Mononuclear phagocytes mediate blood-brain ... - Semantic Scholar

Mononuclear phagocytes mediate blood-brain barrier compromise and neuronal injury during HIV-1-associated dementia Yuri Persidsky,*† Jialin Zheng,*† Donald Miller,㛳 and Howard E. Gendelman*†‡§ *Center for Neurovirology and Neurodegenerative Disorders, the Departments of †Pathology and Microbiology, ‡ Medicine, §The Eppley Institute for Cancer and Allied Diseases, and 储College of Pharmacy, University of Nebraska Medical Center, Omaha

Abstract: The neuropathogenesis of HIV-1 infection revolves around the production of secretory factors from immune-activated brain mononuclear phagocytes (MP). MP-secreted chemokines may play several roles in HIV-1 encephalitis (HIVE). These can promote macrophage brain infiltration, blood-brain barrier (BBB) and neuronal dysfunction during HIV-1-associated dementia. We investigate how HIV-1-infected MP regulates the production of chemokines and how they influence HIV-1 neuropathogenesis. We demonstrate that HIV-1-infected and immune-activated MP (for example, microglia) and astrocytes produce ␤-chemokines in abundance, as shown in both laboratory assays and within infected brain tissue. HIV-1-infected microglia significantly modulate monocyte migration in a BBB model system and in brains of SCID mice with HIVE. HIV-1-infected MP downregulate tight junction protein and special polarized transport systems on brain microvascular endothelial cells as shown in human autopsy brain tissue and in SCID mice with HIVE. Chemokines can damage neurons directly. Toxicity caused by binding of stromal-derived factor-1␣ to its receptor on neurons exemplifies such mechanism. In toto, these works underscore the diverse roles of chemokines in HIV-1 neuropathogenesis and lay the foundation for future therapeutic interventions. J. Leukoc. Biol. 68: 413– 422; 2000. Key Words: chemokine 䡠 chemokine receptor microglia 䡠 astrocyte 䡠 tight junction

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INTRODUCTION Central nervous system (CNS) disease is a late complication of HIV-1 infection. Nearly a quarter of infected persons develop cognitive, behavioral, and/or motor abnormalities commonly called HIV-1-associated dementia (HAD). The clinical manifestations of this subcortical dementia range from mild forgetfulness to frank mental deficits including hallucinations and incontinence, which often leads to death

[1, 2]. CNS effects of virus infection are likely more common than those manifest clinically because half of virus-infected individuals show neuropathological changes at autopsy. Despite the high incidence of neurological involvement, the host-viral interactions leading to HAD are just beginning to be well understood. It is now agreed, among most experts in the field, that mononuclear phagocyte (MP; perivascular and parenchymal macrophages and microglia) activation drives neural dysfunction in HAD through secretion of neurotoxins. Productive viral replication in brain occurs nearly exclusively in MP [3], although limited evidence also exists for infection in macroglia (astrocytes and oligodendrocytes) or neurons [4, 5]. Overt disease is clearly associated with the number of activated CNS macrophages [6] but not always with neuropathological features of HIV-1 encephalitis (HIVE). These observations suggest that even minimal increase in number and, more importantly, state of immune activation of macrophages/microglia could be sufficient to cause neurological dysfunction. Moreover, disease can be reversed by highly active anti-retroviral therapies accompanied by reduction of neurotoxin production [7]. These observations provide direct evidence that MP secretory neurotoxins drive a metabolic encephalopathy characteristic of HAD. The hallmark of HIVE is productive viral replication in brain macrophages and microglia associated with giant cell formation and macrophage infiltration from blood to brain. Other features of HIVE include widespread microglial activation and accompanying reactive astrogliosis, which are found in the areas of pronounced dendritic alterations [8, 9]. Synaptic loss appears to be the best correlate of mental deterioration [10]. Three-dimensional stereological measures showed a significant correlation between reduced synaptic density and poor neuropsychological performance. These data underscore the significance of microglial activation in HIV-1-associated neurodegeneration. Macrophage brain infiltration is associated with neurological decline [11]. Morphometric studies suggested that there is a significant decrease in the size of all cortical areas, in the cerebral white matter, and in the basal

Correspondence: Yuri Persidsky, M.D., Ph.D., Center for Neurovirology and Neurodegenerative Disorders, 985215 Nebraska Medical Center, Omaha, NE 68198-5215. E-mail: [email protected]

