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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Brain Pathol. Author manuscript; available in PMC Apr 27, 2006.
Published in final edited form as:
Brain Pathol. Jan 2004; 14(1): 97–108.
PMCID: PMC1449744
NIHMSID: NIHMS8901

Human Immunodeficiency Virus Infection of the Brain: Pitfalls in Evaluating Infected/Affected Cell Populations

Abstract

Monocyte/macrophages and CD4 T-cells are the primary hematopoietic targets of productive HIV infection. In the brain, potential cellular targets for HIV infection include perivascular and parenchymal macrophages/microglia, oligodendrocytes, endothelia, neurons, and astrocytes. We examine evidence of productive and non-productive infection for each cell type in the brains of HIV-infected patients with and without HIV encephalitis. Despite the voluminous literature and substantial experimental effort over the past two decades, evidence for productive infection of any brain cell other than macrophages is left wanting.

INTRODUCTION

It has been over 20 years since the AIDS epidemic began. During this time, considerable strides have been made in understanding the life cycle of the causative agent of AIDS, the lentivirus human immunodeficiency virus (HIV). All mammalian lentiviruses infect monocytes/macrophages. HIV and simian immunodeficiency virus (SIV) are peculiar amongst the lentiviruses in that they also infect CD4 T-cells and this substantially amplifies viral production. This “peculiarity” mediates CD4 T-cell loss and immunosuppression that characterize AIDS. Because immunosuppression was the clinically defining symptom of AIDS, early studies of HIV infection focused on infection of CD4 T-cells. However, it was soon recognized that macrophage tropic strains are the predominant viruses transmitted between hosts (105) and the principal strains associated with neurological disease (30, 54).

Viral receptors

To efficiently infect cells, surface viral proteins (ligands) bind to host cell proteins (receptors). Host cells do not express surface proteins to act simply as viral receptors, but rather the virus co-opts specific host protein(s) to facilitate cell attachment and entry. Initial studies showed that HIV bound to and infected cells bearing the CD4 receptor (31, 69). Subsequently, it was discovered that infection was more efficient in the context of a co-receptor. The designation of receptor versus co-receptor is arbitrary and predominantly based upon historical considerations that CD4 was discovered first (68, 151). HIV and SIV co-receptors include the chemokine receptors CXCR4 (46) or CCR5 (35, 38). In the case of SIV, CCR5 appears to play a greater role in promoting infection than the originally discovered CD4 molecule (39, 40, 85). In vitro, it was recognized that viral strains utilizing CCR5 are macrophage tropic (2), while strains utilizing CXCR4 are T-cell tropic (38). Most viral strains are able to use both receptors for entry with varying efficiency. Other co-receptors, including CCR2, CCR3, CCR8, BOB, and AJP, can be used by some viral strains in vitro (Reviewed in (27)). These latter co-receptors have reduced infection efficiency compared to CCR5, and it is still debatable whether they are important during in vivo infection (For review, see 14, 101).

Productive versus non-productive lentiviral infection

Due to the complex life cycle of lentiviruses, cell entry does not necessarily lead to replication and assembly of viral progeny (89). After entry into the cell, the HIV capsid disassembles and lentiviral RNA enters the cytoplasm along with viral proteins that are incorporated into the virion. HIV and SIV use these incorporated viral proteins to reverse transcribe viral complementary DNA (cDNA). The cDNA preintegration complex is synthesized into double stranded viral DNA, and this pre-integration complex is transported to the nucleus where it is integrated into the host genome as a provirus. Inside host DNA, the HIV promoter long terminal repeat (LTR) can be activated by host transcriptional machinery. Some viral cDNA is not integrated into host DNA and remains as an episomal circle that is thought to be replication incompetent (For review, see 89).

Lentiviral infected lymphocytes and monocyte/macrophages can be characterized as exhibiting the following infection states:

  • productive infection—actively producing new virions
  • latent infection—harboring proviral DNA without producing virus (blocked at the level of transcription) with potential for production of new progeny virus
  • non-productive infection or restricted infection—containing unintegrated or integrated viral DNA that will not lead to production of progeny virus. Transcription or translation of select viral genes (eg, tat, nef, and rev) may occur.

HIV or SIV either productively or latently infects CD4 T-cells and monocyte/macrophages, and replication competent provirus found in these cells is expected to be in an integrated form. Non-productive or restricted infection occurs if viral particles are defective or host cells are not permissive (e.g. viral cDNA is not integrated).

