HIV Neuropathogenesis in the Presence of a Disrupted Dopamine System.

Antiretroviral therapy (ART) has transformed HIV into a chronic condition, lengthening and improving the lives of individuals living with this virus. Despite successful suppression of HIV replication, people living with HIV (PLWH) are susceptible to a growing number of comorbidities, including neuroHIV that results from infection of the central nervous system (CNS). Alterations in the dopaminergic system have long been associated with HIV infection of the CNS. Studies indicate that changes in dopamine concentrations not only alter neurotransmission, but also significantly impact the function of immune cells, contributing to neuroinflammation and neuronal dysfunction. Monocytes/macrophages, which are a major target for HIV in the CNS, are responsive to dopamine. Therefore, defining more precisely the mechanisms by which dopamine acts on these cells, and the changes in cellular function elicited by this neurotransmitter are necessary to develop therapeutic strategies to treat neuroHIV. This is especially important for vulnerable populations of PLWH with chemically altered dopamine concentrations, such as individuals with substance use disorder (SUD), or aging individuals using dopamine-altering medications. The specific neuropathologic and neurocognitive consequences of increased CNS dopamine remain unclear. This is due to the complex nature of HIV neuropathogenesis, and logistical and technical challenges that contribute to inconsistencies among cohort studies, animal models and in vitro studies, as well as lack of demographic data and access to human CNS samples and cells. This review summarizes current understanding of the impact of dopamine on HIV neuropathogenesis, and proposes new experimental approaches to examine the role of dopamine in CNS HIV infection. Graphical abstract HIV Neuropathogenesis in the Presence of a Disrupted Dopamine System. Both substance abuse disorders and the use of dopaminergic medications for age-related diseases are associated with changes in CNS dopamine concentrations and dopaminergic neurotransmission. These changes can lead to aberrant immune function, particularly in myeloid cells, which contributes to the neuroinflammation, neuropathology and dysfunctional neurotransmission observed in dopamine-rich regions in HIV+ individuals. These changes, which are seen despite the use antiretroviral therapy (ART), in turn lead to further dysregulation of the dopamine system. Thus, in individuals with elevated dopamine, the bi-directional interaction between aberrant dopaminergic neurotransmission and HIV infection creates a feedback loop contributing to HIV associated neurocognitive dysfunction and neuroHIV. However, the distinct contributions and interactions made by HIV infection, inflammatory mediators, ART, drugs of abuse, and age-related therapeutics are poorly understood. Defining more precisely the mechanisms by which these factors influence the development of neurological disease is critical to addressing the continued presence of neuroHIV in vulnerable populations, such as HIV-infected older adults or drug abusers. Due to the complexity of this system, understanding these effects will require a combination of novel experimental modalities in the context of ART. These will include more rigorous epidemiological studies, relevant animal models, and in vitro cellular and molecular mechanistic analysis.

Shedding of PECAM-1 during HIV infection: a potential role for soluble PECAM-1 in the pathogenesis of NeuroAIDS.