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ganglia/thalamus (subcortical gray matter) [12]. Activation of microglia and diffuse microgliosis were shown to correlate with ventricular expansion and neuropathological changes [13]. It is important to note that MP activation also causes the production of pro-inflammatory factors, which can lead to neural injury. Microglial activation [detected as expression of class II major histocompatibility complex (MHC) antigen HLA-DR] and, to a lesser extent, HIV-1 infection and reactive astrogliosis results in prominent chemokine secretion [11]. Once infected with HIV, MP populations are predisposed to immune activation. Accumulating data suggests that HIV-1 proteins Tat and gp120 interact with brain MP, perhaps via viral cellular receptors, to induce a cascade of intracellular events altering the threshold required for activation [14 –19]. HIV-1 proteins, productive viral replication, or other stimuli may “prime” the MP for activation by pro-inflammatory cytokines, chemokines, or T cell-expressed factors, for example CD40 ligand (CD40L) [14 –23, and R. Cotter et al., unpublished observations]. The combination of viral replication and immune activation ultimately results in neurotoxin production by MP [J. Zheng et al., unpublished results; 17, 19, 20, 24]. MP neurotoxins include arachidonic acid and its metabolites [25], platelet-activating factor (PAF) [26], pro-inflammatory cytokines [tumor necrosis factor ␣ (TNF-␣) or interleukin-1␤ (IL-1␤)], quinolinic acid [27], NTox [28], and nitric oxide (NO) [29]. Viral proteins such as gp120 [30], gp41 [29], and Tat [31] secreted by infected brain macrophages can directly affect neuronal viability and/or function. Most recently, studies from our laboratories, and those of others, indicated that progeny virions or HIV-1 gp120 may bind to chemokine receptors (for example, CXCR4) expressed on neurons, and alter intracellular signaling, perhaps leading to apoptosis [32–34]. These findings, taken together, underscore the significance of MP activation in HIV-1-associated neurodegeneration. The mechanism of HIV-1-associated brain injury probably involves cell-cell interactions (glial, neuronal, and/or endothelial) with a number of neurotoxins. The regulation of MP effector cell responses may play both a positive and a negative role in disease pathogenesis. Although increases in cytokine and chemokine production can regulate virus replication in macrophages [17; R. Cotter et al., unpublished results], they also mediate chronic cellular inflammatory cascades involved with the recruitment of additional macrophages to the site of injury, leading to an amplification in neural injury [11; J. Zheng et al., unpublished results]. It is interesting that chemokine secretion, macrophage infiltration, blood-brain barrier (BBB) compromise, and neuronal damage have all been linked, one with the other, and are prominent features of HAD. It is important to note that all appear to be regulated through MP secretory responses and thus serve as a focal point for our laboratory’s research efforts and this article.

Impairments of BBB function in HAD Alteration of BBB function is a common feature of HIV-1 CNS infection [35]. Significant structural and functional abnormalities of the microvasculature have been demonstrated during HIV-1 infection of the brain. This includes serum protein leakage and morphological alterations in capillary endothelial cells and basement membranes [35–37]. In support of the idea 414