Infection of hematopoietic cells

CD4 T-cells and monocyte/macrophages are the primary cellular targets of productive HIV infection (28), but several other hematopoietic cell types have been reported to be HIV-infected including dendritic cells (109), CD8 T-cells (79), natural killer cells (139), and natural killer T-cells (48). In vitro, B cells, megakaryocytes, eosinophils, promyelocytes, stem cells, thymocytes, langerhans cells, and follicular dendritic cells have been reported to be susceptible to HIV infection (For review, see 76). The number of peripheral blood mononuclear cells that harbor HIV DNA is variable (123) (approximately 0.1%-15% [8, 103]). It has been estimated using PCR techniques that asymptomatic patients carry 1 in 100 to 40 000 infected CD4 T-cells (20, 106, 119), while patients with AIDS carry approximately 1 in 100 to 10 000 infected CD4 T-cells (119) or up to 10% of blood CD4 T-cells (64). Using in situ PCR, others have observed that either 0.2% to 69% of CD4 T-cells (11) or 4% to 15% peripheral blood mononuclear cells (103) harbor proviral DNA. Of the few reports that have examined blood monocyte infection, it seems relatively few peripheral monocytes are infected (11). This issue has never been fully illuminated.

During early stages of HIV infection, it is widely thought that macrophages and dendritic cells are the key tissue elements that propagate virus (22, 140, 155). However, others have recently observed that CD4 T-cells are the predominant infected cells in lymphoid tissues during acute infection (118, 125). During early time points in infection, it is estimated that a median of 90% of productively infected lymphoid cells are CD4 T-cells while a median of 7% are macrophages. The frequency of productively infected cells per gram of lymphoid tissue has been reported to be approximately 500 000 to 5 000 000 (118). During late stages of infection, most of the remaining CD4 T-cells in the lymph nodes are infected (99). In early and late stages of infection, variable numbers of latently infected CD4 T-cells and macrophages are found throughout all lymphoid tissues (41).

Infection of non-hematopoietic non-CNS cells

Many non-hematopoietic cells have also been reported to be infected by HIV including: epithelia (84, 95, 143), endothelia (34, 92), cardiomyocytes (111), striated myocytes (120), podocytes (112), hepatocytes (25), and others. In vitro, some cells such as columnar and goblet cells, fibroblasts, renal tubular cells, synovial membrane, retina, prostate, testes, and uretha have been reported to be susceptible to HIV infection (For review, see 76). If one indiscriminately accepts all of the literature, it would appear that HIV infection could be detected to some extent in most human cells. Requiring more rigorous proof of infection (ie, consistent findings among multiple groups) would suggest a more limited infection.

Infection of CNS cells

In the brain, potential cellular targets for HIV infection include perivascular and parenchymal macrophages/microglia, oligodendrocytes, neurons, endothelia, and astrocytes. The literature is replete with confusing and conflicting reports claiming HIV infection of brain cells of multiple lineages. In this review, we examine evidence of productive and non-productive infection for each cell type in the brains of HIV-infected patients with and without HIV encephalitis. Our emphasis will not be on cataloging potentially infected cells, but rather on defining the strengths and weaknesses of current claims.

MICROGLIA/MACROPHAGES (m/M)

In vivo observations of microglial/macrophage infection

Early infection

Soon after lentiviral infection, virus can be recovered from the brain in addition to most body compartments. The source of this early virus is not clear. In the human disease, with rare exception (33), studies of primary HIV infection are limited to assessment of the cerebrospinal fluid (CSF), at best an imperfect measure of the brain. Studies of virus isolated from the CSF during acute infection have demonstrated both macrophage and T-cell tropism, while isolations from brain tissue were predominantly macrophage tropic (71). The origin of CSF virus is difficult to pinpoint. Early CSF infection is probably a reflection of trafficking infected lymphocytes from the blood stream to the CSF, consistent with lymphocytic CSF pleocytosis observed at this period. It seems unlikely that substantial amounts of parenchymal virus would percolate into the CSF; however, infected cells in the perivascular and meningeal spaces would certainly contribute to CSF virus.

In the brain parenchyma, acute lentiviral infection does not seem to robustly seed nervous system cells (51). Theoretically, the CNS (both meningeal and parenchymal) could act as a “reservoir” of lentiviral infection. However, as little provirus can be detected in the CNS during non-encephalitic phases (147), this reservoir would appear to be quantitatively small.

Late infection

After the development of severe immunodeficiency, approximately one quarter of HIV infected individuals develop HIV encephalitis (7, 32, 82, 86). Since the advent of highly active anti-retroviral therapy, the incidence of HIV encephalitis is decreasing (55), yet the prevalence of the disease is increasing (55, 73) with one estimate citing 45% of AIDS autopsies demonstrating HIV encephalitis (73). Pathologically, HIV encephalitis is characterized by the presence of microglial nodules, multinucleated giant cells, and abundant HIV-infected macrophages as determined by immunocytochemistry, in situ hybridization (ISH), or quantitative HIV RNA assessment (21, 147) (Figure 1). The majority of HIV strains isolated from the CNS are macrophage tropic (30, 54, 71), and there is a consensus that microglia/macrophages (m/M) are the predominant infected cell in brains with HIV encephalitis (21) (Figure 1). It is still unexplained why only a fraction of individuals develop HIV encephalitis, though length of survival with severe immunosuppression may be a factor (123a, 144).

