Brian Tomkowicz

Macrophage activation through CCR5‐ and CXCR4‐mediated gp120‐elicited signaling pathways

Macrophages are major targets for infection by human immunodeficiency virus type 1 (HIV‐1). In addition to their role as productive viral reservoirs, inappropriate activation of infected and uninfected macrophages appears to contribute to pathogenesis. HIV‐1 infection requires initial interactions between the viral envelope surface glycoprotein gp120, the cell‐surface protein CD4, and a chemokine receptor CCR5 or CXCR4. Besides their role in HIV‐1 entry, CCR5 and CXCR4 are G protein‐coupled receptors that can activate multiple intracellular signaling pathways. HIV‐1 gp120 has been shown to activate signaling pathways through the chemokine receptors in several cell types including lymphocytes, neurons, and astrocytes. In some cell types, these consequences may cause cellular injury. In this review, we highlight our data demonstrating diverse signaling events that occur in primary human macrophages in response to gp120/chemokine receptor interactions. These responses include K+, Cl–, and nonselective cation currents, intracellular Ca2+ increases, and activation of several kinases including the focal adhesion‐related tyrosine kinase Pyk2, mitogen‐activated protein kinases (MAPK), and phosphoinositol‐3 kinase. Activation of the MAPK leads to gp120‐induced expression of chemokines such as monocyte chemoattractant protein‐1 and macrophage‐inflammatory protein‐1β and the proinflammatory cytokine tumor necrosis factor α. These responses establish a complex cytokine network, which may enhance or suppress HIV‐1 replication. In addition, dysregulation of macrophage function by gp120/chemokine receptor signaling may contribute to local inflammation and injury and further recruit additional inflammatory and/or target cells. Targeting these cellular signaling pathways may have benefit in controlling inflammatory sequelae of HIV infection such as in neurological disease.

HIV‐1 gp120‐induced TNF‐α production by primary human macrophages is mediated by phosphatidylinositol‐3 (PI‐3) kinase and mitogen‐activated protein (MAP) kinase pathways

Human immunodeficiency virus type 1 (HIV‐1) infection is initiated by binding of the viral envelope glycoprotein gp120 to CD4 followed by a chemokine receptor, but these interactions may also take place independently from infection. gp120 stimulation of primary human macrophages is known to trigger production of cytokines implicated in pathogenesis, particularly tumor necrosis factor α (TNF‐α), but the mechanisms have not been determined. We sought to define the pathways responsible for TNF‐α secretion by monocyte‐derived macrophages (MDM) following HIV‐1 gp120 stimulation. MDM exposure to recombinant macrophage‐tropic (R5) gp120 led to dose‐ and donor‐dependent release of TNF‐α, which was cyclohexamide‐sensitive and associated with up‐regulated message. Pretreatment with specific inhibitors of the mitogen‐activated protein kinases (MAPK) extracellular signal‐regulated kinase 1/2 (ERK‐1/2; PD98059, U0126) and p38 (SB202190, PD169316) inhibited the secretion of TNF‐α. gp120‐elicited TNF‐α production was also blocked by phosphatidylinositol‐3 kinase (PI‐3K) inhibitors (wortmannin, LY294002). Moreover, PI‐3K inhibition ablated gp120‐induced phosphorylation of p38 and ERK‐1/2. The response was inhibited by a CC chemokine receptor 5 (CCR5)‐specific antagonist, indicating that CCR5 was in large part responsible. These results indicate that gp120‐elicited TNF‐α production by macrophages involves chemokine receptor‐mediated PI‐3K and MAPK activation, that PI‐3K is an upstream regulator of MAPK in this pathway, and that p38 and ERK‐1/2 independently regulate TNF‐α production. These gp120‐triggered signaling pathways may be responsible for inappropriate production of proinflammatory cytokines by macrophages, which are believed to play a role in immunopathogenesis and in neurological sequelae of AIDS.

Role of HIV-1 Vpr in AIDS pathogenesis: relevance and implications of intravirion, intracellular and free Vpr

Vpr, a 14-kDa, 96 amino acid protein, is conserved among the primate lentiviruses HIV-1, HIV-2 and Simian Immunodeficiency virus supporting the notion that it plays an important role in virus life cycle in vivo. Vpr appears to have several functions including cell cycle arrest at G2 stage, apoptosis, nuclear localization, nuclear import of the pre-integration complex, cation selective channel activity and transcriptionally activate HIV-1 LTR and other heterologous promoters. Over the years, we have addressed several issues pertaining to Vpr including the amount of Vpr present in the virus particles and structure-function relationship of Vpr. Here, we have reviewed the sources of Vpr that may potentially contribute to the cytopathic features observed in the context of HIV-1 infection. There are three different sources of Vpr available in the infected individuals to initiate the pathogenic effects. These include cell-associated, virion-associated (infectious, infectious-non productive, and non-infectious defective viruses) and free Vpr (cell-free and virus-free). A potential role of Vpr in neuropathogenesis of HIV infection in CNS was also suggested by early studies demonstrating neurotoxicity of recombinant Vpr protein. Interestingly, free Vpr (cell-free and virus-free) has been demonstrated in the serum of HIV-1 infected individuals and in the CSF of AIDS patients with neurological dysfunctions. Based on the toxic effects of extra-cellular Vpr on cells noted in several studies, it is likely that free Vpr could contribute to the bystander cell depletion in lymphoid tissues, peripheral blood, and the CNS. These results led us to propose a model for the role of Vpr in AIDS pathogenesis.