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that BBB breakdown can be induced by HIV-1 infection of the brain, higher levels of neurotoxins were found in peripheral blood than in the cerebrospinal fluid (CSF) in a patient with HAD. These were associated with signs of BBB compromise as observed by magnetic resonance imaging (MRI) of the brain [5]. Highly active anti-retroviral therapy can result in a significant reduction of viral replication, neurotoxin levels, and improvement of BBB function all associated with reversal of HAD symptoms. Functional changes in the BBB have also been shown during other lentiviral infections of the nervous system [38]. An inverse relationship between severity of simian immunodeficiency virus (SIV), encephalitis, and expression of the endothelial 55-kDa isoform of BBB glucose transporter 1 was shown in cortical gray matter, the caudate nucleus, and the cerebellum of virus-infected macaques. Under normal conditions, the BBB efficiently restricts movement of ions, proteins, and other polar organic molecules to the brain. Structurally, the BBB is composed of specialized nonfenestrated microvascular endothelial cells (BMVEC) connected by tight junctions (TJ) and devoid of transcellular pores [39]. TJ and TJ proteins [outer membrane protein, zonula occludens (ZO-1) and integral membrane protein, occludin] formed by BMVEC ensure the BBB’s structural integrity. The development of tight junctions seems to depend on two primary processes: the appearance of high levels of the occludin and intracellular signaling processes that control the state of phosphorylation of junctional proteins [40]. Additional components of the barrier are the surrounding capillary basement membrane and astrocytes. Astrocyte end-feet are in close apposition to the abluminal surface of the brain endothelium and assist in the barrier function by coordinating the functional activities of BMVEC [41– 43]. Structural impermeability of BBB is further enhanced by a number of special transport systems expressed on BMVEC. One of them is P-glycoprotein (P-gp), a transmembrane glycoprotein located on the apical/luminal membrane of BMVEC that transports endothelium-penetrating lipophilic molecules back into the blood [46]. We hypothesize that altered P-gp expression in HIVE may further disrupt BBB integrity with resulting increased toxicity to blood-borne molecules into brain [45]. HAD pathogenesis is influenced by BBB dysfunction documented by MRI and single-proton emission computed tomography as perfusion defects and white matter changes [46, 47]. Increased BBB permeability was shown by increased levels of quinolinic acid, metalloproteinases, and NO in the CSF of HIV-1-infected patients [7, 48 –50]. How BBB dysfunction occurs is not well understood. TJ or P-gp, if disrupted, could affect BBB function and influence HAD progression. To evaluate the regulation of P-gp and TJ proteins during HIVE, we performed a series of crossvalidating experiments. First, we examined expression of P-gp, ZO-1, occludin, markers of MP activation/infection (CD68, HLA-DR, HIV-1 p24), and astrogliosis (GFAP) by immunohistochemistry in different brain regions (seven with HIVE, four HIV-1 seropositive without HIVE/HAD, and three seronegative controls) as previously described [11]. Analyses of HIVE brain tissue showed a focal decrease of ZO-1/occludin antigen expression associated with MP brain infiltration. Although both TJ proteins showed continuous http://www.jleukbio.org

membrane immunoreactivity on BMVEC in control HIV-1seronegative cases, both medium-size and small microvessels featured fragmented or weak immunoreactivity for ZO-1 occludin in brain tissue with HIVE (Fig. 1, A–F and G–J). Such diminished TJ expression was strongly associated with macrophage perivascular infiltration. Microvessels in the areas without significant macrophage accumulation usually had weak but continuous ZO-1 or occludin endothelial immunostaining. Consistent with intensity of HIVE, white matter and deep gray matter showed the most prominent decrease in TJ antigen expression. Cortical gray matter was affected only in the two most severe HIVE cases that demonstrated focal monocyte perivascular infiltrate in cortical neuropil. Microglial activation (but not MP numbers) was associated with lowered P-gp immunostaining in HIVE (Fig. 1, E and F). Only P-gp expression was decreased in HIV-1-infected patients without HIVE when compared with controls. Its decrease appeared to correlate with focal microglia activation (as detected by increased HLA-DR expression). Dalastra et al. [51] recently demonstrated significant TJ disruption (fragmentation or absence of immunoreactivity for occludin and ZO-1) within vessels from subcortical white matter, basal ganglia, and, to a lesser extent, cortical gray matter in HIVE patients. Second, we examined expression of P-gp, ZO-1, occludin, and CD14 (a marker for MP) by reverse-transcriptase polymerase chain reaction (RT-PCR) in brain regions (adjacent to those investigated immunohistochemically for TJ protein and P-gp expression). Levels of mRNAs were examined after reverse transcription with antisense primers and PCR amplification of the initially amplified cDNA. RNA for the cellular gene actin served as an internal standard for these studies. TJ and P-gp mRNAs were significantly down-regulated in HIVE and strongly associated with MP CD14 expression. P-gp was lower both in HIV-1-seropositive patients without neurological impairment and HIVE patients when compared with controls. Overall, immunolabeling for TJ and P-gp correlated with their mRNA expression in the same brain regions. Third, we studied expression of ZO-1, occludin, and P-gp in our SCID mouse model of HIVE [52]. HIV-1ADA-infected MDM were stereotactically inoculated into basal ganglia of SCID mice. Animals were killed 1 week later. Immunohistochemical analysis of brain tissue showed focal decrease in TJ and P-gp expression in microvessels surrounded by virus-infected MP. Areas around human cells featured microglia activation and reactive astrocytosis. Expression of TJ proteins and P-gp was unchanged in brains of sham-operated animals. Fourth, we examined functional activity of P-gp in primary cultures of human BMVEC through the use of rhodamine 123 uptake assay as previously described [53]. When pro-inflammatory cytokines [TNF-␣, IL-1␤, interfer-

on-␥ (IFN-␥)] or supernatants from HIV-1-infected, activated MP were placed onto BMVEC P-gp was down-regulated (seen functionally as an increase in rhodamine 123 accumulation). Supernatants from HIV-1-infected and immune-stimulated MDM elicited the most prominent rhodamine accumulation. Overall, these findings demonstrate both structural and functional impairments of BBB during HIV-1 infection.