Figure 1
Confocal microscopy of histological sections from rhesus macaque brains with SIV encephalitis shows neurons and astrocytes are not productively infected by SIV. A. Immunofluorescent staining for neuronal marker microtubule associated protein-2 (blue), ...

Parenchymal versus perivascular microglia/macrophage infection

In HIV encephalitis, there is some controversy as to whether perivascular or parenchymal microglia/macrophages comprise the majority of infected cells (29, 150). Studies of human autopsy tissues suggest that while there is an angiocentricity to the brain lesions, m/M throughout the brain support productive infection. Given the abundance of brain vasculature and the disruption of tissue morphology during encephalitis, it is frequently not possible to morphologically distinguish perivascular from parenchymal m/M. One attempt to address this issue utilized the primate model for HIV infection of humans: SIV infection of Asian macaques. Assuming that CD14 and CD45 were present only on macrophages that have differentiated from recently trafficked monocytes and are not present on parenchymal microglia, it was concluded that perivascular macrophages and not parenchymal microglia are infected by SIV (150). While it is true that a large number of infected cells in lentiviral encephalitis are in a perivascular position (Figure 1B), trying to tease apart the distinction between parenchymal and perivascular cells is highly problematic. Operationally, when does a perivascular macrophage become a parenchymal microglia? In this highly vascularized organ, how far does an infiltrating macrophage need to traffic into the parenchyma to be labeled as a resident cell? If CD14 and CD45 define recently differentiated macrophages, what is the length of time needed in the brain to lose expression of these markers? Can expression of these surface markers change during the course of disease as observed with other markers, including more crucial surface proteins such as the viral receptors CD4 and CCR5? Perhaps more germane to the issue of pathogenesis, how long after migrating into the CNS is a macrophage capable of supporting lentiviral infection? The claim that only recently trafficked macrophages were infected with SIV was recently disputed with the finding that parenchymal microglia expressed both CD14 and CD45 in HIV encephalitic brains (29). It was also observed that parenchymal microglia accounted for ⅔ of HIV p24+ cells in HIV encephalitic brains (29). Is this a difference between human and non-human primates? Since microglia and macrophages are derived from the same monocyte lineage precursors, this distinction may be more a question of semantics. Finally, there remains controversy regarding whether the sheer presence of increased numbers of perivascular and parenchymal macrophages mediate neuronal damage, or whether the total number of infected CNS m/M are greater contributors to neuronal damage.

Viral receptors on microglia/macrophages

Microglia/macrophages express the HIV receptors CD4 and CCR5 (63), and some reports have shown CXCR4 expression on m/M (152, 154). When initially isolated from the peripheral blood, monocytes express high levels of CD4 and low levels of CCR5 (75). Depending upon culture conditions, CCR5 surface expression and infectability of monocyte derived macrophages peaks between 1 and 2 weeks in vitro. Isolated directly from brain tissue, m/M express a differentiated phenotype similar to that of cultured adherent monocyte derived macrophages. Compared to their monocytic progenitor cells, m/M express more CCR5 and less CD4 (75), thus would be expected to be more susceptible to HIV-strains expressing CCR5/macrophage specific V3 envelope sequences.

Summary of microglia/macrophage infection

In summary, lentiviruses clearly infect brain m/M. Prior to the development of encephalitis, CSF viral isolates are pre-dominantly macrophage tropic with minor quantities of T-cell-tropic strains. With the development of encephalitis both perivascular and parenchymal m/M produce virus. The relative role of infection of recently trafficked monocytes versus resident microglia is controversial, however, this distinction is probably not an impediment to our understanding the pathogenesis of HIV mediated neurodegeneration. Certainly monocytes acting as “Trojan horses” trafficking HIV into the brain remains the most attractive hypothesis for viral entry, however, extending these findings to understanding the time course of brain infection will require novel experimentation beyond descriptive studies.

Non-macrophage CNS cells

Leaving the hematopoietic cells and considering infection of endogenous CNS elements is fraught with as much uncertainty as Ulysses' journey home from Troy. Many Sirens beckon the unsuspecting investigator onto rocky shoals. Early in the epidemic, there were numerous published reports, based upon cell morphology, of HIV infecting neuroglial cells other than m/M. Many double label studies utilizing a wide variety of cellular and viral probes followed these early reports. While the variety of reagents might explain some of the discrepant findings, certainly the art of histopathology could also account for the differences. Distinguishing between technical and interpretive issues and true biological issues of pathogenesis has plagued the field. As will be described, some of the debate has deteriorated to quite indirect evidence followed by highly artificial experimental systems of questionable relevance to AIDS.

