Neuroimmunity and the Blood–Brain Barrier: Molecular Regulation of Leukocyte Transmigration and Viral Entry into the Nervous System with a Focus on NeuroAIDS

Abstract

Introduction

An early consequence of human immunodeficiency virus type 1 (HIV-1, which we will refer to as “HIV”) infection is entry of the virus into the central nervous system (CNS), resulting in several neurological sequelae. Neuropathological conditions that result from HIV entry into the CNS include HIV-associated encephalitis (HIVE), which is characterized by the formation of multinucleated giant cells, inflammation, microglial activation, astrogliosis, myelin pallor, loss of neurons and astrocytes, and the development of a syndrome of cognitive and motor dysfunction often leading to HIV-associated dementia (HAD) (Gendelman et al. 2005). Prior to the availability of highly active antiretroviral treatment (HAART), HAD was detected in approximately 20% of individuals with AIDS (McArthur et al. 1993). Initially, there was a drop in the incidence of dementia and other neurological manifestations as a result of HAART. However, the prevalence of neurologic disease in people with HIV has actually increased because individuals are living much longer, and therapeutic drugs are relatively ineffective in crossing the blood–brain barrier (BBB) (McArthur et al. 2003; Anthony et al. 2005). Thus infection and inflammation persist in the CNS despite HAART, suggesting that CNS infection and inflammation are independent of peripheral HIV status. The mechanism of HIV-induced CNS infection and inflammation is complex. In this review, we summarize several reports that provide an understanding of leukocyte transmigration across the BBB and viral entry into the CNS, and their role in the pathogenesis of cognitive impairment. Lastly, we describe some animal models that have contributed to our understanding of HIV entry into the CNS, therapeutics that could potentially improve or limit some of the neurological complications induced by HIV infection, and the contributions of substances of abuse to the pathogenesis of NeuroAIDS.

Mechanisms of viral entry across the BBB

The molecular mechanisms by which HIV enters the CNS and contributes to both acute and chronic inflammatory processes that may culminate in neurologic dysfunction are still unclear. The most accepted hypothesis on viral entry into the CNS is the “Trojan Horse” model, in which infected monocytes and lymphocytes transport the virus from the blood into the CNS. After entry into the CNS, these infected cells release toxic molecules, including viral proteins, cytokines, and chemokines that recruit resident CNS cells, especially microglia and astrocytes, to areas of viral infiltration (Fig. 1). This recruitment allows for subsequent infection of these resident cells and the spread of HIV within the CNS. HIV infects perivascular macrophages and microglia productively, while astrocyte infection is restricted, resulting in the formation of viral reservoirs within CNS cells in which replication competent viral genomes persist in a stable state (Lavi et al. 1998). There is no evidence of direct HIV infection of neurons; therefore, the neuronal cell damage and death that occurs in HAD must be mediated by production and release of neurotoxic factors by other infected or uninfected cells within the CNS. In fact, neuronal dysfunction correlates more closely with inflammation and activated monocytes/microglia than with viral load (Sevigny et al. 2004).

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Fig. 1

Sequence of inflammatory events in NeuroAIDS. Gap junctions, integrins, adhesion molecules, tight junction proteins (TJPs), adherens junction proteins (AJPs), matrix metalloproteases (MMPs), and viral proteins such as Tat, gp120, Nef, and Vpr, participate in HIV-infected leukocyte adhesion to and transmigration across EC of the BBB. CCL2, and possibly other chemoattractants, significantly enhances this transmigration. Once within the CNS, the leukocytes produce soluble viral proteins, cytokines, chemokines, and other factors, thus recruiting, activating, and facilitating the infection of microglia and astrocytes. HIV-associated cognitive impairment begins with the initial transmigration of infected leukocytes into the CNS, which leads to BBB disruption, causing acute inflammation. During the subsequent chronic phase of inflammation and infection, leukocyte transmigration is an ongoing process, resulting in NeuroAIDS. Substances of abuse and alcohol contribute to the progression of this neuropathogenesis.

