Abstract

uman immunodeficiency virus type-1 associated dementia (HAD) is a devastating complication of advanced viral infection. Although the incidence of dementia is decreasing, in the era of highly active antiretroviral therapy (HAART), the increased longevity of HIV infected individuals might positively effect disease prevalence. Clinical manifestations of the disease include both motor and cognitive impairment. Neurological symptoms begin with difficulty concentrating, followed by forgetfulness and behavioral abnormalities which progress to florid dementia, motor impairments, seizures, coma and then death (1)(2). This progression can occur within a matter of months, and is generally seen late in the course HIV-1 disease. Many of the most severely affected patients have HIV-1 encephalitis (HIVE). The HIVE neuropathology consists of a multinucleated giant cell encephalitis with astrogliosis, microglial nodule formation and neuronal injury/dropout (2). Yet, HIVE and HAD are not directly correlated, as some patients with a pathologic diagnosis of HIVE may not have shown clinical signs of dementia, and vice versa. Unlike other types of viral encephalitis, in which either neural cells are infected directly with virus or autoimmune mechanisms affect neuronal damage, HIV-1 replication is limited, nearly exclusively, to mononuclear phagocytes (MP) (brain macrophages and microglia). Although limited nonproductive infection occurs in glial and/or endothelial cells, the biological significance of these virus-cell interactions remains uncertain. In some significant measure, the pathogenesis of HAD involves viral infection and immune activation of MP. The absolute level of virus load is not a clear predictor of neurological impairment. Although CSF viral load may correlate with dementia symptoms in individual patients, there is no absolute level that predicts or indicates the presence of disease. Indeed, the number of immune competent macrophages, not the levels of virus per se appear to be the best correlate of central nervous system (CNS) disease (3). A brain inflammatory reaction triggered by viral infection of MP likely affects HAD onset and progression.

Introduction

Inflammatory reactions in the brain are perpetrated, in measure, through MP. For instance, following mechanical trauma to the cortex or spinal cord a transient early influx of neutrophils is followed by a secondary, more vigorous, inflammatory reaction of macrophages (4). In turn, excitotoxic neuronal injury affects microglia and macrophage secretory functions (5). Transgenic mouse studies have suggested that the failure of the CNS to recruit neutrophils may be related to select deficits in neutrophil chemokine production by brain MP (6). In contrast, microglia and astrocytes produce high levels of monocyte chemoattractants following CNS injury (7). The exact mechanisms which initiate HAD are unknown, but not related to altered neutrophil function/migration, but rather an influx of infected macrophages into the brain that become immune competent.

Immune stimulated macrophages secrete numerous neurotoxic products, many of which have been found in the CSF or brain tissue of patients with HAD. Some or all of these produces may be implicated in HAD pathogenesis. HIV-1 infection of macrophages "primes" the cells to secrete even higher levels of inflammatory products upon immune stimulation (8)(9). Thus the role of virus in inducing neurotoxic responses of MP may be related to lowering the threshold of the cells to subsequent activation signals. The stimuli for the brain immune activation in HAD, however, remain unknown. Recent works from our laboratory suggest that the CD40 ligand produced by activated CD4+ T lymphocytes may be one such stimulatory response (10). Here lymphocytes expressing CD40L may activate brain perivascular macrophages eliciting an amplified paracrine and autocrine inflammatory reaction. Such a reaction could initiate secretory immune responses in neighboring microglia/astroglia and, in turn, affect neuronal function (9)(11). The putative neurotoxic factors that activated infected MPs secrete include cytokines, arachidonic acid and its metabolites, reactive oxygen intermediates, excitatory amino acids, and virotoxins (gp120, gp41, tat, rev, nef) (8)(11)(12)(13)(14)(15). The cellular immune products, the focus of this paper, are discussed in further detail below. In addition to the secretion of inflammatory products (putative neurotoxins), MP can also secrete trophins which promote homeostasis of neurons (unpublished data) or astrocytes (16) and maintenance of the CNS microenvironment. MP can also regulate astrocyte inflammatory secretions. Therefore, the regulation of brain macrophage/microglia neuroprotective and neurotrophic activities in the CNS may be the singular event that affects cognitive impairments in infected subjects. This idea is supported by recent evidence that HAD may be nearly completely reversible in some patients. Indeed, since the advent of HAART, there have been reports of reversal of dementia in affected patients (with documented clinical, laboratory and radiologic improvement)(17). However, numerous other reports have shown failure of patients with HAD to respond to HAART, leading to ACTG trials utilizing adjunctive therapies for HAD. This lends even more credence to the hypothesis that the disease is a metabolic encephalopathy perpetrated by secretory products of HIV-1 infected MP, rather than just an effect of the virus itself.