OLIGODENDROCYTES

In vivo observations of oligodendrocyte infection

Simple identification and labeling of oligodendrocytes in vivo has a long and complicated history. Even today oligodendrocyte surface labeling is problematic in vivo and in vitro. Few reports of in vivo oligodendrocyte infection by HIV exist (58, 66). Most investigators have not found evidence of either restricted or productive infection of this cell lineage (122, 131, 141). Given the difficulty of surface labeling these cells, electron microscopy has been used to study infection of oligodendrocytes. As there is no morphological structure distinguishing intracellular lentiviruses, only budding virus and extracellular virions can be assessed by standard electron microscopy. In one report brain tissue from AIDS patients showed structures that were ultrastructurally similar to retrovirus budding from a possible oligodendrocyte (58). Virions have also been detected in extracellular spaces surrounded by myelin sheath (66) consistent with HIVgp120 envelope protein binding to oligodendrocytes (16). Without optimal morphological preservation or immunolabeling with cell specific markers, it is difficult to use electron microscopy and resolve the lineage of cells containing budding virus. Even with rare brain biopsies, preservation of human brain tissue is never optimal. Autopsy tissue compounds preservation problems and is mostly unsuitable for fine ultrastructural discriminations. Brain tissue is dense with little apparent extracellular space. Cell processes can be long and difficult to attribute to a specific cell soma. Lastly, virion structure is polymorphic and except for mature lentiviral particles, most intermediate forms can be confused with secretory vesicles or other subcellular structures, while membrane budding can readily be confused with synaptic membrane specializations.

Immunostaining for HIV p24 and ISH for HIV nucleic acids in brain tissue has been reported in rare cells with the morphological appearance of oligodendrocytes (45, 107, 127), but to date there are no reports utilizing double labels co-localizing virus and oligodendrocyte markers. Using morphological features to identify oligodendrocytes that immunostain for HIV antigens at the light microscopy level is even more subjective than at the ultrastructural level. Worse in conditions of tissue pathology, cell morphology may be distorted or changed. Staining cells with cell specific markers in conjunction with viral antigens is absolutely necessary to draw conclusions regarding whether a cell is productively infected.

In vivo/in vitro viral receptors on oligodendrocytes

Neither CD4 protein nor mRNA has been detected in oligodendrocytes (122). There are little data on chemokine receptors expressed by human or simian oligodendrocytes; however, rat oligodendrocytes have been reported to express CXCR1 and CXCR2 (98). Since oligodendrocytes do not appear to express HIV receptors, the burden-of-proof rests even more prominently on investigators trying to confirm that HIV can infect oligodendrocytes.

In vitro infection of oligodendrocytes

Despite the paucity of evidence to support the idea that HIV infects oligodendrocytes in vivo, several groups have attempted to address the issue in vitro. Oligodendrocytes are a difficult cell to isolate and culture. Nevertheless, there is one report of purified human adult brain oligodendrocytes that immunostained for HIVp24 gag protein after infection with HIVBAL and HIVIIIB (1). Early passages of primary cells are seldom pure, and oligodendrocytes are difficult to maintain for late passages. Most studies have utilized leucine methyl ester (LME) to remove contaminating phagocytic cells from the cultures. While LME is an excellent method for killing phagocytic cells (eg, m/M) in vitro, phagocytic cells found in mixed cultures are partially protected from LME toxic effects. This interjects a note of caution in interpreting tissue culture experiments claiming productive oligodendrocyte infection when the assay is limited to analysis of supernatant viral production. Even low frequencies of contaminating m/M in oligodendrocyte cultures could account for viral production in the supernatant. Double labeling for virus and cellular markers would be essential to claim infection of oligodendrocytes in vitro.

To circumvent problems with primary cells, it is possible to use transformed cells (either from neoplasms or from in vitro transformations). Such cell lines eliminate m/M contaminating initial isolations and can be grown for extended passages. With the caveat that such cell lines may not replicate in vivo conditions of non-dividing cells, there is sparse evidence to suggest that even oligodendroglial cell lines support productive HIV infection. Circumventing infection permits addressing the question: does a cell's transcriptional machinery permit utilization of the HIV genome? One study demonstrated that transfecting the whole HIV genome or select regions can lead to HIV transcription (24). This finding is not surprising given that most cells would be expected to contain transcription factors capable of replicating HIV, but without viral entry or integration it is difficult to imagine productive infection. It is clear from such experiments that oligodendroglial cell lines can transcribe HIV genetic material, however, there is no evidence that viral integration and productive infection transpires even in this artificial system.