A second proposed mechanism of HIV entry into the CNS is the direct infection of BBB cells by cell-free HIV, providing a source of infectious particles in close proximity to the CNS parenchyma, thus promoting viral infection of resident CNS cells as well as the further release free virus. However, this theory does not explain how the extracellular virus in the circulation survives long enough to infect BBB cells. Recent studies demonstrated that proteoglycans on the surface of endothelial cells (EC) bound and protected circulating HIV from degradation for a prolonged period of time, providing an opportunity for EC to become infected (Argyris et al. 2003; Bobardt et al. 2004) through a CD4 and galactosylceramide-independent mechanism (Moses et al. 1993; Mankowski et al. 1994). This mechanism, however, still does not address how HIV crosses the structural components of the BBB, which includes astrocyte foot processes and pericytes, after EC infection, nor does it distinguish between viral uptake and productive infection. Furthermore, the issue on whether EC are productively infected by HIV remains controversial.

A third model of viral entry into the CNS is the internalization of the HIV virion by EC or astrocytic foot processes by macropinocytosis or endocytosis, with subsequent transfer of the virus to parenchymal CNS cells (Banks et al. 1998, 2001; Marechal et al. 2001; Liu et al. 2002a). Macropinocytosis in BBB EC is dependent on intact lipid rafts and MAPK signaling (Liu et al. 2002a). The possibility of macropinocytotic entry of HIV into CNS EC is supported by the presence of increased surface microvilli on EC, abundance of cytoplasmic vesicles, and inhibition of HIV entry by dimethylamiloride, an inhibitor of the Na⁺/H⁺ pump in membranes (Liu et al. 2002a). However, the presence of virions within EC or BBB cells may also be due to direct infection of these cell types, transcytosis of virions through the BBB, or the presence of HIV-infected leukocytes trapped in the BBB during the process of transmigration.

The last proposed mechanism for viral entry into the CNS is the nonspecific passage of HIV through the BBB and into the brain. This mechanism is a combination of the other mechanisms (previously described) that is dependent on the loss of BBB integrity due to exposure to cytokines, chemokines, viral proteins, or neurotransmitters, and/or the induction of apoptosis in EC and/or astrocytes. However, this theory is the least likely explanation, because it requires extensive BBB disruption early in the course of infection without repair. This seems unlikely given that HIV-infected individuals now live longer, in a chronic phase of infection, as a result of HAART.

In all of these proposed models, the integrity and function of the BBB is essential in regulating the passage of the virus into the CNS parenchyma. Alterations in the structural integrity of the BBB result in increased EC permeability. The mechanisms involved in this increase in permeability are unknown, but there is a close correlation between elevated blood and cerebral spinal fluid (CSF) cytokine levels induced by injury and the breakdown of the BBB, again suggesting that inflammation plays an important role (Martiney et al. 1992; Hurwitz et al. 1994; Fiala et al. 1997, 1998; Dallasta et al. 1999; Rubin and Staddon 1999; Gloor et al. 2001; Strelow et al. 2001; Eugenin et al. 2006a).

Blood–brain barrier function

BBB is a physical and metabolic barrier that separates the CNS from the periphery (see Persidsky et al. in this issue for details on BBB biology). It is not rigid and is composed of dynamic vessels that are capable of responding to rapid changes in the brain or blood (Huber et al. 2001). BBB is mainly composed of EC in close contact with astrocytic end foot processes (Fig. 1). BBB cells express very specific systems of transport of metabolites, as well as tight junction proteins (TJP), which seal the intercellular gaps between EC–EC and EC–astrocytes, resulting in impermeability to most macromolecules and blood cells (Pardridge and Choi 1986; Goldstein 1988; Pardridge et al. 1990; Risau and Wolburg 1990; Risau 1991; Rubin and Staddon 1999).

The major functions of BBB are to transmit biochemical signals from the blood to the brain, to exclude leukocytes and soluble factors, and to select specific nutrients to transport into or out of the brain (Pardridge and Choi 1986; Pardridge et al. 1990; Risau and Wolburg 1990). The concept of the CNS as an “immune privileged” site requires that the cells of the BBB and CNS parenchyma be shielded from physical and chemical interactions with circulating cells of the immune system. However, cells of the BBB, CNS parenchyma, and circulating leukocytes all express many common cell surface receptors that respond to soluble factors released by these cell types. Many studies demonstrated crosstalk among BBB cells, cells within the CNS expressing immune cell receptors, and immune cells expressing receptors common to CNS cells, warranting the reevaluation of the extent to which the CNS is “immune privileged” (Fig. 2).