Human immunodeficiency virus (HIV) infection is characterized by viral entry into the central nervous system (CNS), which is mediated, in part, by the transmigration of HIV-infected monocytes into the brain. The elaboration of chemokines and other factors by these infected cells contributes to CNS inflammation and cognitive impairment in a significant number of HIV-infected individuals. Recently, we demonstrated that HIV-infected monocyte transmigration into the CNS is enhanced greatly by the chemokine CC chemokine ligand 2 (CCL2)/monocyte chemoattractant protein-1. Platelet endothelial cell adhesion molecule-1 (PECAM-1) plays an important role in leukocyte transmigration across the endothelium of the systemic vasculature by mediating homophilic interactions between endothelial cells (EC)-EC and EC-leukocytes, thus preserving vessel integrity. The role of PECAM-1 in HIV-infected leukocyte transmigration across the blood brain barrier (BBB) and NeuroAIDS has not been characterized. We demonstrate that in brain tissue from individuals with HIV encephalitis, there is an accumulation of cleaved, soluble forms of the extracellular region of PECAM-1 (sPECAM-1). In addition, HIV-infected individuals have elevated levels of sPECAM-1 in their sera. Our in vitro data demonstrate that HIV-infected leukocytes, when treated with CCL2, shed sPECAM-1, suggesting a mechanism of extracellular PECAM-1 cleavage and release dependent on HIV infection and CCL2. We hypothesize that sPECAM-1 production by HIV-infected leukocytes, resulting in the accumulation of sPECAM-1 within the CNS vasculature and the generation of truncated, intracellular forms of PECAM-1 within leukocytes, alters PECAM-1 interactions between EC-EC and EC-leukocytes, thus contributing to enhanced transmigration of HIV-infected leukocytes into the CNS and changes in BBB permeability during the pathogenesis of NeuroAIDS.

MCP-1 (CCL2) protects human neurons and astrocytes from NMDA or HIV-tat-induced apoptosis.

Acquired immunodeficiency syndrome (AIDS)-associated dementia is often characterized by chronic inflammation, with infected macrophage infiltration of the CNS resulting in the production of human immunodeficiency virus type 1 (HIV-1) products, including tat, and neurotoxins that contribute to neuronal loss. In addition to their established role in leukocyte recruitment and activation, we identified an additional role for chemokines in the CNS. Monocyte chemoattractant protein-1 (MCP-1 or CCL2) and regulated upon activation normal T cell expressed and secreted (RANTES) were found to protect mixed cultures of human neurons and astrocytes from tat or NMDA-induced apoptosis. Neuronal and astrocytic apoptosis in these cultures was significantly inhibited by co-treatment with MCP-1 or RANTES but not IP-10. The protective effect of RANTES was blocked by antibodies to MCP-1, indicating that RANTES protection is mediated by the induction of MCP-1. The NMDA blocker, MK801, also abolished the toxic effects of both tat and NMDA. Tat or NMDA treatment of mixed cultures for 24 h resulted in increased extracellular glutamate ([Glu]e) and NMDA receptor 1 (NMDAR1) expression, potential contributors to apoptosis. Co-treatment with MCP-1 inhibited tat and NMDA-induced increases in [Glu]e and NMDAR1, and also reduced the levels and number of neurons containing intracellular tat. These data indicate that MCP-1 may play a novel role as a protective agent against the toxic effects of glutamate and tat.

Mycophenolate mofetil treatment reduces atherosclerosis in the cholesterol-fed rabbit.

Immune/inflammatory responses of arterial vessel wall constituents to lipid metabolic disturbances have been postulated to contribute to the pathogenesis of atherosclerosis. Mycophenolate mofetil (MMF), an antiproliferative agent used in clinical transplantation, has been shown to inhibit smooth muscle cell (SMC) proliferation and decrease the recruitment of monocytes into sites of chronic inflammation. This study was conducted to determine the effect of MMF on atherosclerotic plaque development after cholesterol-induced injury. New Zealand white rabbits were fed a high-cholesterol diet containing 0.5% cholesterol and 8% peanut oil. The experimental group (n = 10) was given MMF (80 mg/kg/day subcutaneously); the control group (n = 10) received placebo injections. The aortas were harvested at 12 weeks for immunohistochemical analyses. SMCs were identified by reactivity with a monoclonal antibody (mAb) to alpha smooth muscle actin. Monocytes/macrophages were detected with mAb RAM 11. Cross-sectional areas of the media and neointima were measured using computer-assisted image analysis. The density of SMCs and macrophage/foam cells within the neointima was calculated by dividing the number of cells by the area of the plaque. Total cholesterol, triglyceride, high density lipoprotein, and low density lipoprotein were significantly increased compared with levels before the initiation of a high-cholesterol diet, but there were no significant differences between the MMF-treated and untreated groups. Neointimal area in aortic tissue sections of the MMF-treated group (0.586 +/- 0.602 mm(2)) was significantly lower when compared with that in control animals (1.082 +/- 0.621 mm(2)) (P < 0.05). The densities of neointimal SMCs and monocytes/macrophages in the control group were 778 +/- 293 and 341 +/- 90 cells/mm(2), respectively. MMF treatment significantly reduced the number of neointimal SMCs (506 +/- 185 cells/mm(2)) (P < 0.05). The number of monocytes/macrophages was also reduced after MMF treatment (260 +/- 124 cells/mm(2)) but not significantly. Our results demonstrate that the administration of MMF significantly reduced neointimal SMC accumulation and plaque development in a hypercholesterolemic model of atherosclerosis.

