HIV-1 gp120 primes lymphocytes for opioid-induced, beta-arrestin 2-dependent apoptosis.

The mechanisms by which opioids affect progression of human immunodeficiency virus type 1 (HIV-1) infection are not well-defined. HIV-1 gp120 is important in the apoptotic death of uninfected, bystander T cells. In this study, we show that co-treatment of human peripheral blood mononuclear cells (PBMC) with HIV-1 gp120/morphine synergistically induces apoptosis in PBMC. Co-treatment of murine splenocytes from mu opiate receptor knockout mice with gp120/morphine resulted in decreased apoptosis when compared to splenocytes from wild type mice. Co-treatment of human PBMC or murine splenocytes with gp120/morphine led to decreased expression of beta-arrestin 2, a protein required for opioid-mediated signaling. The role of beta-arrestin 2 was confirmed in Jurkat lymphocytes, in which 1) over-expression of beta-arrestin 2 inhibited gp120/morphine-induced apoptosis and 2) RNA interference of beta-arrestin 2 expression enhanced gp120/morphine-induced apoptosis. These data suggest a novel mechanism by which HIV-1 gp120 and opioids induce lymphocyte cell death.

Phosphoethanolamine-complexed C-reactive protein: a pharmacological-like macromolecule that binds to native low-density lipoprotein in human serum.

BACKGROUND: C-reactive protein (CRP) is an acute phase plasma protein. An important binding specificity of CRP is for the modified forms of low-density lipoprotein (LDL) in which the phosphocholine-binding sites of CRP participate. CRP, however, does not bind to native LDL. METHODS: We investigated the interaction of CRP with native LDL using sucrose density gradient ultracentrifugation. RESULTS: We found that the blocking of the phosphocholine-binding sites of CRP with phosphoethanolamine (PEt) converted CRP into a potent molecule for binding to native LDL. In the presence of PEt, CRP acquired the ability to bind to fluid-phase purified native LDL. Because purified native LDL may undergo subtle modifications, we also used whole human serum as the source of native LDL. In the presence of PEt, CRP bound to native LDL in serum also. The effect of PEt on CRP was selective for LDL because PEt-complexed CRP did not bind to high-density lipoprotein in the serum. CONCLUSIONS: The pharmacologic intervention of endogenous CRP by PEt-based compounds, or the use of exogenously prepared CRP-PEt complexes, may turn out to be an effective approach to capture native LDL cholesterol in vivo to prevent the development of atherosclerosis.

Differential regulation of SOCS-1 signalling in B and T lymphocytes by hepatitis C virus core protein.

Hepatitis C virus (HCV) infection is characterized by a strong propensity toward chronicity, autoimmune phenomena and lymphomagenesis, supporting a role for lymphocyte dysregulation during persistent viral infection. We have shown that HCV core protein inhibits T-cell functions through interaction with a complement receptor, gC1qR. Here, we further report that B cells also express gC1qR that can be bound by HCV core protein. Importantly, using flow cytometry, we demonstrated differential regulation of B and T lymphocytes by the HCV core-gC1qR interaction, with down-regulation of CD69 activation in T cells but up-regulation of CD69 activation and cell proliferation in B cells. HCV core treatment led to decreased interferon-gamma production in CD8+ T cells but to increased immunoglobulin M and immunoglobulin G production as well as cell surface expression of costimulatory and chemokine receptors, including CD86 (B7-2), CD154 (CD40L) and CD195 (CCR5), in CD20+ B cells. Finally, we showed down-regulation of suppressor of cytokine signalling-1 (SOCS-1) using real-time reverse transcription-polymerase chain reaction, accompanied by up-regulation of signal transducer and activator of transcription-1 (STAT1) phosphorylation in B cells in response to HCV core protein, with the opposite pattern observed in HCV core-treated T cells. This study demonstrates differential regulation of B and T lymphocytes by HCV core and supports a mechanism by which lymphocyte dysregulation occurs in the course of persistent HCV infection.

C-reactive protein-bound enzymatically modified low-density lipoprotein does not transform macrophages into foam cells.