MP and astrocytes as a source of ␤-chemokines in HIVE As shown above, MP activation and its resultant secretory products underlie many features of HIV-1 neuropathogenesis. Because the number of immune-activated MP is the best correlate of HAD [6], understanding the mechanism for MP infiltration into brain is of critical importance for elucidating disease mechanisms. Chemokines and chemotactic cytokines play an important part in this process. They mediate migration, recruitment, accumulation, and activation of leukocytes into the CNS. Each subfamily of chemokines has a specific variation of a conserved structural cysteine motif: ␣-subfamily (one amino acid separates the first two cysteine residues, CXC), ␤(cysteine residues are adjacent, CC), ␥- (only two cysteine residues are present, C), and ␦- (three amino acids separate the first two cysteine residues, CX3C) [54]. Members of a subfamily show considerable homology in amino acid sequence and overlapping cell-specific chemoattractive properties. Because ␤ chemokines [macrophage inflammatory proteins-1␣ and -1␤ (MIP-1␣ and MIP-1␤), macrophage chemotactic protein-1 (MCP-1), MCP-2, MCP-3, and regulated upon activation normal T cell expressed and secreted (RANTES)] specifically promote monocyte migration, they received special attention in studies of HIV-1 neuropathogenesis. Indeed, ␤-chemokines were detected in both HIVE brain tissue [55, 56] and CSF of HIV-1-infected patients with neurocognitive impairment [21]. Microglia, resident brain MP, and astrocytes produce ␤-chemokines in vitro after stimulation with viral proteins, inflammatory cytokines, lipopolysaccharide (LPS) or CD40L [18, 21, 23, 57, 58]. Weiss and colleagues [54] using an in vitro BBB system (umbilical vein endothelial cells and fetal astrocytes seeded on opposite sides of a porous membrane) showed that MCP-1 was the primary chemoattractant for monocytes and that astrocytes stimulated by pro-inflammatory cytokines were the major source of this chemokine. Although the works described above provide circumstantial evidence of ␤-chemokine involvement in HIV-1 neuropathogenesis, the exact composition of chemokines secreted, their cellular sources, and the role of MP infection and immune activation remain unclear. To address these questions, we developed an integrated approach utilizing an in vitro model for the BBB (human BMVEC and astrocytes seeded on the oppo-

3 Fig. 1. Expression of TJ proteins and P-gp in BBB in HIVE. Microglial cells are activated (shown by HLA-DR expression) in HIVE brain tissue (B) compared with controls (A). Perivascular macrophages and parenchymal microglia expressed HIV-1 p24 (D) but not in controls (C). Expression of P-gp was significantly diminished on BMVEC in HIVE (F) compared with controls (E). Down-regulation of ZO-1 was found on BMVEC in HIVE (H) compared with controls (G). Similarly, occludin showed a continuous immunoreactive pattern in control brain (I) but was weakly reactive in HIVE (J). Frozen sections were immunostained using antibodies to HLA-DR (A, B), HIV-1 p24 (C, D), P-gp (E, F), ZO-1 (H, G), and occludin (I, J). Frozen tissue sections were counterstained with Mayer’s hematoxylin. Original magnification: panels A and B, ⫻100; panels C–J ⫻200.