Induction of IG9 monocyte adhesion molecule expression in smooth muscle and endothelial cells after balloon arterial injury in cholesterol-fed rabbits.

The expression of monocyte-specific adhesion molecules and chemokines by cell types within the vessel wall plays an important role in foam cell accumulation during atherosclerotic plaque development. We previously identified IG9, a novel monocyte adhesion protein that is expressed on endothelial cells (ECs) overlying human and rabbit advanced atherosclerotic plaques. The present study was designed to determine the temporal and spatial expression of IG9 and the chemokine, monocyte chemoattractant protein-1 (MCP-1), after balloon injury with (double injury) or without (single injury) prior air desiccation EC injury in the femoral arteries of rabbits fed a high-cholesterol diet. By immunohistochemical analyses, intense reactivity with monoclonal antibodies to IG9 and MCP-1 was detected 24 hours after single injury in medial smooth muscle cells (SMCs) and in SMCs of adventitial microvessels. However, monocyte infiltration of the tunica media was minimal or not detected in these sections. IG9 and MCP-1 antibody reactivity in vessel sections 28 days after single injury and 24 hours, 7 days, and 28 days after double injury was localized to medial and neointimal SMCs, foam cells, and luminal ECs overlying the plaques. Uninjured rabbit (cholesterol or normal diet) vessel sections exhibited minimal IG9 and MCP-1 immunostaining. In vitro studies using human aortic SMCs demonstrated IG9 protein induction after 24 hours of treatment with platelet-derived growth factor-BB and interferon-gamma or epidermal growth factor. IG9 expression was further increased by pretreatment of SMCs with the proatherogenic lipid, minimally oxidized low density lipoprotein. After balloon injury (24 hours), IG9 is induced in vascular SMCs before the detectable accumulation of monocytes within the vessel wall. Thus, the expression of IG9 by SMCs as well as by ECs may be an important factor in the accumulation of foam cells in atherosclerotic plaque development after arterial injury.

Human vascular smooth muscle cells possess functional CCR5.

CC chemokine receptors are important modulators of inflammation. Although CC chemokine receptors have been found predominantly on leukocytes, recent studies have suggested that vascular smooth muscle cells respond to CC chemokines. We now report that human smooth muscle cells express CCR5, a co-receptor for human immunodeficiency virus. CCR5 mRNA was detectable by RNA blot hybridization in human aortic and coronary artery smooth muscle cells. The cDNA generated by reverse transcription-polymerase chain reaction from aortic smooth muscle cells had 100% identity throughout the entire coding region with the CCR5 cloned from THP-1 cells. By immunohistochemistry, CCR5 and the CCR5 ligand, macrophage inflammatory protein-1beta (MIP-1beta), were detected in smooth muscle cells and macrophages of the atherosclerotic plaque. In smooth muscle cell culture, MIP-1beta induced a significant increase in intracellular calcium concentrations, which was blocked by an antibody to CCR5. In addition, MIP-1beta caused a calcium-dependent increase in tissue factor activity. Tissue factor is the initiator of coagulation and is thought to play a key role in arterial thrombosis. These data suggest that human arterial smooth muscle cells express functional CCR5 receptors and MIP-1beta is an agonist for these cells.

