HIV-1 Env-Chemokine Receptor Interactions in Primary Human Macrophages: Entry and Beyond
While HIV has subverted the chemokine receptors CCR5 and CXCR4 for its own use as an entry co-receptor, their normal functions are to transduce signals in response to extracellular ligands. Our lab is interested in understanding how HIV-1 glycoprotein 120 (gp120) may activate intracellular signals through these receptors in primary human macrophages, and how these responses may contribute to pathogenesis. Our studies demonstrate HIV-1 gp120 elicits several different types of signals in macrophages through CXCR4 and CCR5, including calcium elevations, ionic channel activation, non-receptor protein tyrosine kinase activation, and activation of MAP kinases. Receptor activation is triggered by both monomeric gp120 and whole HIV virus. Furthermore, gp120 elicits a number of functional responses in macrophages, such as secretion of chemokines and other soluble products, and we demonstrate that specific pathways linked to the chemokine receptors are responsible. These studies help illuminate the pathways through which chemokine receptors are coupled in primary macrophages, and provide a mechanistic basis for effects that HIV has on macrophage function. These signaling responses may play a role in the pathogenesis of organ dysfunction such as HIV encephalopathy and lymphocytic interstitial pneumonitis where macrophages are the principal infected cell type and inappropriate immune activation plays a central role.
Ronald Collman, M.D., was the first speaker of the day. Collman's research focuses on interactions between the viral envelope and the receptor/co-receptor complex on the surface of the cell, specifically in macrophages. Though not as extensively studied as CD4 lymphocytes, macrophages play a critical role in HIV pathology. HIV-1 strains that infect macrophages (macrophage-tropic isolates) are responsible for person-to-person transmission and are the predominant virus type during the clinical latency phase. In addition, macrophages are the main infected cells in organs such as the brain and lungs, and appear to be responsible for inflammation and injury in these sites, which lead to neurological dysfunction and AIDS Dementia Complex (ADC), or pulmonary inflammation and Lymphoid Interstitial Pneumonitis (LIP).
Collman began his presentation by reviewing the structure of HIV and the specific interactions between the viral envelope and the cellular receptor complex, which comprises CD4 plus a chemokine receptor. While the CD4 receptor is required for infection by all naturally occurring HIV-1 strains, specific cell surface molecules called chemokine receptors also are necessary. He went on to discuss how HIV strains are defined on the basis of their tropism. Macrophage-tropic (M-tropic) strains infect primary macrophages and primary lymphocytes and use the chemokine receptor CCR5 as a co-receptor. T-cell-tropic (T-tropic) strains infect primary lymphocytes but not macrophages and use CXCR4 as the co-receptor. Dual-tropic strains infect all 3 types of cells and use both co-receptors as entry pathways. In addition, some strains of HIV-1 can use a variety of other chemokine receptors in studies in vitro, however it does not appear that they have major relevance to HIV infection in vivo and pathogenesis. The use of CCR5 by M-tropic strains, CXCR4 by T-tropic strains, and both receptors by dual-tropic strains had suggested a simple paradigm for the cellular determinants of tropism whereby CCR5 would be expressed on macrophages, CXCR4 on T-cells, and both types of co-receptors on primary lymphocytes. Collman's group has questioned to what extent this paradigm holds true for macrophages derived from in vivo samples, as well as the consequences of these interactions in macrophages.
To test the validity of this paradigm, Collman's lab performed a series of experiments using CCR5-deficient macrophages from individuals homozygous for the CCR5 32 polymorphism. This genetic polymorphism leads to an absence of CCR5 expression on cells from these individuals. M-tropic and T-tropic strains were unable to replicate in these cells. Surprisingly, 89.6, a dual-tropic strain, was able to replicate and blockade of the CXCR4 co-receptor prevented infection, demonstrating that macrophages have functional CXCR4 co-receptors that can be used for viral entry. This result was not specific to 89.6 and was observed with DH12, another dual-tropic strain. Furthermore, some strains that use CXCR4 only, such as UG021, could also infect macrophages through the CXCR4 co-receptor. Cell-cell fusion experiments confirmed that 89.6 uses both CCR5 and CXCR4 co-receptors on macrophages, while UGO21 uses macrophage CXCR4 exclusively. Collman explained that the co-receptor determinants of tropism are more complicated than originally believed, which led him and his colleagues to formulate a revised paradigm of tropism (see Figure 1). Macrophages express both CCR5 and CXCR4 and can be infected by M-tropic and dual-tropic strains, but not T-tropic strains. Some strains of HIV use both receptors in vivo, others use CCR5 or CXCR4 exclusively, and others may predominantly use one co-receptor in macrophages and another co-receptor in lymphocytes. It appears that use of co-receptors on target cells differs depending on the virus and that factors other than co-receptor expression determine tropism.
Figure 1. A model for the cellular determinants of HIV-1 tropism. Primary macrophages and primary lymphocytes express both CCR5 and CXCR4 (in addition to CD4), while T-cell lines express only CXCR4. Macrophage (M)-tropic HIV-1 strains infect macrophages and lymphocytes via CCR5. T-cell line (T)-tropic HIV-1 strains infect lymphocytes and T-cell lines via CXCR4 but cannot use macrophage CXCR4 for entry. Dual-tropic strains infect all 3 cell types, either by using CCR5 and CXCR4 on macrophages and T-cell lines, respectively, or through an ability to use CXCR4 on all target cell types.