Inflammatory products

(1) Cytokines
One of the most well studied inflammatory factors in the CNS is the cytokine tumor necrosis factor-alpha (TNF-a), produced by MP. TNF-a gene products have been detected in post-mortem brain tissue from patients with HAD (12)(18)(19)(20). In general the levels of TNF-a were higher in HIV-1 infected than in non-infected controls and highest in HAD brains. TNF-a has also been found in CSF of patients with HAD (12)(17)(20). In one case study, levels of TNF-a decreased following HAART therapy in a patient with severe HAD (17). The drop in TNF-a was associated with clinical neurological improvement in cognitive function (measured by neuropsychological testing) and a reversal of magnetic resonance spectroscopy (MRS) abnormalities.

Interleukin-1 (IL-1), produced by astrocytes and microglia, and interferon-gamma (IFN-g), produced by cytotoxic T cells, have been found in both brain tissue and CSF of HIV-1 infected patients (12)(19)(21). Cytotoxic T cells likely play a pivotal role, early on, in controlling viral replication in brain. Elevated levels of neopterin (a byproduct of guanosine triphosphate metabolism in macrophages, an indirect measure of IFN-g activity) have been found in CSF of HAD patients (12)(21)(22)(23). Unlike TNF-a, however, levels of IL-1 and IFN-g do not appear to correlate with neurological impairment.

The transforming growth factor beta (TGFb) family of polypeptides are potent immunoregulators which influence multiple aspects of immune function, including chemotaxis, immunosuppression and production of cell adhesion molecules. TGFb 1, 2 and 3 are detected in deep cortical and subcortical white matter in patients with HIV-1 encephalitis (HIVE) at sites of inflammation (24), while in normal brains expression is confined to the leptomeninges and blood vessels. In addition, in the same study, all three isoforms were detected in macrophages in cryptococcal meningitis, and in reactive astrocytes associated with progressive multifocal leukoencephalopathy (PML), toxoplasma encephalitis and HIVE. This suggests that TGFb may participate in both HIVE and other CNS inflammatory diseases associated with advanced HIV infection. Certainly the chemotactic role of TGFb in recruiting monocytes to the brain of HAD patients may be a potential feature of the overall disease process (see below). Of note, numerous publications have shown that both TNF and TGFb have neurotrophic as well as neurotoxic properties, and it is likely that overproduction or dysregulation of the factors accounts for associated neurotoxicity.

(2) Chemokines
Although HAD correlates with the numbers of immune activated MP in brain, it is unclear what factors lead to monocyte infiltration into the CNS during the disease. One possibility is that HIV-1 infected and/or activated MPs and astrocytes within the CNS secrete monocyte chemoattractants. These may include RANTES, monocyte chemoattractant proteins (MCP-1, MCP-2, MCP-3), and macrophage inflammatory proteins (MIP) 1a and 1b. The production of chemokines may follow glial exposure to viral and/or cellular factors. For example, HIV-1 tat released by infected cells, has been shown to increase NF-kB binding to its consensus sequence in the viral promoter in astrocytes (25). This could influence the expression MCP-1 from astrocytes or MP. A recent study has shown elevated MCP-1 levels in the CSF of HIV-1 infected patients with HAD as compared to patients with other neurodegenerative disorders (25). Interestingly, the CSF levels of MCP-1 in HAD patients were in the range that could affect monocyte chemotaxis. In the same study MCP-1 was detected in brain tissue of HAD patients by in situ hybridization, but was not seen in normal brain tissue or in HIV-1 infected non-demented tissue. In another study, transcripts for MIP-1a and MIP-1b were detected in HAD brain tissues by either reverse transcriptase polymerase chain reaction (RT-PCR) or RT in situ PCR techniques, and were higher than found in HIV-1 infected non-demented brains (26). Recent works have demonstrated both neurotoxic and/or neuroprotective roles of chemokines (27)(28). These works were, in measure, associated with HIV-1 gp120-induced neuronal injury. The functional roles of any/all of these chemokines during disease progression await further investigation.