Infection of endothelial cells with Trypanosoma cruzi activates NF-kappaB and induces vascular adhesion molecule expression.

Transcriptional activation of vascular adhesion molecule expression, a major component of an inflammatory response, is regulated, in part, by the nuclear factor-kappaB/Rel (NF-kappaB) family of transcription factors. We therefore determined whether Trypanosoma cruzi infection of endothelial cells resulted in the activation of NF-kappaB and the induction or increased expression of adhesion molecules. Human umbilical vein endothelial cells (HUVEC) were infected with trypomastigotes of the Tulahuen strain of T. cruzi. Electrophoretic mobility shift assays with an NF-kappaB-specific oligonucleotide and nuclear extracts from T. cruzi-infected HUVEC (6 to 48 h postinfection) detected two major shifted complexes. Pretreatment with 50x cold NF-kappaB consensus sequence abolished both gel-shifted complexes while excess SP-1 consensus sequence had no effect. These data indicate that nuclear extracts from T. cruzi-infected HUVEC specifically bound to the NF-kappaB consensus DNA sequence. Supershift analysis revealed that the gel-shifted complexes were comprised of p65 (RelA) and p50 (NF-kappaB1). Northern blot analyses demonstrated both the induction of vascular cell adhesion molecule 1 and E-selectin and the upregulation of intercellular adhesion molecule 1 mRNA in HUVEC infected with T. cruzi. Immunocytochemical staining confirmed adhesion molecule expression in response to T. cruzi infection. These findings are consistent with the hypothesis that the activation of the NF-kappaB pathway in endothelial cells associated with T. cruzi infection may be an important factor in the inflammatory response and subsequent vascular injury and endothelial dysfunction that lead to chronic cardiomyopathy.

An endothelial cell adhesion protein for monocytes recognized by monoclonal antibody IG9. Expression in vivo in inflamed human vessels and atherosclerotic human and Watanabe rabbit vessels.

BACKGROUND: Monocyte adhesion to the vascular endothelium, an important component of an inflammatory response, is one of the earliest detected events in the pathogenesis of atherosclerosis. We have identified a monocyte adhesion molecule, recognized by monoclonal antibody (mAb) IG9, on the cell surface of human umbilical vein endothelial cells (HUVEC) treated with tumor necrosis factor-alpha (TNF-alpha), interleukin-1, or lipopolysaccharide. Endothelial cell expression in vitro and in vivo of the protein recognized by mAb IG9 (IG9 protein) was further characterized. EXPERIMENTAL DESIGN: The kinetics of cytokine-induced IG9 protein expression on HUVEC were evaluated by enzyme-linked immunosorbent assay. TNF-alpha-treated HUVEC surface proteins, labeled with [125I]Na, were solubilized in NP-40 detergent and immunoprecipitated with mAb IG9 to determine the molecular weight of the IG9 protein. The functional role of the IG9 protein in monocyte binding in vitro to cytokine-activated endothelial cells was established in adhesion assays utilizing U937 cells (human promyelomonocytic cell line) and human peripheral blood monocytes. Minimally oxidized or modified low density lipoproteins (MM-LDL) have previously been shown to induce monocyte adhesion to endothelial cells for up to 48 hours after exposure. In order to characterize the adhesion molecule(s) contributing to this increase in monocyte binding, MM-LDL-treated HUVEC and human aortic endothelial cells were assayed for monocyte adhesion molecule expression by enzyme-linked immunosorbent assay. In addition, mAb IG9-mediated alterations in MM-LDL-induced monocyte binding were studied in endothelial-monocyte adhesion assays. To assess IG9 protein expression in vivo, formalin-fixed, paraffin-embedded sections of inflamed human tissues obtained from lung and healing myocardial infarctions, in addition to sections of human atherosclerotic coronary arteries, were analyzed by immunohistochemistry. Tissue sections from atherosclerotic Watanabe heritable hyperlipidemic rabbit aortas were also included in these studies. RESULTS: The IG9 protein, undetected on untreated HUVEC, was expressed on their cell surface within 3 hours of treatment with TNF-alpha, peaked at 4 to 9 hours, and persisted for up to 48 hours as determined by enzyme-linked immunosorbent assay. A similar kinetic profile was elicited by interleukin-1 and lipopolysaccharide, whereas interferon-gamma (IFN-gamma) had minimal effect on IG9 expression. The IG9 protein has a molecular weight of 105,000 as determined by immunoprecipitation studies with TNF-alpha-treated HUVEC protein lysates. mAb IG9 significantly inhibited the binding of U937 cells and human peripheral blood monocytes to TNF-alpha-treated HUVEC and had no effect on peripheral blood lymphocyte or granulocyte adhesion. Treatment of human aortic endothelial cells or HUVEC with MM-LDL for 24 hours induced IG9 protein expression 3-fold above background with no concomitant increase in binding of antibodies to intercellular adhesion molecule-1 (ICAM-1), E-selectin, or vascular cell adhesion molecule-1 (VCAM-1), endothelial cell adhesion proteins involved in monocyte binding. mAb IG9 F(ab')2 inhibited MM-LDL-induced monocyte adhesion to HAEC by 23%. Immunohistochemical analysis demonstrated that the endothelial cell lining of vessels in human lung and heart with evidence of inflammation characterized by an extensive mononuclear cell infiltration exhibited reactivity with mAb IG9, whereas vessels with no evidence of inflammation in the same sections as well as in sections from normal lung and heart were nonreactive.(ABSTRACT TRUNCATED AT 250 WORDS)