Summary of oligodendrocyte infection

In summary, there is sparse evidence to indicate that HIV or SIV infects oligodendrocytes either in vivo or in vitro. In the absence of cell-surface viral receptors, if the infection process were to occur rarely, it would not be expected to be very efficient or biologically important.

ENDOTHELIAL CELLS

In vivo observations of endothelial infection

A number of early studies reported infection of endothelial cells in adult brain tissue (113, 143, 148). These studies were based upon interpretation of the morphological appearance and vascular position of cells stained for HIV antigen by immunohistochemistry or HIV RNA by ISH. With rare exception (10), double labels were not performed to determine if the HIV antigen positive cells were demonstrably endothelial cells by immunohistochemistry. Interpreting in vivo infection of vascular endothelial cells is complicated by the life cycle of HIV. Plasma viral titers, particularly in untreated subjects, are very high (>108 virions per ml). As human tissues are seldom perfused with saline prior to histological preparation, virus present in plasma or adherent to endothelial cell surfaces will complicate interpretation of immunocytochemistry and ISH results. Trafficking of infected blood monocytes also confounds interpretation of endothelial cell infection. Light microscopy does not permit resolution of true endothelial cell infection from either abundant adherent virus or emperipolesis of infected monocytes across endothelial cell membranes. With the advent of highly active antiretroviral therapy, post mortem tissues can be examined in the absence of contaminating high plasma viral loads. Analysis of such more recent tissues has not supported significant endothelial cell infection (131).

To circumvent technical issues of sensitivity, several investigators have employed PCR in situ hybridization (PCR-ISH). Publications using this method have reported detecting DNA in macrophages, endothelial cells, astrocytes, and oligodendrocytes and RNA in macrophages, endothelial cells, and astrocytes using RT-PCR-ISH (4, 5, 83). Given the technical difficulties of carrying out such studies, most identifications are based on morphological appearance of cells without double labels (4, 5). Beyond the difficulties of interpreting morphological data from a technique where tissue preservation is severely compromised, there is the additional difficulty of interpreting the biological meaning of PCR-ISH results. In theory PCR-ISH has the capacity to detect solitary molecules of HIV RNA or DNA. Without biological amplification, does detection of solitary molecules have biological meaning or has PCR-ISH opened Pandora's box (145)?

In vitro infection of endothelial cells

Overall, in vivo data suggesting endothelial cells are infected has not been rigorously demonstrated. With the complexities and caveats associated with in vivo studies, several investigators have turned to in vitro systems to address the issue of infectability of endothelial cells by HIV. Detection of HIV products has been shown in several primary brain capillary endothelial cell cultures infected with HIV. In one report, 40% of primary endothelial cells derived from human brain were productively infected by HIV (90). Interestingly, brain derived HIV strains were unable to infect these endothelial cells (91). Even more strangely, the opposite has been reported in simian endothelial cells, where only macrophage tropic SIV (83) with certain sequences in the transmembrane portion of the envelope protein successfully infected these cells (47). Others claim endothelial cells are not productively infected but that virus can be rescued by co-culture with cells susceptible to HIV infection (104). Of course with rescue experiments it is difficult to distinguish detection of membrane adherent input virus from true infection. This could explain why some have concluded that brain-derived endothelial cells are not infectable (65, 78).

Recently, it has been observed that brain derived capillary endothelial cells are susceptible to dual tropic viral isolates (viral strains that can use both CXCR4 and CCR5) but not monotropic strains that predominantly only gain entry by one co-receptor (149). SIV has also been reported to infect primary rhesus brain endothelial cells (129) in a CD4 independent CCR5 dependent mechanism where RANTES (soluble ligand for CCR5) inhibited infection (40). Others have shown that brain passaged SIV strains had greater in vitro infection efficiency; however, less than 5% of the endothelial cells immunostained for SIV p27 (129). SIV infection has been demonstrated in a blood brain barrier model of macaque endothelial cells and fetal astrocytes under flow conditions (0.1-1 ng SIV/ml) (128). Double labels were not performed to verify the identity of infected cells, and it is possible that m/M contaminated the cultures.

Some researchers postulate that endothelial cells (infected or not) are the portals through which cell-free HIV enters the brain. Since HIV gp120 envelope binds to sialic acid and n-acetylglucosamine (12), it is hypothesized that gp120 can bind to endothelial cells, facilitating the crossing of free virus through the blood brain barrier. HIV could penetrate a monolayer of coronary artery derived (57) and brain derived capillary (78) endothelial cells with tight junctions. Finally, HIV virions were identified near clathrin-coated pits in brain derived capillary endothelial cells where disrupting lipid rafts and glycosylaminoglycan inhibited virion uptake (78). These observations suggest that cultured endothelial cells might macropinocytose HIV. Although HIV seems to penetrate brain endothelial monolayers in vitro, little evidence supports this possibility in vivo.

