RESUMEN
We report that cocaine may act as cofactor in HIV pathogenesis by increasing dendritic cell-specific C type ICAM-3-grabbing nonintegrin (DC-SIGN) expression on dendritic cells (DC). Our results show that cocaine-using, long-term nonprogressors and normal progressors of HIV infection manifest significantly higher levels of DC-SIGN compared with cocaine-nonusing long-term nonprogressors and normal progressors, respectively. Furthermore, in vitro HIV infection of MDC from normal subjects cultured with cocaine and/or HIV peptides up-regulated DC-SIGN, confirming our in vivo finding. Cocaine, in synergy with HIV peptides, also up-regulates DC-SIGN gene expression by MDC. Furthermore, the cocaine-induced effects were reversed by a D1 receptor antagonist demonstrating the specificity of the reaction. Our results indicate that cocaine exacerbates HIV infection by up-regulating DC-SIGN on DC and these effects are mediated via dysregulation of MAPKs. These data are the first evidence that cocaine up-regulates the expression of DC-SIGN on DC. A better understanding of the role of DC-SIGN in HIV infection may help to design novel therapeutic strategies against the progression of HIV disease in the drug-using population.
Asunto(s)
Antígenos CD/metabolismo , Moléculas de Adhesión Celular/biosíntesis , Cocaína/farmacología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Infecciones por VIH/inmunología , Infecciones por VIH/metabolismo , VIH-1/inmunología , Lectinas Tipo C/biosíntesis , Receptores de Superficie Celular/biosíntesis , Moléculas de Adhesión Celular/genética , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , Células Dendríticas/inmunología , Células Dendríticas/virología , Antagonistas de Dopamina/farmacología , Sinergismo Farmacológico , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Productos del Gen tat/farmacología , Proteína gp120 de Envoltorio del VIH/farmacología , Infecciones por VIH/enzimología , Infecciones por VIH/patología , VIH-1/efectos de los fármacos , Humanos , Lectinas Tipo C/genética , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/inmunología , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/fisiología , Receptores de Superficie Celular/genética , Receptores Dopaminérgicos/metabolismo , Replicación Viral/efectos de los fármacos , Replicación Viral/inmunología , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
Chemokines and their receptors have been implicated in the pathogenesis of neuroAIDS. Herein we describe the effects of morphine on the gene expression of beta chemokines and their receptors by primary normal human astrocytes (NHA). Our results show that NHA treated with morphine showed significant downregulation of the gene expression of beta chemokines, MCP-1, and MIP-1 beta, while reciprocally upregulating the expression of their specific receptors, CCR2b, CCR3, and CCR5 as detected by real-time quantitative PCR. These morphine-induced effects on NHA cells were reversed by the opioid mu receptor antagonist, naloxone. Further, our results indicate that morphine-induced effects are mediated via the modulation of MAPK and CREB signaling pathways. These results support our hypothesis that opiates act as co-factors in the neuropathogenesis of HIV infection.
Asunto(s)
Astrocitos/efectos de los fármacos , Quimiocina CCL2/metabolismo , Regulación de la Expresión Génica , Proteínas Inflamatorias de Macrófagos/metabolismo , Morfina/farmacología , Astrocitos/inmunología , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CCL4 , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Regulación hacia Abajo , Humanos , Proteínas Inflamatorias de Macrófagos/genética , Naloxona/farmacología , Receptores Opioides mu/antagonistas & inhibidores , Receptores Opioides mu/biosíntesis , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Dendritic cells are the critical mediators of various immune responses and are the first line of defense against any infection including HIV. They play a major role in harboring HIV and the subsequent infection of T cells and passage of virus through the blood-brain barrier (BBB). The recently discovered DC-specific, CD4-independent HIV attachment receptor, DC-SIGN, and T-cell suppressing factor, indolamine 2,3-dioxygenase (IDO), are known to play a critical role in the immuno-neuropathogenesis of HIV infection. Since brain microvascular cells (BMVEC) express dendritic cell (DC)-specific C type ICAM-3 grabbing nonintegrin (DC-SIGN), it is possible that DC-SIGN may play a critical role in human immunodeficiency virus-type 1 (HIV-1) infection and migration of infected DC across BBB. Matrix metalloproteinases (MMPs) are proteolytic enzymes known to be responsible for maintenance, turnover and integrity of extracellular matrix. Our results show that cocaine upregulates IDO and DC-SIGN expression by DC. Further, cocaine upregulates DC-SIGN and MMPs in BMVEC supporting the hypothesis that cocaine causes membrane permeability facilitating endothelial transmigration of infected DC in to the CNS. Targeting DC-SIGN and IDO with specific monoclonal antibodies, inexpensive synthetic antagonists, antisense oligonucleotides and siRNA may lead to develop novel treatment strategies particularly in high-risk populations such as cocaine users.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Cocaína/farmacología , Células Dendríticas/efectos de los fármacos , Dioxigenasas/metabolismo , Infecciones por VIH/complicaciones , Lectinas Tipo C/metabolismo , Receptores de Superficie Celular/metabolismo , Trastornos Relacionados con Sustancias/complicaciones , Anestésicos Locales/farmacología , Moléculas de Adhesión Celular/genética , Células Cultivadas , Células Dendríticas/metabolismo , Dioxigenasas/genética , Relación Dosis-Respuesta a Droga , Infecciones por VIH/enzimología , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa , Lectinas Tipo C/genética , Metaloproteinasas de la Matriz/genética , Metaloproteinasas de la Matriz/metabolismo , Metaloproteinasas de la Matriz Asociadas a la Membrana , Metaloendopeptidasas/genética , Metaloendopeptidasas/metabolismo , ARN Mensajero/biosíntesis , Receptores de Superficie Celular/genéticaRESUMEN