Tumor necrosis factor alpha induces adhesion molecule expression on human fetal astrocytes.

Leukocyte adhesion molecules on endothelial cells of the blood-brain barrier may participate in the entry of leukocytes into the central nervous system. Because astrocytes are also a component of the blood-brain barrier and have been associated with inflammation, we studied the ability of astrocytes to express leukocyte adhesion molecules using Northern blot and immunocytochemical techniques. Astrocytes treated with the proinflammatory cytokine tumor necrosis factor alpha (TNF) expressed messenger RNA for the adhesion molecules E-selectin, vascular cell adhesion molecule 1, and intercellular adhesion molecule 1, as well as their corresponding proteins. In addition, TNF-treated astrocytes expressed a monocyte adhesion protein identified by our laboratory, recognized by the monoclonal antibody IG9. These results indicate that under inflammatory conditions in the central nervous system, such as multiple sclerosis and acquired immune deficiency syndrome, astrocyte expression of adhesion molecules may facilitate the migration of leukocytes and contribute to the disease process.

Cytokine gene expression of endothelial cells infected with Trypanosoma cruzi.

Coronary microvascular spasm and platelet hyperreactivity have been implicated in the pathogenesis of Chagas' cardiomyopathy. To clarify further the role of the microvasculature in this disease, alterations in cytokine gene expression due to Trypanosoma cruzi infection of human umbilical vein endothelial cells were examined. Northern blot analysis of total RNA from endothelial cells demonstrated that interleukin (IL)-1 beta, IL-6, and colony-stimulating factor 1 (CSF-1) mRNA expression was absent or minimal in uninfected cells but significantly increased in infected cells. c-sis mRNA levels diminished with increased time of infection. In situ hybridization studies also demonstrated high levels of IL-6 mRNA in individual infected cells. Significant levels of IL-6 and IL-1 beta protein were detected in the supernatants of infected endothelial cells. The serum of an acutely infected individual contained high levels of IL-6 protein, suggesting the potential importance of cytokines secreted by the vascular endothelium in the pathogenesis of Chagas' cardiomyopathy.

Interleukin 6 modulates c-sis gene expression in cultured human endothelial cells.