In vivo/in vitro viral receptors on endothelial cells

To examine the theoretical infectability of endothelial cells, several groups have looked for the presence of viral receptors on these cells. Endothelia in the brains of adults express the chemokine co-receptors required for HIV entry, CXCR4 (74, 117) and CCR5 (114). However, CXCR4 might only be expressed during pathological conditions such as HIV encephalitis (117). Brain endothelial cells also express CCR1 (117) and CXCR3 (52). In vitro, adult human brain capillary endothelial cells express several potential HIV co-receptors including CXCR4 (15, 93, 149), CCR5 (15, 93, 149), CCR8 (149), AJP (93, 149), DC-Sign (93), GPR1 (149), and CCR3 (15, 93, 149). Brain capillary endothelial cells derived from pediatric brains have been reported by some to express CD4 (126); however, others have not detected CD4 expression (93). Many of these reports lack double labels to provide evidence that the cells expressing the chemokine receptors are endothelial cells, however, some double labels have been reported and morphologically endothelial cells are fairly readily identified in vivo. The consensus of opinion appears to be that chemokine receptors provide a potential avenue for HIV entry to endothelial cells independent of CD4. Whether these receptors are used for infection or trans-cellular viral movement remains to be determined.

Summary of endothelial infection by HIV

Despite early suggestions that HIV or SIV infects endothelial cells in vivo, no double label experiments have been published that convincingly demonstrate co-localization of HIV and endothelial cell markers. Despite the presence of demonstrable viral receptors on the surface of endothelial cells or even in vitro evidence of infectability, it seems most likely that endothelia might be more important as a conduit of HIV infection than as a nidus of infection.

NEURONS

In vivo observations of neuronal infection

Early in the HIV epidemic, cells with neuronal morphology were reported to be immunolabelled with antibodies to HIV p25 gag protein in a subset of patients (107). Others claim that only immature, mitotically active neurons and glia found during fetal development support a productive infection (44). In addition to the above caveats regarding interpreting cell lineage without double labeling, HIV immunohistochemistry of brain tissue should be interpreted with caution since several neuronal proteins have the potential to cross-react with HIV “specific” reagents. The neurotrophic factor neuroleukin shares partial sequence homology with a highly conserved region of gp120 (87) and normal dentate gyrus and pyramidal neurons stain with monoclonal antibody to p24 in Pick disease and in Alzheimer disease (153). Immunocytochemical studies from the past decade have not been able to confirm lentivirus infection of neurons in vivo (17) (Figure 1A).

In an attempt to employ the theoretically most sensitive technology, there is one report using PCR-ISH on HIV-infected patient brains. HIV gag DNA was detected in a high percentage of cells, 90% in some regions, that stained for neuron specific enolase (100). This astounding high percentage has never been replicated by other groups and given the abundance of signal, might be attributed to an uncontrolled artifact. Even if the majority of neurons truly contained one copy of viral DNA, in the absence of viral RNA, it is unclear what biological import can be attributed to the molecular state of the viral DNA. Over the intervening years, localization of HIV to neuronal elements has not been replicated.

Laser capture microscopy is the most recent technique employed to detect HIV DNA in individual brain cells. Using this technique HIV DNA has been detected in pyramidal neurons of the hippocampus (135) and in microtubule associated protein-2 (MAP-2) containing neurons in the frontal cortex or basal ganglia in patients with HIV encephalitis or HIV dementia (136). Unfortunately while this method is an attractive means of capturing individual cells from a homogenous tissue, the brain is the antithesis of a homogenous tissue. Despite claims that HIV DNA can be detected at the single cell level, as employed in these reports, 30 to 100 neurons were captured in order to detect a signal (137, 146). The brain is notable for the paucity of extracellular space and the highest cell density of any organ. Numerous sub-micron cell processes contribute to the synaptic neuropil and perisomal space. Sampling a field with MAP-2 staining does not assure that the entire sample originates from a neuron. Until laser capture microscopy can microdissect a cell body/nucleus rather than a pre-defined circular area, unequivocal identification of material from a single cell (let alone 100) seems improbable.

In vivo/in vitro viral receptors on neurons

To assess the theoretical possibility of neuronal infection or binding of HIV, several studies have examined the presence of viral receptors on the neuronal surface. Like endothelial cells, some neurons in adult human brains express the 2 co-receptors required for HIV entry, CXCR4 (74, 117) and CCR5 (114). CD4 was detected in cerebellar, thalamic, pons, and hippocampal neurons (50), but other reports were unable to detect CD4 at the protein or message level. Neurons also express the HIV co-receptor CCR3 (117), thus, it is possible that HIV could enter neurons via a CD4 independent mechanism.