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Fig. 2

Neuroimmune interactions in NeuroAIDS. A simplified representation of extensive interactions among BBB cells, and immune and central nervous systems. These interactions allow each system to detect changes in others. Most of the communication among these systems is between gap junctions, ions (sodium, potassium, and calcium), receptor–ligand interactions, and soluble factors, including cytokines, chemokines, neurotransmitters, neurotrophic factors, and adhesion molecules, both intact on the cell surface and/or in soluble forms. HIV infection in both the CNS and the periphery can alter the ability of these systems to detect change.

The role of BBB as an active participant in CNS function is still not completely understood. However, the recent findings that leukocytes and CNS cells share proteins originally believed to be expressed uniquely on one or the other, may enhance our understanding of viral entry into the CNS, as well as explain how the neurological complications seen in NeuroAIDS are continuing during the chronic phase of infection and become highly prominent during the late stages of HIV disease pathogenesis. Some of the newly described mechanisms by which BBB cells may have a proactive role in the modulation of CNS function are: (1) calcium waves in BBB cells, supported by ATP receptors and gap junctions; (2) neurotransmitters originating from the CNS or blood acting on BBB cells; (3) intercellular communication between BBB cells and leukocytes through gap junctions; (4) BBB activation of accessory cells, such as pericytes and perivascular macrophages.

Transmigration of HIV-infected leukocytes across the BBB

Leukocyte transmigration across the BBB during normal immune surveillance is an active process not only for the leukocyte but also for the BBB cells (Fig. 1). It has been characterized as a dynamic multistep process involving the initial “rolling” of cells on vessel endothelium in response to locally produced proinflammatory mediators, and subsequent firm adhesion to, and diapedesis across the vasculature. The rolling of leukocytes along the endothelial surface is mediated by weak interactions of selectin molecules and their corresponding glycoprotein ligands, expressed by activated EC and leukocytes. Rolling leukocytes are then stimulated by chemokines and other chemotactic molecules, resulting in activation of the β1 integrin, VLA-4, and the β2 integrin, LFA-1. Ultimately, leukocyte arrest occurs, mediated by strong interactions between LFA-1 and its EC counter receptor, intercellular adhesion molecule-1 (ICAM-1), and between VLA-4 and its EC counter receptor, vascular cell adhesion molecule-1 (VCAM-1) (Muller 2003; Liu et al. 2004; van Buul and Hordijk 2004). Leukocyte diapedesis across the blood vessel endothelium is a process mediated, in part, by homophilic and heterophilic binding molecules including junctional adhesion molecules (JAM), platelet endothelial cell adhesion molecule 1 (PECAM-1), and CD99 (Martin-Padura et al. 1998; Del Maschio et al. 1999; Ostermann et al. 2002; Schenkel et al. 2002; Ebnet et al. 2004). Although other adhesion proteins and integrins are involved in leukocyte transmigration, data suggest that those mentioned above may be associated with the development of HIV CNS pathology (Seilhean et al. 1997; Eugenin et al. 2006b).

The BBB excludes most circulating leukocytes, although there is baseline trafficking of lymphocytes and monocytes. Therefore regulatory mechanisms must exist to minimize the deleterious effects of inflammatory events in the CNS. There are a few studies that address how HIV infection alters the dynamic interaction between leukocytes and EC. An immunohistochemical study of HIVE tissue showed increased expression of adhesion proteins on astrocytes (Seilhean et al. 1997). HIV infection of human monocytes increased their expression of LFA-1 (Stent and Crowe 1997), and another study showed that the adhesion of HIV-infected monocytes to EC induced the expression of E selectin and VCAM-1 (Nottet et al. 1996). Recently, we demonstrated that in brain tissue from individuals with HIVE there is an accumulation of cleaved, soluble forms of the extracellular region of PECAM-1 (sPECAM-1) (Eugenin et al. 2006b). In addition, we assayed sera from individuals with HIV for sPECAM, and found elevated levels. These findings suggest that this sPECAM-1 production by HIV-infected cells can alter PECAM interactions between EC–EC and EC–leukocytes and thus contribute to enhanced transmigration of HIV-infected leukocytes into the CNS and changes in BBB permeability during the pathogenesis of NeuroAIDS.