Human vascular endothelial cells secrete platelet-derived growth factor (PDGF)-like polypeptides which may mediate some of the vascular effects in the inflammatory process. We have demonstrated that IL-6 caused a significant increase in the mRNA level of the c-sis gene (PDGF B chain) in cultured human endothelial cells. IL-1 alpha and IL-1 beta also increased c-sis mRNA transcripts after an extended incubation period and both cytokines acted synergistically with IL-6 in increasing c-sis expression. Tumor necrosis factor enhanced the accumulation of c-sis mRNA and interferon-gamma decreased its level. In the inflammatory process specific cytokines can modulate c-sis expression in human endothelial cells. Their subsequent production of PDGF-like polypeptides could stimulate cell migration and proliferation, and cause the release of vascular inflammatory mediators.

The role of endothelial cell adhesion molecules in the development of atherosclerosis.

The vascular endothelium serves as a dynamic interface between circulating blood elements and the interstitial tissues. As such, it communicates to cells within the vessel wall as well as to the surrounding tissue, sensing its environment and responding accordingly. The vasculature must maintain a delicate balance when initiating a functional response by producing both proinflammatory and antiinflammatory mediators, vasoconstrictors and vasodilators, growth stimulators and inhibitors, and prothrombogenic and antithrombogenic factors. Any response to injurious agents could lead to pathology. Confounding this complex interplay is the fact that the very response to injury that may have developed to undo the damage may itself be even more deleterious. One response to injury by the endothelium is the new or increased expression of surface receptors for immune elements. In atherosclerosis, the adhesion of monocytes (and T cells) to the endothelium is a key event triggered by some form of insult. Subsequent events include monocytic infiltration of the vessel wall, alterations in lipid metabolism, and the activation of these cells into foam cells. The presence of large numbers of foam cells in the intima may produce a high concentration of cytokines and growth factors within a localized area, extracellular matrix perturbations, smooth muscle cell proliferation, and ultimately platelet aggregation at the site of stenosis. Endothelial cells themselves will not only elaborate factors after the initial injury to the vessel wall but also in response to the factors produced by foam cells within the plaque. These endothelial cell factors include MCP-1, a chemoattractant for monocytes (179,180), IL-1 (63,64), IL-6 (interleukin-6) (65-67), IL-8 (interleukin 8) (181), and PDGF, a potent smooth muscle mitogen (4,72) (Fig. 3). Endothelial cells will propagate an inflammatory response long after the initial insult to the arterial vessel. A chronic cycle of endothelial cell activation and leukocyte infiltration is constitutively activated. Thus, all of the cellular elements of the vessel wall, as well as the atherosclerotic plaque itself, elaborate cytokines and growth factors that amplify and propagate the pathological process.

5,5'-Dimethyloxazolidine-2,4-dione is a strong inducer of differentiation of human promyelocytic leukemia (HL-60) cells.

5,5'-Dimethyloxazolidine-2,4-dione (DMO), a weak non-metabolizable acid, is commonly utilized for determining intracellular pH. In these studies, DMO was tested as an inducer of differentiation on the basis that its uptake and subsequent dissociation might transiently raise intracellular pH and activate ion-fluxes critical for triggering maturation. After 5 days of exposure to 40 mM DMO, greater than 60% of HL-60 cells displayed phenotypic and functional changes characteristic of mature granulocytes. As with other inducers of HL-60 cell differentiation, commitment to differentiation required culture in the presence of DMO for more than 24 h, indicating that if transient effects on pH or ion-fluxes occurred, they were not sufficient to trigger this process. DMO was either weak or inactive as an inducer of murine erythroleukemia cell (FLC) differentiation. Although other weak acids and bases triggered differentiation of both HL-60 cells and FLC, the spectrum of response differed markedly between the two lines. These results suggest that: (1) a number of common buffering agents have the potential to alter cell phenotype, and (2) their effects must be evaluated for each individual cell type.