Transmigration of macrophages across the choroid plexus epithelium in response to the feline immunodeficiency virus.

Although lentiviruses such as human, feline and simian immunodeficiency viruses (HIV, FIV, SIV) rapidly gain access to cerebrospinal fluid (CSF), the mechanisms that control this entry are not well understood. One possibility is that the virus may be carried into the brain by immune cells that traffic across the blood-CSF barrier in the choroid plexus. Since few studies have directly examined macrophage trafficking across the blood-CSF barrier, we established transwell and explant cultures of feline choroid plexus epithelium and measured trafficking in the presence or absence of FIV. Macrophages in co-culture with the epithelium showed significant proliferation and robust trafficking that was dependent on the presence of epithelium. Macrophage migration to the apical surface of the epithelium was particularly robust in the choroid plexus explants where 3-fold increases were seen over the first 24 h. Addition of FIV to the cultures greatly increased the number of surface macrophages without influencing replication. The epithelium in the transwell cultures was also permissive to PBMC trafficking, which increased from 17 to 26% of total cells after exposure to FIV. Thus, the choroid plexus epithelium supports trafficking of both macrophages and PBMCs. FIV significantly enhanced translocation of macrophages and T cells indicating that the choroid plexus epithelium is likely to be an active site of immune cell trafficking in response to infection.

Astrocytes and microglia differentially regulate trafficking of lymphocyte subsets across brain endothelial cells.

Feline brain endothelial cells (BECs), astrocytes, and microglia were combined in different configurations in a cell culture insert system to assess the effect of different cell types on the trafficking of peripheral blood mononuclear cell (PBMC) subsets in response to feline immunodeficiency virus (FIV). The addition of astrocytes to BECs significantly increased the adherence of PBMCs. This increase in adherence was suppressed by microglia, whereas microglia alone had no effect on PBMC adherence. FIV exposure of the glial cells did not alter PBMC adherence as compared to same configurations with untreated cells. All PBMC subsets showed some level of trafficking across the endothelial cell layer. The level of trafficking of monocytes and B cells was significantly increased if astrocytes were present. The presence of microglia with the astrocytes reduced transmigration across all PBMC subsets. FIV exposure of astrocytes significantly increased the percentage of CD8 T cell transmigration from 24% to 64% of the total CD4 and CD8 numbers. The presence of microglia significantly reversed the preferential trafficking of CD8 cells in the presence of astrocytes. The results suggested that interaction between the triad of endothelial cells, astrocytes, and microglia played an important, but varying, role in the trafficking of different PBMC subsets. In general, astrocytes had a positive effect on trafficking of PBMCs, while microglia had a suppressive effect. Effects of FIV on trafficking were largely restricted to increases seen in CD8 T cells and monocytes.

Development of neuronal sensitivity to toxins in cerebrospinal fluid from HIV-type 1-infected individuals.

HIV infection of the immature nervous system generally results in a rapid progression of neurological disease that cannot easily be explained by the severity of encephalitis, viral burden, systemic immune deficiency, or developmental changes in utero. Rather than the viral infection dictating disease progression, we explored the possibility that immature neurons might be particularly sensitive to toxins secreted in response to HIV. Primary cultures of rat cortical neurons were exposed to toxic cerebrospinal fluid (CSF) from HIV-infected individuals (CSF(tox)) and evaluated for changes in intracellular calcium and cell death. CSF(tox) had no detectable effect on early neurite outgrowth, calcium regulation, or cell death during the first few days in culture. Starting at Day 4, delayed increases in intracellular calcium appeared in response to CSF(tox). The magnitude of the delayed calcium rise and cell death increased with the age of the culture and correlated with the appearance of synaptophysin immunoreactive varicosities. A similar gradual development of sensitivity was seen during exposure of feline neurons to toxins generated by choroid plexus macrophages after exposure to feline immunodeficiency virus. The possibility that toxin sensitivity is dependent on the presence of synaptic activity is consistent with the rapid pathogenesis in the CNS seen during the first postnatal year. Emerging synaptic activity coupled with other factors such as high metabolic demand in the young nervous system may combine to increase the likelihood of calcium overload and neuronal dysfunction in response to HIV-associated toxins.

Perinuclear biogenesis of mutant torsin-A inclusions in cultured cells infected with tetracycline-regulated herpes simplex virus type 1 amplicon vectors.

TorsinA is a novel protein identified in the search for mutations underlying the human neurologic movement disorder, early onset torsion dystonia. Relatively little is understood about the normal function of torsinA or the physiological effects of the codon deletion associated with most cases of disease. Overexpression of wild-type torsinA in cultured cells by DNA transfection results in a reticular distribution of immunoreactive protein that co-localizes with endoplasmic reticulum resident chaperones, while the dystonia-related mutant form accumulates within concentric membrane whorls and nuclear-associated membrane stacks. In this study we examined the biogenesis of mutant torsinA-positive membrane inclusions using tetracycline-regulated herpes simplex virus amplicon vectors. At low expression levels, mutant torsinA was localized predominantly around the nucleus, while at high levels it was also concentrated within cytosolic spheroid inclusions. In contrast, the distribution of wild-type torsinA did not vary, appearing diffuse and reticular at all expression levels. These observations are consistent with descriptions of inducible membrane synthesis in other systems in which cytosolic membrane whorls are derived from multilayered membrane stacks that first form around the nuclear envelope. These results also suggest that formation of mutant torsinA-positive inclusions occurs at high expression levels in culture, whereas the perinuclear accumulation of the mutant protein is present even at low expression levels that are more likely to resemble those of the endogenous protein. These nuclear-associated membrane structures enriched in mutant torsinA may therefore be of greater relevance to understanding how the dystonia-related mutation compromises cellular physiology.

Infection of the choroid plexus by feline immunodeficiency virus.