(3) Quinolinic acid and NTox
A number of published reports have suggested that HIV-1-associated neurotoxicity may be mediated through N-methyl-D-aspartate (NMDA) receptors (29)(30)(31). This may be mediated, in part, through quinolinic acid (QUIN). QUIN is an excitotoxic L-tryptophan metabolite and NMDA receptor agonist which is elevated in the CSF of patients with HIVE (17)(29)(32). Levels of QUIN correlate with severity of neurologic deficits in some but not all studies (32)(33). Additionally, decreases in QUIN have been observed following antiretroviral treatment (32)(33), and were correlated with improvement in neuropsychological testing and MRS abnormalities (17). QUIN mRNA has been found to be elevated in brain tissue of patients with HIVE (34). However, it was also elevated in brains of HIV-1 infected patients with opportunistic infections (cytomegalovirus and histoplasma encephalitis, CNS lymphoma, and PML). These studies suggest that QUIN production is associated with brain inflammation, as opposed to HIV-1 infection, per se. Other investigators have identified another neurotoxic amine, referred to as Ntox (35), released by HIV-1 infected monocytic cells. Ntox may act through NMDA receptors and induce neuronal injury/death. NTox has also been found to be produced by monocytes and microglias from HIV-1 infected individuals, and can be isolated from brains of HIV-1 infected patients (36).

(4) Platelet activating factor
Platelet activating factor (PAF), among other effects, regulates glutamate release, TNF-a and IL-1b, all of which have been implicated in neurotoxicity. PAF is released by HIV-1 infected human monocytes (11) and induces neuronal death in vitro (13). A study of HIV-1 infected subjects found that PAF was detectable in the CSF of HIV-1 infected patients, and was higher in patients with HAD than in those without clinical signs of dementia (13). However, PAF was also found in the CSF of control (uninfected) subjects with neurologic symptoms from other medical conditions (multiple sclerosis, leukemia, disseminated cancer, hepatic failure and cerebrovascular disease), suggesting it is a marker of CNS inflammation rather than being specific for HIV-1 induced neurodegeneration.

(5) Nitric oxide
Nitric oxide (NO) and its metabolite peroxynitrite have been shown to be toxic to glial and neuronal cells in vitro (37)(38). The inducible form of nitric oxide synthase is regulated mainly by IFN-g, and augmented by TNF-a and IL-1b produced predominantly by astrocytes. CSF and serum levels of nitrite and nitrate (metabolites of NO, but not a direct measure of NO activity), have been found to be elevated in patients with HIV-1 (17)(39)(40)(41), and are higher in AIDS patients with neurological disease than in HIV-1 negative patients with other inflammatory neurologic diseases (42). In addition, elevated levels if iNOS mRNA have been detected in brain tissue of patients with severe HAD as compared to non- or mildly demented HIV-1 positive patients and seronegative controls (43). In one case study, treatment with antiretrovirals led to decrease in serum NO levels and correlated with improvement in dementia (17). CSF levels of NO were undetectable at all times.

(6) Prostaglandins
Prostaglandin E₂ (PGE₂) is the primary eicosanoid product of activated macrophages, and has been observed to be elevated in the CSF of HIV-1 positive patients with dementia and/or myelopathy. Levels of PGE₂ correlate with cognitive impairment in HAD. CSF levels of thromboxane B₂ (TxB₂) and PGF2a (other products of the cyclooxygenase pathway of arachidonic acid metabolism) were also found to be elevated in HIV-1 patients with dementia (14).

Conclusions

Multiple inflammatory factors have been found in the CSF and/or brain tissue of patients with HAD. While some of these factors correlate with HIV-1 dementia, others are less specific and correlate more with inflammation in the CNS than with HIV-1. For example, elevated levels of PGE₂ and iNOS are found in CSF and brains of patients with multiple sclerosis (MS) (44)(45). Elevated levels of QUIN have been found in CSF and brain tissue of patients with meningitis, autoimmune disorders, and septicemia (46). Lastly, TNFa has been found in brains afected by Alzheimer’s disease (47) and in the CSF and brain of patients with Parkinson’s disease (48)(49). While the inciting events in these disorders are different, they likely share some similar pathogenic mechanisms revolving around secretions from immune stimulated macrophages. Future works in HAD will decipher the mechanisms for monocyte influx and immune stimulation of the CNS. Moreover, the elucidation of MP toxic and trophic regulatory activities and the viral strain variation permit an understanding of how the CNS microenvironment is altering the disease.

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