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site sides of porous membrane), a SCID mouse model of HIVE, and the use of brain autopsy tissue to confirm the observations made in cell culture and animals [52, 59, 60]. First, to mimic BBB compromise during HIVE we placed MDM or microglia with or without HIV-1 infection in the bottom of our transwell BBB inserts (“brain” side). Migration of peripheral blood monocytes (isolated at the time of the experiment and placed in the upper chamber of inserts) were evaluated. Microglia-containing BBB models showed the most prominent monocyte migration (2.5–3 times more than the MDM-seeded inserts). Second, we explored the idea that production of ␤-chemokines or cytokines (TNF-␣) was distinct between MDM and microglial cells. This proved correct because microglia secreted 5–20 times more of both TNF-␣ and chemokines [shown by enzyme-linked immunosorbent assay (ELISA)] than the same numbers of MDM. Immune stimulation further enhanced their secretion in both cell types. It is interesting that there was no difference in chemokine and TNF-␣ production between HIV1-infected and uninfected cells. Because these data did not correlate with the results observed in the BBB model, we hypothesized that cells other than microglia or macrophages could be a source of chemokines. Astrocytes are likely candidates. A strocytes were part of our BBB constructs and are known to produce chemokines. Furthermore, signs of astrocyte reactions were detected previously in our BBB models during MDM migration [61]. To study the interactions between virusinfected MP and astrocytes, supernatants derived from HIV1-infected or uninfected activated microglia or MDM were applied to primary human astrocytes. Under these experimental conditions, astrocytes produced significant amounts of ␤-chemokines (in particular MCP-1). Culture fluids collected from immune-activated virus-infected microglia elicited the greatest chemokine production. Collectively, these results indicated that the interplay between activated microglia and astrocytes may be of critical importance for HIV-1 neuropathogenesis. Third, to determine the possible pro-inflammatory and transendothelial migratory effects of resident brain macrophages in SCID mice with HIVE we inoculated equal numbers of infected or uninfected MDM and microglia into the basal ganglia of the mice. In our previous works we showed that salient features of HIVE are reproduced in such animals, including neuropathological changes, neurotoxin production, and behavioral abnormalities [52, 61, 62]. SCID mice, which received infected microglia, showed the most prominent neuropathological changes (including astrogliosis and mouse microglia reaction) and elicited the greatest accumulation of mouse macrophages in the inoculated areas when compared with animals inoculated with equal numbers of MDM. Fourth, expression of chemokines was compared to the intensity of HIVE in human brain tissues. We found that the severity of HIVE (level of macrophage brain infiltration, formation of microglial nodules, and astrogliosis) correlated with microglia activation (MHC class II HLA-DR expression) and, to a lesser extent, HIV-1 infection and chemokine expression (detected by immunohistochemical tests). Both activated microglial cells and reactive astrocytes were the major sources of chemokines in HIVE. It is important that neuropathological evidence of HIVE severity correlated with neurocognitive impairment. Diffuse microglia activation may explain how relatively small

numbers of HIV-1-infected perivascular MP could cause widespread neurological dysfunction. In support of this notion, microglia activation was shown to be the best predictor of neuronal injury and behavioral abnormalities in SIV-infected macaques [63].

Chemokines and their receptors in HIV-1associated neuronal injury Much of the work described above outlines the role of chemokines in monocyte trafficking into the brain. However, because chemokine receptors are expressed on a wide range of neural cells we hypothesized that such factors might also play roles in neural injury. This was tested in a number of experimental systems in our laboratories. It is interesting to note there are several ways that chemokines and their receptors may perturb neural function and these are delineated below. Chemokines exert their effects by activating members of a seven-transmembrane G-protein-coupled receptor (GPCR) family. These receptors are divided into four categories as described above [64, 65]. ␣-chemokine receptor, CXCR4, and ␤-chemokine receptor, CCR5, are believed to be the important viral co-receptors for HIV-1 infection. Macrophage-tropic (M-tropic) HIV-1 strains use the chemokine receptors CCR5 or CCR3 as coreceptors for viral infection [66 –71], whereas CD4⫹ T lymphocyte-tropic (T-tropic) HIV-1 strains use CXCR4 [72]. It is important that HIV-1 virion-associated gp120 can also instigate signal transduction through binding to chemokine receptors of MP or CD4⫹ T lymphocytes [73–75]. Recently, a variety of chemokine receptors have been found to be shared by leukocytes and neuronal cells. Because viral proteins and chemokines are present in the HIV-1-infected brain tissue, chemokine receptor-chemokine interactions may affect spread of infection and its associated neurodegenerative events [32, 34, 69 –71, 76 – 83]. One of these receptors is CXCR4 [72]. A number of studies have linked CXCR4 to the immune and neuronal compromise that occurs during latestage HIV-1 infection [32, 36, 77, 79, 81– 83]. Several reports document CXCR4 expression by a number of neuroectodermal cell types, including neurons, microglia, and astrocytes [69 – 71, 76 – 82]. Furthermore, CXCR4 also plays a substantial role in neuronal function and apoptosis [32, 34, 84, 85]. We previously demonstrated that the HIV-1 envelope glycoprotein, gp120, or ligand for CXCR4, SDF-1␣, can elicit neuronal apoptosis [32–34]. This response can be inhibited by antibody to CXCR4 (12G5). It is important that progeny virions may also bind to chemokine receptors (for example, CXCR4) expressed on neurons and lead to apoptosis [84, 86] (Fig. 2). Thus, HIV-1 virions or chemokines produced by immuneactivated glial cells may bind to neuronal receptors and alter cellular function. The interaction of these ligands with receptors on neurons may lead to the neuronal loss, alterations in dendritic arborization, and decreased synaptic density observed in tissue specimens of affected individuals [16, 32–34, 84, 87, 88]. However, the exact mechanism for the interaction among chemokines, virion, and neuronal chemokine receptors is still unknown. Alternatively, some chemokines may have a neuroprotective function in the brain [16, 89, 90]. Certainly, the overabundant expression of chemokines in both normal and diseased brain raises an important question regarding the exact