The human, simian, and feline immunodeficiency viruses rapidly penetrate into the brain and trigger an inflammatory process that can lead to significant neurologic disease. However, the mechanisms that permit efficient trafficking of macrophage-tropic and the more neurotoxic lymphocytotropic isolates are still poorly understood. One potential source of virus entry may be the blood-CSF barrier provided by the choroid plexus. Infected cells are often detected within the choroid plexus but it is unclear whether this reflects trafficking cells or infection of the large macrophage population within the choroidal stroma. To address this issue, we cultured fetal feline choroid plexus and evaluated the ability of feline immunodeficiency virus (FIV) to establish a primary infection. Significant provirus was detected in macrophage-enriched choroid plexus cultures as well as in the choroid plexus of cats infected in vivo. FIV p24 antigen production in vitro was very low but detectable. Addition of a feline T-cell line to macrophages inoculated with FIV resulted in a dense clustering of the T cells over macrophages with dendritic cell-like morphologies and a robust productive infection. The direct infection of choroid plexus macrophages with FIV, the efficient transfer of the infection to T cells indicate that the choroid plexus can be a highly efficient site of viral infection and perhaps trafficking of both macrophage-tropic and T-cell-tropic viruses into the CNS.

Choroid plexus macrophages proliferate and release toxic factors in response to feline immunodeficiency virus.

Recent observations have suggested that lentiviruses stimulate the proliferation and activation of microglia. A similar effect within the dense macrophage population of the choroid plexus could have significant implications for trafficking of virus and inflammatory cells into the brain. To explore this possibility, we cultured fetal feline macrophages and examined their response to feline immunodeficiency virus (FIV) or the T-cell-derived protein, recombinant human CD40-ligand trimer (rhuCD40-L). The rhCD40-L was the most potent stimulus for macrophage proliferation, often inducing a dramatic increase in macrophage density. Exposure to FIV resulted in a small increase in the number of macrophages and macrophage nuclei labeled with bromodeoxyuridine. The increase in macrophage density after FIV infection also correlated with an increase in neurotoxic activity of the macrophage-conditioned medium. Starting at 16-18 weeks postinfection, well after the peak of viremia, a similar toxic activity was detected in cerebrospinal fluid (CSF) from FIV-infected cats. Toxicity in the CSF increased over time and was paralleled by strong CD18 staining of macrophages/microglia in the choroid plexus and adjacent parenchyma. These results suggest that lentiviral infection of the choroid plexus can induce a toxic inflammatory response that is fueled by local macrophage proliferation. Together with the observation of increasing toxic activity in the CSF and increased CD18 staining in vivo, these observations suggest that choroid plexus macrophages may contribute to an inflammatory cascade in the brain that progresses independently of systemic and CSF viral load.

Destabilization of neuronal calcium homeostasis by factors secreted from choroid plexus macrophage cultures in response to feline immunodeficiency virus.

The choroid plexus contains a major reservoir of macrophages poised for efficient delivery of virus and neurotoxins to the brain after infection by lentiviruses such as human or feline immunodeficiency virus (FIV). However, their contribution to neurotoxicity is poorly understood. Medium from FIV-infected, choroid plexus macrophages applied to cultured feline cortical neurons induced a small acute calcium rise followed by either a delayed calcium deregulation (41%) or swelling and bursting (23%). NMDA glutamate receptor blockade prevented the acute calcium increase and antagonists to the IP(3) receptor, voltage-gated calcium channels and sodium channels suppressed both the acute and late increases. Analysis of intracellular calcium recovery in toxin-treated neurons after a brief exposure to glutamate, revealed a decrease in the rate and extent of recovery. The apparent diverse pharmacological contributions to intracellular calcium destabilization may be due to the ability of macrophage toxins to interfere with recovery of intracellular calcium homeostasis.

Microglial proliferation in cortical neural cultures exposed to feline immunodeficiency virus.

Microglia are thought to play an important role in neurodegenerative changes due to infection with human or animal immunodeficiency viruses. Using feline immunodeficiency virus and cat neural cultures, we observed a dramatic increase in the accumulation of microglia from a basal rate of 5-7% day(-1) to 25-126% day(-1). Both live virus and heat-inactivated virus induced proliferation. Negligible proliferation was seen in purified microglial cultures. Conditioned medium from astrocytes or mixed neural cultures treated with feline immunodeficiency virus stimulated the proliferation of purified microglia. Disease progression may be facilitated by early non-infectious interactions of lentiviruses with neural tissue that promote the activation and proliferation of microglia.

Neurotoxicity of FIV and FIV envelope protein in feline cortical cultures.

The neurotoxic effects of the feline immunodeficiency virus (FIV) and FIV envelope proteins were measured in primary cultures of feline cortical neurons. Envelope protein from the FIV-PPR strain promoted neuronal swelling and death, whereas envelope protein from the FIV-34TF10 isolate produced intermediate or negligible toxicity. No effect was observed in control cultures treated with envelope protein from the Epstein-Barr virus. A concentration-effect curve showed that FIV-PPR protein produced maximal toxicity at 200 pM protein and decreased toxicity at higher concentrations, which is consistent with previous reports of the HIV-1 surface glycoprotein, gp120. These effects required the presence of low concentrations of glutamate. Using the natural host cells as targets, the effects of envelope protein and infectious virions were directly compared. All of the toxic activity could be attributed to non-infectious interactions between the viral envelope and target cells. Addition of 1 microM tetrodotoxin failed to block the effects of FIV-PPR in the presence of 20 microM glutamate. Toxicity would appear to involve two steps in which the envelope protein first sensitizes neurons through non-synaptic interactions (TTX insensitive) thereby setting the stage for enhanced synaptic activation via glutamate receptors (TTX sensitive).