There is evidence in end-stage HIV disease of abnormal BBB structure and altered expression of the TJP ZO-1 and occludin on sites where leukocyte infiltration and HIV infection were detected (Dallasta et al. 1999; Boven et al. 2000). Using an in vitro model of the BBB and blood-derived primary monocytes infected with the R5 HIV strain, HIV-1_ADA, it was shown that the state of immune activation of cells that constituted the BBB dictated the ability of monocytes to traverse the BBB (Persidsky et al. 1997). Using a similar in vitro model of the BBB, we demonstrated increased BBB permeability and a loss of TJP when HIV-infected monocytes transmigrated across the BBB in response to the chemokine CCL2. The mechanism of BBB disruption and enhanced transmigration of HIV-infected cells was specifically CCL2-dependent, because other chemokines did not elicit the same effect (Eugenin et al. 2006a). We also found that BBB disruption was associated with enhanced expression of matrix metalloprotease-2 (MMP-2) and matrix metalloprotease-9 (MMP-9) (Eugenin et al. 2006a). We propose that during acute infection, HIV-infected cells disrupt the BBB as they gain access to the CNS in vivo. This transient disruption facilitates ongoing inflammation within the CNS. The advent of HAART has created a chronic phase of CNS infection/inflammation in which this ongoing inflammation is subtle, but continues to cause CNS damage over an extended time period. In late-stage disease, extensive inflammation occurs, and may result in overt dementia. Future studies will investigate the role of CCL2 in HIV entry into the CNS by characterizing the transmigration of HIV-infected monocytes in the absence and presence of CCL2, and by examining the contribution of BBB cells and HIV-infected peripheral blood mononuclear cells to CCL2 enhanced transmigration.

Cytokines/chemokines that contribute to neuropathogenicity of HIV

Blood–brain barrier dysfunction during HIV infection may be attributable to many host factors. Cytokines/ chemokines, excitatory amino acids, neuropeptides, and other factors have been shown to cause BBB disruption, increased leukocyte transmigration, and EC and neuron apoptosis. Cytokines are a large and diverse group of small polypeptides that have a broad range of effects on different cell types. Chemokines are a subclass of cytokines that chemoattract and activate immune and nonimmune cells both in vivo and in vitro. Neurons, astrocytes, microglia, and oligodendrocytes produce many of these molecules, and all cell types in the brain express an array of corresponding receptors that provide responsiveness to a variety of different cytokines/chemokines (Cartier et al. 2005). Cytokines including TNF-α, IFN-γ, interleukin (IL)-1β and IL-6, and the chemokine CCL2 have been shown to be increased in sera, brain tissue, and CSF of individuals with HIVE and to modulate the BBB.

Although the signals that mediate the early transmigration of HIV-infected leukocytes into the brain are unknown, it has been proposed by our group and others that chemokines, especially CCL2, play a critical role during this process. Chemokines and their receptors are more highly expressed in HIV and simian immunodeficiency virus (SIV) encephalitic brain tissue as compared to normal brain, as well as in cultured microglia, astrocytes, and neurons in the presence of HIV (Sasseville et al. 1996; Conant et al. 1998; Gabuzda et al. 1998; Kelder et al. 1998; Lavi et al. 1998; Sanders et al. 1998; Albright et al. 1999; McManus et al. 2000). Experiments in transgenic mice and humans indicated that CCL2 is an essential chemokine for monocyte transmigration into the brain parenchyma (Fuentes et al. 1995; Gonzalez et al. 2002). Moreover, it has been demonstrated that HIV-infected individuals homozygous for the MCP-1-2578G allele have elevated levels of MCP-1/CCL2 in the CSF, and a 4.5-fold increased risk of developing HAD (Gonzalez et al.2002; Letendre et al. 2004). In addition, elevated levels of CCL2 have been detected in the CSF of individuals with HAD and HIVE (Conant et al. 1998; Kelder et al. 1998). In SIV, an animal model of HIV (see later sections for more details), CSF levels of CCL2 are biphasic, rising early after infection, and again later during the course of CNS disease (Zink et al. 2001; Mankowski et al. 2004). When minocycline, an antibiotic with potent anti-inflammatory and neuroprotective properties, was given beginning 21 days postinoculation, there was reduced severity of encephalitis, suppressed viral load in the brain, and decreased expression of CNS inflammatory markers (Zink et al. 2005). These SIV studies have shown that the second phase of increased CCL2 CSF levels may correlate with the severity of injury. As noted above, we demonstrated the enhanced transmigration of HIV-infected leukocytes across the BBB, and BBB disruption, in response to CCL2. The expression by EC of CCR2, the CCL2 receptor, may also play a role in this process (Dzenko et al. 2005).