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Fig. 2. Immunocytochemical analysis of CXCR4 antigen expression in human fetal neurons and signal transduction and neuronal apoptosis is induced by SDF-1␣ and gp120. Human neurons were double immunostained with 12G5 for CXCR4 (green, B, C) and antibody for neurofilament (NF 150 kDa, red, B, C). Neuronal networks showed branched dendrites by NF staining (B, C). Neuronal cell bodies and process were double immunostained with 12G5 and NF antibody (B, C). MAP-2 (green, A) and GFAP staining (red, A) showed more than 70% cells identified as neurons and less than 30% were astrocytes. All original magnifications are ⫻200 for A and B. Panel C is a high-power view of B illustrating cell membrane (CXCR4) and dendritic (NF) morphology (⫻600). In panel D, SDF-1␣ (50 nM) and recombinant gp120SF-2 (1 nM) with or without 12G5 (administered at 5 ␮g/mL) were placed on human neurons for 4 days and apoptosis detected by ELISA (D) shows results using anti-histone and anti-DNA antibodies. In panel E, PI hydrolysis induced by SDF-1␣ or gp120 were determined in replicate cultures. These experiments are representative of three replicate assays. Data are expressed as means ⫾ SD. *P ⬍ 0.01 with or without SDF-1␣ or gp120. †P ⬍ 0.01 with or without 12G5.

role of these chemokines and their receptors for neuronal function. In addition to CXCR4, other ␣-chemokine receptors such as CXCR2 [77] or CXCR5 [91] are expressed by neurons. Preliminary data from our laboratory and those of others suggest that CXCR5 are mainly expressed on neuronal dendrites, whereas CXCR2 and CXCR4 are expressed both on neuronal cell bodies and processes [90]. Production of IL-8, ligand for CXCR2, is increased in astrocytes or astrocytoma cell lines under pro-inflammatory conditions [90]. IL-8 is also produced, at lower levels, by astrocytes under normal conditions [92, 93]. It is important to note that our recent preliminary data suggests that IL-8 is also produced by HIV-1-infected lymphocytes. Similarly, HIV-1 infection and immune activation induced the production of high levels of IL-8 by MDM [90]. Similar results were obtained in the primary cultures of human fetal microglia. Importantly, IL-8 production appears to be up-regulated in human brain tissues affected by HIVE, adding in vivo relevance to the laboratory results [Y. Persidsky et al., unpublished observations]. In summary, HIV-1 infection and second418


ary immune activation induce the production of chemokines from MP and/or astrocytes. Some of the chemokines (such as SDF-1␣) or viral proteins (such as gp120) can affect neuronal apoptosis. Alternatively, some of the chemokines may be neuroprotective [89, 90]. How and under what circumstances chemokines exert protective or toxic results are central questions currently being investigated in our laboratories and others.

