-10 antibody; Pierce), blocked (5% nonfat dry milk in PBS), 96-well Immulon plates for 2 h. Bound cytokine was detected with biotinylated anti-IL-10 and streptavidin-HRP (both from BD Pharmingen) by substrate cleavage (tetramethlybenzidine, Sigma) quantified using a 96-well microplate reader (Molecular Devices).…”

Maintenance of Epstein-Barr Virus Latent Status by a Novel Mechanism, Latent Membrane Protein 1-Induced Interleukin-32, via the Protein Kinase Cδ Pathway

“…Previous studies demonstrated that IL-32 enhances IL-6, IL-8, and IL-10 (29,46,47). In addition, these cytokines have been induced by LMP1 (48)(49)(50). To explore the effects of IL-32-induced cytokine expression, we depleted IL-32 expression in LCLs.…”