In vitro infection of neurons

Given the paucity of evidence that HIV can infect neurons in vivo, several studies have examined the question of neuronal infection in vitro. Primary purified neuronal cultures are notoriously difficult to make and essentially impossible to passage, thus most reports have focused on neuronal cell lines. As the highly differentiated state of neurons is their sine qua non, all of the caveats mentioned above (and then some) would apply to the biological relevance of studying systems employing neuronal cell lines. There is one report of primary dorsal root ganglia cultures containing neurons, Schwann cells, and fibroblasts from fetal tissue that replicate virus faster than CD4 T-cells or macrophages at 1 to 2 days post-infection (72). However, in this study virus was not detected at day 7 post-infection, thus, it is possible that the input virus was responsible for the signal observed at 1 to 2 days post-infection. Several reports demonstrate productive infection of neuron tumor cell lines or fetal derived neurons in vitro. HIV infects medulloblastoma, SKN-MC tumor neuronal cells, and fetal neural cells by a CD4 independent mechanism (3, 61, 77). After HIV infection, reverse transcriptase activity is observed and HIV DNA is detected by ISH in neuroblastoma cell lines (121). Infected neuroblastoma cell lines (121) and fetal neuroblasts (42) are able to transmit virus to other HIV permissive cells. Whether this represents true infection or residual input virus binding to cultured cells is not easily resolved. HIV p24 production and HIV gag DNA were detected in HCNA-1 immature cerebral cortical neuronal cell lines (138). It appears that fibroblast growth factor, neuronal growth factor, tumor necrosis factor-β, and HIV tat protein have the ability to either increase HIV p24 production (43, 88) or increase NF-κB binding to the HIV promoter (70, 130) in neuronal cell lines. Others have shown that human cortical neurons are not productively infected by HIV (65). It seems prudent to question the utility of in vitro lentiviral infection of tumor cell lines and impure fetal cultures that are likely to behave differently than adult neurons, especially when one considers how little support there is for such infections in vivo.

Summary of neuronal infection by HIV

In vivo evidence of neuronal infection by HIV is meager (Figure 1A). However, it is theoretically possible that HIV or SIV could infect neurons since lentiviruses can infect non-dividing cells. While neurons do not express CD4, some neuronal subsets do express HIV co-receptors that could mediate signal transduction cascades but are inadequate to support neuronal infection.

ASTROCYTES

In vivo observations of astrocyte infection

Infection of astrocytes is one of the most controversial topics in studies of HIV infection of the brain. Early reports were somewhat inconsistent on whether astrocytes demonstrated productive infection in vivo (127, 148). Some reports showed cells with morphology of astrocytes that stained for HIV structural protein p25 (107) or HIV accessory protein nef (6), and some detected viral particles in cells with intermediate filaments in HIV-infected patients (58). These early reports were technically limited by availability of molecular probes and dependence on cell morphology to determine cell lineage. Unfortunately astrocytes can adopt a variety of morphologies that can be particularly diverse in reactive conditions. Additionally, others have observed cross reactivity between HIV probes and neuroglial antigens (eg, astrocytes stain for HIVp17 monoclonal antibody in Alzheimer disease brains (67) and for HIV gp41 in pathologically normal brains (124)).

The advent of double label protocols, made investigating the question of astrocyte infection more tractable. Using combinations of ISH, PCR-ISH, and immunocytochemistry for glial fibrillary acidic protein (GFAP) several studies have detected co-labeling of astrocytes with HIV markers (100, 108, 131-133). Using laser capture microscopy with 100 samples in a fashion similar to that discussed above for neurons, HIV gag DNA was detected in GFAP immunostained astrocytes in ¾ of patients with HIV encephalitis or HIV dementia (136).

Historically, the keen interest in astrocytes and the abundance of GFAP they express in reactive conditions (particularly in plaques of multiple sclerosis patients) made antibodies to GFAP one of the first immunohistochemical reagents available for neuropathological studies. Those who frequently use the probes are aware that double labeling with GFAP is a double-edged sword. On the positive side, with superb immunohistochemical probes available for GFAP, they are readily deployed in double label experiments. On the negative side, depending upon staining conditions, GFAP antibodies can stain intermediate filaments in a variety of cells. To make matters worse, reactive astrocytes can non-specifically bind a variety of antibodies (perhaps through the intermediate filaments themselves). Conservative interpretation of “real” staining is critical particularly when detection systems are amplified and pushed to their limits.

This historical background distills to a practical philosophy that immunostaining of astrocytes for GFAP and other antibodies needs to be interpreted with caution. Despite the plethora of reactive astrocytes in HIV encephalitis, few investigators have found convincing evidence of productive infection of astrocytes using either double label IHC, ISH, or electron microscopy. This led to the hypothesis that only early viral proteins (tat, nef, rev) were translated in “abortively” infected astrocytes (non-productive infection), while translation of later structural proteins was blocked. Numerous antibodies were developed to these HIV antigens or their SIV counterparts and used to stain brain tissues (102, 116). Many of these probes were well characterized from a molecular standpoint, but staining protocols were poorly designed or controlled.