The second part of Collman's presentation concentrated on inappropriate macrophage activation in HIV pathogenesis. Since the normal function of the chemokine receptors used by HIV-1 for entry is to activate cells in response to various stimuli, he hypothesized that the virus itself (or its envelope glycoprotein gp120 that mediates attachment to cells) might mimic natural activation signals and be responsible for inflammation and injury in organs like brain and lung where macrophages are infected and also activated. The researchers examined what effects gp120 has on intracellular signals in macrophages and how these changes relate to HIV pathogenesis. Specifically, Collman and his group investigated the effects of gp120 on ion channel activation, intracellular Ca++ levels, protein kinase activation, and the functional consequences of these changes on the target macrophage. Their data revealed that CCR5-binding ("R5") gp120 and CXCR4-binding ("X4") gp120 trigger a multitude of ion channel responses, including activation of Cl- channels, Ca++ channels (in conjunction with the release of Ca++ from intracellular stores), non-selective cation channels, and Ca++-activated K+ channels. These ionic changes are mediated by the specific chemokine co-receptor, rather than the CD4 receptor, as shown by experiments using CXCR4 antagonists and CCR5-deficient macrophages. Whether the opening of these channels is necessary for viral entry has not been determined; these experiments are difficult to interpret since pharmacological blockade of these channels would have a dramatic effect on cell function, regardless of Env binding.
To explore further how interactions between the virus and these receptors might elicit cellular changes that contribute to HIV pathogenesis, his laboratory proceeded to run a series of experiments looking at protein kinases, enzymes important in relaying cellular signals. Exposure to R5 gp120 and X4 gp120 resulted in activation of proline-rich tyrosine kinase 2 (Pyk2), a non-receptor tyrosine kinase related to focal adhesion kinase. They showed that activation of Pyk2 is Ca++-dependent and is mediated by CCR5 and CXCR4; binding only to CD4 is not sufficient for activation. Collman's laboratory also investigated effects of gp120 on the mitogen-activated protein (MAP) kinase family of signaling proteins. MAP kinases exert their effects through upregulation of transcription factors and function downstream of Pyk2 in several cell types. Both R5 gp120 and X4 gp120 activate the MAP kinases p38 and c-Jun amino terminal kinase/stress-activated protein kinase (JNK/SAPK), though it is unclear whether a third member of the MAPK family, extracellular regulated kinase (ERK 1/2), is activated. Collman's group has also observed that chemokines such as macrophage-inflammatory protein-1ß (MIP-1ß) and macrophage chemoattractant protein-1 (MCP-1) are secreted by macrophages in response to gp120, and that secretion of these inflammatory factors is dependent on MAP kinase activation. Exposure to gp120 also resulted in upregulation of tumor necrosis factor-a (TNF-a) mRNA expression that was partially inhibited by a MAP kinase antagonist. Though the exact mechanisms regulating macrophage secretion have not yet been elucidated, these data demonstrate that these intracellular cascades, specifically MAP kinase activation, are involved and necessary (see Figure 2).
Figure 2. Intracellular signaling pathways that are activated by HIV-1 gp120 through the chemokine receptors. In addition to mediating entry and infection, interaction of gp120 with CCR5 and CXCR4 can induce functional changes in the cell. Several ion channels are triggered, intracellular calcium is elevated, and protein kinase pathways including Pyk2 and the MAP kinases are activated. Triggering of these pathways leads to activation of macrophages and secretion of soluble products, including products that can injure neurons.
Nevertheless, the question remains as to how these cellular changes relate to the pathogenesis of HIV. In ADC, infected macrophages, increased macrophage activation, reactive gliosis (activated astrocytes) and apoptotic neuronal death are manifest. Much work has been performed examining the mechanisms of AIDS dementia, though the majority of this work has concentrated on the neurotoxic factors secreted by macrophages in response to HIV. Little work has examined the mechanisms within macrophages that are responsible for the production of these toxic factors. Using a neuronal cell line, NT2.N, Collman's lab showed that neurons die in response to supernatant from macrophages exposed to gp120. Cell death was significantly decreased by treating the macrophages with a MAP kinase inhibitor before being exposed to gp120. Thus, the pathways for MAP kinase activation seem to be responsible for the specific products secreted by macrophages and the resulting neurotoxicity.
Collman concluded his presentation by describing how these pathways could contribute to HIV encephalopathy by causing continued inflammation, astrocytosis, activation of other macrophages, and recruitment of uninfected and infected cells into the brain (see Figure 3). He reminds us that the best correlate of clinical dementia is not the amount of HIV in the brain, but rather the amount of macrophage/microglia activation. This activation may be responsible for astrocyte activation and subsequent production of neurotoxins. Collman admits that many questions remain. His laboratory continues to examine the relationship between the intracellular changes in macrophages exposed to HIV and the resulting pathogenesis.
Figure 3. A model for how HIV-1 signaling through CCR5 and CXCR4 on macrophages (and the closely related microglial cells) in the brain can contribute to the pathogenesis of AIDS Dementia Complex (ADC). The viral glycoprotein gp120 (either as a free protein or on the surface of virus particles) can trigger activation of both infected and uninfected macrophages. This leads to secretion of mediators that attract inflammatory cells (such as lymphocytes and more macrophages from blood), that can activate astrocytes, and that may injure neurons directly.
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Ronald G. Collman, M.D. is from the University of Pennsylvania School of Medicine and Penn Center for AIDS Research.