The formation of low-density lipoprotein (LDL) cholesterol-loaded macrophage foam cells contributes to the development of atherosclerosis. C-reactive protein (CRP) binds to atherogenic forms of LDL, but the role of CRP in foam cell formation is unclear. In this study, we first explored the binding site on CRP for enzymatically modified LDL (E-LDL), a model of atherogenic LDL to which CRP binds. As reported previously, phosphocholine (PCh) inhibited CRP-E-LDL interaction, indicating the involvement of the PCh-binding site of CRP in binding to E-LDL. However, the amino acids Phe66 and Glu81 in CRP that participate in CRP-PCh interaction were not required for CRP-E-LDL interaction. Surprisingly, blocking of the PCh-binding site with phosphoethanolamine (PEt) dramatically increased the binding of CRP to E-LDL. The PEt-mediated enhancement in the binding of CRP to E-LDL was selective for E-LDL because PEt inhibited the binding of CRP to another PCh-binding site-ligand pneumococcal C-polysaccharide. Next, we investigated foam cell formation by CRP-bound E-LDL. We found that, unlike free E-LDL, CRP-bound E-LDL was inactive because it did not transform macrophages into foam cells. The function of CRP in eliminating the activity of E-LDL to form foam cells was not impaired by the presence of PEt. Combined data lead us to two conclusions. First, PEt is a useful compound because it potentiates the binding of CRP to E-LDL and, therefore, increases the efficiency of CRP to prevent transformation of macrophages into E-LDL-loaded foam cells. Second, the function of CRP to prevent formation of foam cells may influence the process of atherogenesis.

T cell dysfunction by hepatitis C virus core protein involves PD-1/PDL-1 signaling.

Reports have shown that a negative T cell costimulatory pathway mediated by PD-1 (programmed death-1) and PDL-1 (programmed death ligand-1) is associated with T cell exhaustion and persistent viral infection. Persistent hepatitis C virus (HCV) infection in humans is also characterized by impaired T lymphocyte function, but the role of the PD-1 and PDL-1 pathway in HCV infection is unknown. Here we report that T cells isolated from chronically HCV-infected patients express significantly higher levels of PD-1 when compared with healthy donors. In addition, PD-1 and PDL-1 expression is upregulated on healthy donor T cells exposed to HCV core, a nucleocapsid protein that is immunosuppressive; upregulation of PD-1 is mediated through interaction of HCV core with the complement receptor, gC1qR. Importantly, T cell functions that are dysregulated by HCV core, including T cell activation, proliferation, and apoptosis, can be restored by blocking PD-1 and PDL-1 engagement. Our results indicate that HCV core can upregulate a key negative T cell signaling pathway associated with viral persistence and highly expressed on the T cells of persistently infected individuals. This upregulation of the PD-1 and PDL-1 pathway in humans represents a novel and perhaps common mechanism by which a virus usurps host machinery to facilitate persistence.

Induction of p38- and gC1qR-dependent IL-8 expression in pulmonary fibroblasts by soluble hepatitis C core protein.

BACKGROUND: Recent studies suggest that HCV infection is associated with progressive declines in pulmonary function in patients with underlying pulmonary diseases such as asthma and chronic obstructive pulmonary disease. Few molecular studies have addressed the inflammatory aspects of HCV-associated pulmonary disease. Because IL-8 plays a fundamental role in reactive airway diseases, we examined IL-8 signaling in normal human lung fibroblasts (NHLF) in response to the HCV nucleocapsid core protein, a viral antigen shown to modulate intracellular signaling pathways involved in cell proliferation, apoptosis and inflammation. METHODS: NHLF were treated with HCV core protein and assayed for IL-8 expression, phosphorylation of the p38 MAPK pathway, and for the effect of p38 inhibition. RESULTS: Our studies demonstrate that soluble HCV core protein induces significant increases in both IL-8 mRNA and protein expression in a dose- and time-dependent manner. Treatment with HCV core led to phosphorylation of p38 MAPK, and expression of IL-8 was dependent upon p38 activation. Using TNFalpha as a co-stimulant, we observed additive increases in IL-8 expression. HCV core-mediated expression of IL-8 was inhibited by blocking gC1qR, a known receptor for soluble HCV core linked to MAPK signaling. CONCLUSION: These studies suggest that HCV core protein can lead to enhanced p38- and gC1qR-dependent IL-8 expression. Such a pro-inflammatory role may contribute to the progressive deterioration in pulmonary function recently recognized in individuals chronically infected with HCV.

The C-terminal region of hepatitis C core protein is required for Fas-ligand independent apoptosis in Jurkat cells by facilitating Fas oligomerization.

Hepatitis C virus (HCV) is remarkable for its ability to establish persistent infection. Studies suggest that HCV core protein modulates immune responses to viral infection and can bind Fas receptor in vitro. To further examine the role of HCV core protein in Fas signaling, full-length (aa 1-192) and truncated (aa 1-152) HCV core proteins were expressed in Jurkat lymphocytes and cells were assayed for apoptotic response, caspase activation, and Fas activation. Jurkat expressing full-length but not truncated core protein exhibited ligand-independent apoptosis. Cytoplasmic targeting of truncated core protein recapitulated its ability to induce apoptosis. Activation of caspases 8 and 3 was necessary and sufficient for full-length core to induce apoptosis. Jurkat cells expressing full-length but not truncated core protein induced Fas receptor aggregation. HCV core activates apoptotic pathways in Jurkat via Fas and requires cytoplasmic localization of core. Infection of host lymphocytes by HCV may alter apoptotic signaling and skew host responses to acute infection.