Chemokine receptor signaling may link inflammation to neuronal apoptosis Our data and others show that chemokine receptor (such as CXCR4)-ligand binding stimulates at least three distinct intracellular signaling cascades in neuronal cells under normal physiological conditions. First, binding of SDF-1␣ to CXCR4 causes transient increases in cytosolic inositol triphosphate (IP3) and [Ca2⫹] levels in cultured neurons and astrocytes. Second, SDF-1␣ binding to CXCR4 inhibits adenylate cyclase activity, leading to decreased cytosolic cAMP levels. Third, CXCR4 stimulation induces MAP kinase phosphorylation [J. http://www.jleukbio.org

Zheng et al., unpublished observations]. The IP3 adenylate cyclase and MAP kinase pathways are likely to have an important role in the normal neuronal homeostasis. Because SDF-1␣ can also induce neuronal apoptosis through binding of CXCR4 receptors expressed on neurons, these same pathways may provide potential mechanisms through which virion or gp120-chemokine receptor interactions trigger neuronal apoptosis. How these physiological signal transduction pathways are linked to apoptosis signaling certainly awaits elucidation. Much work has been done to explore the apoptotic mechanisms mediating brain cell injury in HAD. Apoptosis of neurons, astrocytes, and/or MP is a major feature of cellular injury in HIVE [52, 94 –97]. These observations are supported by work performed in human neuronal culture systems where MP-secretory products induce neuronal apoptosis. Binding of neuronal CXCR4 by cellular and viral factors [34, 84] can instigate intracellular signaling. In general, apoptosis is induced by extracellular signals such as the Fas ligand, TNF-␣, or others, and/or by deprivation of neurotrophic factors. The final steps in neuronal gene expression leading to apoptosis in brains of HAD patients is not known. The newly characterized caspase system provides a suitable starting point for understanding the molecular mechanisms through which chemokine receptor binding and apoptosis may be linked in neurons. Caspases are a family of proteases that stimulate apoptosis. Ten caspases have been identified [98, 99] and have been grouped according to sequence homology as being either ICE- (caspases 1, 4, and 5) or ced-3 like (caspases 3, 6, 7, 9, and 10) [98, 99]. Caspases are synthesized as inactive “pro-enzymes” that are processed by proteolytic cleavage to form an active enzyme. For example, caspase-3 is a key protease that becomes activated during the early stages of apoptosis [100]. Active caspase-3, found in cells undergoing apoptosis, consists of a heterodimer of 17- and 12-kDa subunits, which are derived from the 32-kDa proenzyme. In its active form, caspase-3 proteolytically cleaves and activates other caspases, as well as relevant targets in the cytoplasm (D4-GDI) and nucleus (PARP) [98, 99]. It is interesting to note that we have demonstrated that caspase-3 is activated by HIV-1 in human neurons [34]. However, it is still not clear how G-protein-coupled chemokine receptors stimulate signaling pathways linked to caspase activation. Nevertheless, although chemokine and chemokine receptors are widely expressed in affected brain, understanding the expression and function of chemokine and chemokine receptors on neurons holds great importance. The determination of chemokine, viral-associated or secreted protein and chemokine receptor interactions and influence on neuronal second messengers may help the elucidation of such important questions. It is possible that these ligand-bound chemokine receptors may directly stimulate the apoptosis-promoting signaling machinery. On the other hand, neuroprotective factors present in the healthy brain may operate through abrogation of the same events or stimulation of antiapoptotic pathways. This research will certainly help to explain why some of the chemokines play a neurotoxic role, whereas other chemokines are neuroprotective. Taken together, these data indicate the central role for HIV-1-infected and immune-activated MP in the neuropathogenesis of HAD. Clearly, MP orchestrates many biological and

biochemical pathways in brain that regulate cell trafficking, viral infection, and compromise of the neuropil. Substantial levels of chemokines and pro-inflammatory cytokines are produced by MP as well as by astrocytes. These two cell types may communicate with each other in steady state and disease, resulting in defined physiological outcomes. The rapidly developing field of neural chemokine-ligand interactions and cell signaling has led to the delineation of how neurons may be destroyed during the progression of HAD. The outcome of such chemokine-chemokine receptor interactions for neural cells is of pivotal importance because under certain sets of circumstances the process may lead to apoptosis, whereas in others it may lead to neuroprotection.

ACKNOWLEDGMENTS This work was supported in part by National Institutes of Health Grants AI42404-03R29 (to Y. P.) and P01NS31492-01, R01NS34239-01, R01NS34239-02, R01NS36126-01, and P01MH57556-01 (to H. E. G.). We thank Ms. Julie Ditter and Ms. Robin Taylor for excellent editorial support; Ms. Xiaojun Liu, Ms. Alicia Lopez, Ms. Clancy Williams, Ms. Rhadika Suryadevara, Ms. Jennifer Rasmussen, Mr. Michel Bauer, and Mr. Tim Moran for excellent technical support.

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