We hypothesize that expression of proangiogenic genes correlates with the metastatic potential of prostate cancer cells. LNCaP, DU-145, and PC-3 are prostate cancer cell lines with low, moderate, and high metastatic potential, respectively, as we demonstrated by their capacity to invade an extracellular matrix, an established tumor invasion assay. The constitutive gene expression of the proangiogenic factors, vascular endothelial growth factor, intercellular adhesion molecule-1, interleukin-8, and transforming growth factor-beta2, was significantly greater in the more metastatic DU-145 and PC-3 cells as compared with LNCaP cells. Matrix metalloproteinase (MMP)-9 is thought to contribute to the invasive phenotype of tumor cells. PC-3 cells showed increased expression of MMP-9 and membrane type 4-MMP as compared with LNCaP and DU-145. Tissue inhibitors of metalloproteinase 1 and 4 gene expression were elevated in DU-145 and PC-3 cells, but paradoxically, LNCaP cells had undetectable levels of these genes. We transfected and overexpressed MMP-9 in poorly metastatic LNCaP cells and measured their invasive activity. Transient expression of human MMP-9 in LNCaP cells produced a 3-5-fold increase in MMP-9 activity with a comparable increase in invasiveness. Antisense ablation of the expression of MMP-9 in DU-145 and PC-3 cells produced concomitant inhibition of the gene expression of the proangiogenic factors, vascular endothelial growth factor, and intercellular adhesion molecule-1 (ICAM-1). Treatment of DU-145 and PC-3 cells with a selective chemical inhibitor of MMP-9 proteinase activity also inhibited their invasive activity. These results support our hypothesis that metastatic potential of prostate cancer cells correlates with expression of proangiogenic factors.
Asunto(s)
Inductores de la Angiogénesis/metabolismo , Proteínas Angiogénicas/genética , Regulación Neoplásica de la Expresión Génica/fisiología , Metástasis de la Neoplasia , Neoplasias de la Próstata/patología , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Interleucina-8/antagonistas & inhibidores , Interleucina-8/metabolismo , Masculino , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Inhibidores de la Metaloproteinasa de la Matriz , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta2 , Células Tumorales CultivadasRESUMEN
CD8+ cytotoxic T lymphocyte (CTL) activity is currently believed to be one of the key immunologic mechanisms responsible for the prevention or attenuation of HIV-1 infection. The induction of CD8+ T cell activation may also result in the production of soluble or non-classical lytic factors that are associated with protection from infection or slower disease progression. Traditionally, CD8+ CTL responses have been measured by the classic chromium release assay, monitoring the ability of T cells (Effector cells) to lyse radiolabelled HLA - matched "target cells" that express the appropriate antigen-MHC complex. This method is not only labor intensive, semi quantitative assay at best, but also needs fresh, non-cryopreserved cells. Recently, cytokine specific ELISPOT assays or tetrameric MHC-I/ peptide complexes have utilized to directly quantitate circulating CD8+ effector cells, and these assays are more sensitive, quantitative and reproducible than the traditional CTL lysis assay and can also be performed on cryopreserved cells. Although these are reproducible assays for the assessment of soluble antiviral activity secreted by activated T cell populations they can be extremely expensive to perform. We have used FACS Analysis to measure Granzyme B release as a function of cell mediated cytotoxicity. This method helps quantitate the CTL activity and also identifies the phenotype of the cells elucidating this immune response. The method described not only monitors immunological response but also is also simple to perform, precise and extremely time efficient and is ideal for screening a large number of samples.
RESUMEN
The brain is a target organ for recreational drugs and HIV-1. Epidemiological data demonstrate that opioid abuse is a risk factor for HIV-1 infection and progression to AIDS. Chemokines and their receptors have been implicated in the neuropathogenesis of HIV-1 infections. However, little is known about the effects of opioids on the expression of chemokines and their receptors (the latter also are HIV-1 coreceptors) by cells of the CNS. Herein we describe the effects of morphine on gene expression of the alpha- and beta-chemokines and their receptors by the astrocytoma cell line U87 and by primary normal human astrocyte (NHA) cultures. U87 cells treated with morphine showed significant down-regulation of IL-8 gene expression, whereas expression of the IL-8 receptor CXCR2 was reciprocally up-regulated as detected by RT-PCR. Treatment of NHAs with morphine suppressed IL-8 and macrophage-inflammatory protein-1beta gene expression, whereas expression of their receptor genes, CCR3 and CCR5, was simultaneously enhanced. These morphine-induced effects on U87 and NHA cells were reversed by the opioid mu receptor antagonist beta-funaltrexamine. Morphine also enhanced the constitutive expression of the opioid mu receptor on astroglial cells. Our results support the hypothesis that opioids play a significant role in the susceptibility of the CNS to HIV-1 infection and subsequent encephalopathy by inhibiting local production of HIV-1-protective chemokines (IL-8 and macrophage-inflammatory protein-1beta) and enhancing expression of HIV-1 entry coreceptor genes (CCR3, CCR5, and CXCR2) within the CNS. These effects of opioids appear to be mediated through the opioid mu receptor that we demonstrated on astroglial cells.