Interestingly, CCL2 may also have a novel role as a protective factor in human neurons and astrocytes from NMDA and HIV-Tat induced apoptosis (Eugenin et al. 2003b). Thus, CCL2 has a complex pivotal role in the pathogenesis of NeuroAIDS. For a detailed discussion on cytokine and chemokine expression induced or modulated by HIV in the CNS, see Speth et al. (2005).

Viral factors and the BBB

Many laboratories examined the direct effects of HIV-specific proteins on the human BBB and CNS cells. Viral proteins in the brain are released or secreted from free viral particles and HIV-infected cells. These proteins induce functional changes in their host cells that further contribute to neuronal injury and BBB disruption (Fig. 1).

Stress and HIV

There have been several studies examining the effects of depression and stress on AIDS progression. Although there are conflicting data due to differences in research design, duration of follow-up, number and quality of control variables, and measurement of depression or study outcome, evidence suggests that depression and stress may be predictive of, or contribute to, HIV disease progression (Burack et al. 1993; Lyketsos et al. 1993; Leserman et al. 1997, 1999, 2000; Ickovics et al. 2001; Leserman et al. 2002). Many studies demonstrated the negative effects of stress and depression on cells of the immune system that are also affected by HIV (Evans et al. 1989; Weisse 1992). Comorbid clinical depression and some symptoms of AIDS are similar, such as poor appetite, weight loss, and cognitive impairment. It has been shown that inflammatory cytokines can contribute to these symptoms (Dantzer and Kelley 1989; Clerici et al. 1997).

Inflammation within the CNS results in the elaboration of cytokines that may act on neurons and other cells within the CNS to cause changes in neuronal function, thereby contributing to cognitive impairment or dementia. HAART may not be effective in reducing neuroinflammation (Anthony et al. 2005), so psychiatric disorders may increase. In addition, the hypothalamic– pituitary–adrenal (HPA) axis and sympathetic nervous system (SNS) have been implicated in mediating the relationship among stress, depression, and HIV. It is proposed that hormones of the HPA axis and SNS are affected by stress and depression, and that dysregulation of these systems may, in turn, have negative immunologic consequences in HIV infection and contribute to disease progression (Leserman 2003). Further research is needed to address these hypotheses as well as to determine if treatment of depression, in addition to enhancing the mental health of individuals with HIV, may also limit disease progression and mortality.

Animal models of NeuroAIDS

Macaque, mouse, and feline animal models have advanced the study of the progression and pathology of NeuroAIDS. In this section, we discuss recent findings using these models, placing emphasis on their contributions to the understanding of neuroimmunity and the BBB in NeuroAIDS, with the recognition that none of these models fully replicate the disease condition exhibited in humans.

Pharmacology of HIV infection and the BBB

Substance abuse, HIV, and the BBB

There is significant comorbidity in HIV-infected individuals who use substances of abuse. Thus, the effects of HIV infection and drug abuse on the BBB and viral entry into the CNS are a target of study (Fig. 1). (For a more comprehensive review, see Nath et al. 2002.)

Conclusion

Alterations in signaling between components of the BBB with either HIV proteins or factors produced in response to HIV infection, or with HIV-infected cells, disrupt BBB integrity and result in compromise, thereby promoting continued transmigration of activated monocytes and/or HIV-infected cells into the brain. A more complete understanding of the interactions among the BBB, nervous system, and immune system is critical to identification of the mechanisms that mediate NeuroAIDS. The use of animal models will facilitate this understanding as well as assist in the development of therapeutic strategies that will limit CNS dysfunction. Clarification of the contribution of substances of abuse to BBB disruption will enhance our understanding of the effects of HIV and drugs on neuroinflammation and comorbidity.

Acknowledgements

Contributor Information

Clarisa M. Buckner, Department of Pathology, F727, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461, USA.

Aimée J. Luers, Department of Pathology, F727, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461, USA.

Tina M. Calderon, Department of Pathology, F727, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461, USA.

Eliseo A. Eugenin, Department of Pathology, F727, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461, USA.

Joan W. Berman, Department of Pathology, F727, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461, USA, ude.uy.mocea@namreb. Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY, USA.

References