Even in the best of situations, screening antibodies on fixed human autopsy tissues is difficult. Because of safety concerns regarding infection control, AIDS tissues tended to be over fixed. Along with the sticky character of reactive astrocytes and stringent treatments associated with antigen recovery or nucleic acid denaturation necessary for ISH, testing the scientifically intriguing hypothesis that astrocytes support non-productive HIV infection using this broad spectrum of incompletely defined antibodies has been a nightmare. Images of in vivo staining were unconvincing and next to impossible to reproduce given the home-grown character of most of the reagents. As of now the field has broken into 2 camps; those who believe HIV infects astrocytes in vivo and those who do not. The believers are further divided into those who believe that astrocytes support productive infection (a dwindling number) and those who believe astrocytes support some yet to be defined abortive infection. Despite the importance of distinguishing between hypotheses assuming productive versus non-productive astrocyte infection, mutually incompatible evidence is shared between these believers in an indiscriminate fashion. Greek mythology was blessed with only one Pandora's box; the field of neuro-AIDS seems to be plagued with several.

In vivo/in vitro viral receptors on astrocytes

Astrocytes from normal human adult brains have immunocytochemically stained for the CCR5 HIV co-receptor (114), while reactive and fetal astrocytes have stained for the other important HIV co-receptor, CXCR4 (18, 110, 117, 149). As CD4 has not been detected on astrocytes, these findings raise the possibility that HIV could infect astrocytes in a CD4 independent fashion.

In vitro infection of astrocytes

Deciphering whether astrocytes were infected in vivo became even more twisted when investigators attempted to address the question in vitro. Simple infection experiments were replaced by more intricate transfection experiments that frequently begged the question of whether HIV could actually enter astrocytes. This article will not attempt to comprehensively review transfection experiments (9, 53), but rather will focus on published results addressing in vitro infectability of astrocytes and astrocyte cell lines.

Several in vitro models of astroglioma or astrocyte cell lines have suggested these cells can be either productively (26, 36, 43) or non-productively infected (53, 97). Those investigators claiming non-productive infection differ on the point of blockage in the virus life cycle. Some claim that the virus enters cell lines by a CD4 independent mechanism (61), while others claim it enters but transcription is blocked (53). Various and conflicting detailed molecular regulatory systems have been proposed to explain why astrocytes are not productively infected. Data from in vitro models of fetal or adult astrocyte infection are similarly confusing and contradicting. Some investigators claim astrocytes produce abundant HIV mRNA and/or structural proteins (19, 23, 62, 94, 102, 134, 142) or that only a subset of the astrocytes are infected (0.5%) (134). As with astrocytoma cell lines, some find non-productive infection of astrocytes (37), while some claim HIV is barred from entering astrocytes (13, 18). Some reports observed that HIV gp120 binds astrocytes in a CD4 and GalCer independent manner (81), while others observe that infection is decreased when cultures are pretreated with soluble CD4 (59, 60). Some claim that HIV produces a latent infection of fetal astrocytes in a CD4 and CCR5 independent manner (115), others claim that RANTES completely blocks productive infection (CCR5 dependent) (102), while still others claim that astrocyte infection is abortive (56). Finally, some hypothesize that astrocyte infection is restricted due to a block in HIV rev function (transport of mRNA variants to the cytoplasm). For example, restricted infection of astrocytomas and human astrocytes show rev in the cytoplasm and nucleus instead of exclusively in the nucleus as seen in permissive cell lines (80, 96).

In summary, the in vitro studies of astrocyte infection cannot be distilled to a coherent theory. The published findings are so conflicting that one can pick data to support any pet hypothesis related to the pathogenesis of astrocyte infection. Selective sampling of this topic lends little to deciphering the original question: are astrocytes infected in vivo?

Summary of astrocyte infection by HIV

In vivo evidence of astrocyte infection is not strong. While early studies suggested the possibility of such an infection, critical evaluation of later studies employing double labeling techniques do not support the theory that HIV or SIV productively infects astrocytes in the brain (Figure 1B). Theories of non-productive astrocyte infection have yet to achieve a coherence that can be rigorously tested. In vitro studies of astrocyte infection are little less than a quagmire of irreconcilable findings.

CONCLUSIONS

Despite the voluminous literature and substantial experimental effort over the past two decades, evidence for productive infection of any brain cell other than macrophages is left wanting. Whether there is biological import or relevance to studying an abortive infection of neuroglial elements awaits construction of a coherent theory of pathogenesis. In the mean time the very real problem of HIV infection of brain macrophages and how this leads to neuronal degeneration remains sadly unattended.

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