Protector, Prey, or Perpetrator:
The Pathophysiology of the Blood-Brain Barrier in NeuroAIDS

William A. Banks<sup>1

1</sup>GRECC, Veterans Affairs Medical Center-St. Louis and Saint Louis University School of Medicine, Div. of Geriatrics, Dept. of Internal Medicine, 915 N. Grand Blvd, St. Louis, MO 63106; USA

E-mail: bankswa@slu.edu

Keywords: Blood-brain barrier, endothelium, adsorptive endocytosis, gp120, HIV-1, choroid plexus, efflux, microglia, p-glycoprotein, lymphatics

What are the Possible Portals of Entry for HIV into the CNS?
The most widely suspected mechanism by which HIV enters the CNS is with the entry of infected monocytes crossing the vascular barrier. This pathway has recently been thoroughly reviewed in a Science Online NeuroAIDS article (1). But other mechanisms are available. HIV could cross at the choroid plexus, which is infected by HIV (2). HIV also could cross by extracellular pathways (3). Although circulating substances such as serum albumin are largely excluded by the BBB, such exclusion is not absolute since serum-derived albumin can be detected in the CSF. Whether viruses in general or HIV in particular can use these pathways is unknown. Viruses could also enter the brain tissue at the circumventricular organs (CVO), small areas of the brain with an absent or diminished BBB (4). Although these areas are separated from the rest of the brain, they do provide a possible site of CNS infection that does not require penetration of the BBB. This could lead to the disturbance of the CNS functions controlled by the CVO. For example, disturbance of the thirst mechanism (5) could lead to the unmasking of diabetes insipidus, which could lead to hypernatremia and altered mental status (6). Furthermore, these areas have neural connections to the rest of the CNS, and the delimiting layer of cells can secrete neuroactive substances into the CNS. Of the other mechanisms of entry into brain used by some viruses, retrograde axonal transport can be excluded since HIV is not neuronotropic, whereas entry through the olfactory mucosa has apparently not been considered as a route for HIV.

How Does HIV Cross Brain Endothelial Cells?
Brain endothelial cells are CD4 and galactosylceramide negative, the two glycoprotein complexes to which HIV-1 typically binds to initiate its entry into immune and other cells (7). So one would assume that HIV could not cross the brain endothelium. However, evidence for several other mechanisms has been presented. Infected macrophages bind to activated brain endothelial cells (8). This may be the first stage in a process that eventually leads to the macrophage crossing the BBB and taking up residence in the CNS as an infected microglial cell (9). Another hypothesis is that brain endothelial cells themselves first become infected with HIV and so could then shed virus into the brain, a mechanism used by several viruses including SIV (10). Brain endothelial cells have been infected with HIV in vitro (7) and some studies have found HIV in brain endothelial cells from AIDS patients (11, 12, 13, 14 ). Cell-free virus may also be able to cross the BBB. Viral uptake by cells is vesicular-dependent and may occur across non-activated brain endothelial cells. It has been shown that gp120 crosses the BBB by inducing adsorptive endocytosis in brain endothelial cells (15, 16). This has led to the hypothesis that gp120 is key to the ability of infected immune cells, which express gp120, and virus to cross the BBB.

Does HIV Have to Enter the CNS to Produce CNS Effects?
HIV could exert effects on the CNS without crossing the BBB through several mechanisms. Actions at the CVO have been outlined above.

Viral products, rather than whole virus or infected immune cells, could cross the BBB. The viral coat gp120 crosses the BBB at a modest rate by the mechanism of adsorptive endocytosis (15). Since gp120 is very neurotoxic, its entry could induce CNS effects. Circulating gp120 has not been demonstrated in the blood, even though it is readily shed from virus. The ability of Tat and other viral products to cross the BBB has not been investigated. However, brain endothelial cells respond to tat treatment (17) suggesting that binding of tat and possibly internalization occurs.

The levels of many of the proinflammatory cytokines are elevated in the blood of patients with AIDS (18, 19) and their decrease with antiretroviral therapy is associated with cognitive improvement (20). TNF, IL-1, and IL-6 are all transported across the BBB and so could directly affect CNS function (21). These cytokines also have direct effects on BBB function, including disrupting it. In addition, cytokines have more subtle effects, which would likely be more important in AIDS dementia. Altered transport of glucose, amino acids, vitamins, or regulatory substances contributes to CNS dysfunction in other disease states and could do so in AIDS (22).

Much work has focused on the release of neurotoxins from microglia and astrocytes. However, the brain endothelial cell is also a source of nitric oxide, cytokines, and other substances with neurotoxic effects (23, 24). Circulating substances associated with HIV infection, such as Tat and the cytokines themselves, can induce the brain endothelial cells to release these substances. The endothelial cell does not require that HIV be within the CNS to produce these effects, as half of the endothelial cell surface faces the circulation and half the CNS. Endothelial cells have the potential to react to blood-borne substances at their luminal surface and to release neurotoxic substances at their abluminal surface.

Why Do Anti-viral Drugs Concentrate in the Brain so Poorly?
Once within the CNS, the BBB may protect HIV from blood-borne endogenous and administered anti-virals. Accumulation of drugs is a balance between their influx (blood to CNS) and efflux rates. The rate of influx is determined largely by whether the drug is lipid soluble and whether there is a saturable transport system located located at the BBB that it can utilize. Efflux depends on reabsorption of the cerebrospinal fluid and whether saturable transport in the CNS to blood direction occurs for that substance. Accumulation in the CNS of a large number of drugs is prevented by a variety of efflux transporters. A classic example is the efflux of penicillin by the probenicid-sensitive organic acid efflux system (25). The p-glycoprotein and multi-drug transporter systems have emerged as major efflux systems for many drugs (26). These efflux systems characteristically reduce accumulation by 3 to 15 fold. In animals without the p-glycoprotein transporter, loperamide, an opiate anti-diarrheal noted for its lack of CNS effects, exerts analgesic properties (27). Some of the HIV anti-virals are substrates for the p-glycoprotein system, in particular the protease inhibitors (28). AZT is transported out of the brain by a probenicid-sensitive efflux system (29). Despite this transportation, AZT treatment temporarily reduces the incidence of neurological complaints (30). Inhibition of efflux from the brain or development of anti-virals that are not substrates for efflux systems would likely make these drugs effective within the CNS. For example, a single dose of 40 mg of stavudine results in a peak concentration in CSF of 61 ng/ml (31), whereas the ED50 for stavudine is 52 ng/ml. Decreasing efflux by even 1/3 would both increase the peak concentration and the half time in the CNS by 3 fold, greatly enhancing the therapeutic effect of this anti-viral drug.

How Does Virus Reenter the Circulation?
If the CNS acts as a reservoir for virus capable of reinfecting the periphery, then how HIV exits the CNS is as vital a question as how it enters. If HIV enters primarily inside infected macrophages, then it might be expected to exit by a similar mechanism. However, the luminal and abluminal surfaces of the BBB have a great number of differences, including a different glycoprotein composition (32, 33). As a result, many of the vesicular mechanisms related to diapedesis do not apparently operate in the brain to blood direction. Cell-free virus would likely be reabsorbed with the cerebrospinal fluid back into blood. Another pathway, at least for gp120, is reabsorption by the primitive lymphatics of the brain (34). These drain directly into the cervical nodes. This provides a pathway by which virus or infected immune cells could travel directly from the CNS to lymph nodes without having to be exposed to blood-borne antivirals.

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