An arrestin-dependent multi-kinase signaling complex mediates MIP-1β/CCL4 signaling and chemotaxis of primary human macrophages

MIP‐1β/CCL4 is a principal regulator of macrophage migration and signals through CCR5. Several protein kinases are linked to CCR5 in macrophages including the src kinase Lyn, PI3K, focal adhesion related kinase Pyk2, and members of the MAPK family, but whether and how these kinases regulate macrophage chemotaxis are not known. To define the role of these signaling molecules, we examined the functions and interactions of endogenous proteins in primary human macrophages. Using siRNA gene silencing and pharmacologic inhibition, we show that chemotaxis in response to CCR5 stimulation by MIP‐1β requires activation of Pyk2, PI3K p85, and Lyn, as well as MAPK ERK. MIP‐1β activation of CCR5 triggered translocation of Pyk2 and PI3K p85 from the cytoplasm to colocalize with Lyn at the plasma membrane with formation of a multimolecular complex. We show further that arrestins were recruited into the complex, and arrestin down‐regulation impaired complex formation and macrophage chemotaxis toward MIP‐1β. Together, these results identify a novel mechanism of chemokine receptor regulation of chemotaxis and suggest that arrestins may serve as scaffolding proteins linking CCR5 to multiple downstream signaling molecules in a biologically important primary human cell type.

Monocyte Migration and LFA-1-Mediated Attachment to Brain Microvascular Endothelia Is Regulated by SDF-1α through Lyn Kinase

Infiltration of activated monocytes into the brain is a prerequisite for the development of various neurological disorders such as HIV-associated dementia, multiple sclerosis, and other inflammatory processes. In these pathologies, the chemokine SDF-1α (CXCL12) is over-expressed and might attract monocytes into the CNS. We demonstrate here that SDF-1α stimulates migration of monocytes through its receptor, CXCR4, and decreases monocyte adherence to surfaces coated with ICAM-1, a ligand for β2 integrins. SDF-1α also decreases monocyte adherence to brain microvascular endothelial cells (BMVEC) that are activated with TNF-α, IL-1β, or recombinant envelope glycoprotein from HIV-1, which increase BMVEC expression of ICAM-1. The decreased adherence is linked to down-regulation on monocytes of the activation-dependent epitope of the β2 integrin LFA-1 by SDF-1α. Knockdown of Lyn in monocytes using small interfering RNA decreases SDF-1α-mediated migration and prevents the inhibition of monocyte attachment to ICAM-1 and activated BMVEC. Thus, in SDF-1α-stimulated monocytes, Lyn acts as a positive regulator of migration and a negative regulator of adhesion to BMVEC through the LFA-1 integrin. These results provide a novel Lyn-mediated signaling mechanism for the regulation of monocyte movement at the blood-brain barrier.

Human herpesvirus-8 encoded Kaposin: subcellular localization using immunofluorescence and biochemical approaches.

Human herpesvirus-8 (HHV-8) has been causally linked to the development of Kaposis sarcoma (KS). DNA sequence analysis of the viral genome revealed a total of 81 open reading frames (ORF). Interestingly, only a small subset of these ORFs has been shown to be transcribed in cells latently infected with HHV-8 and in cells of the KS lesions. Among the genes active during latency, kaposin, is noted for its abundance and ability to transform cells in culture, thus implicating a potential role in KS pathogenesis. This has prompted us to undertake an investigation on elucidating the mechanism(s) by which Kaposin brings about transformation of cells. Towards this goal, we have generated an eukaryotic expression plasmid encoding Kaposin (Kap). As Kaposin is predicted to be a type II membrane protein, several strategies were utilized to address this, including the generation of Kaposin with the Flag (FL) epitope (DYKDDDDK) at the C-terminus of the protein (Kap-C-FL). Antibodies specific for Kaposin (kap-2), recognized both Kaposin and Kaposin-Flag, while antibodies against the Flag epitope recognized only Kaposin-Flag. Transfection of Kap and Kap-C-FL expression plasmid DNA into NIH3T3 cells resulted in cellular clones that exhibited a phenotypic property of transformation by forming large, multiclustered cells, when grown on soft agar. Because there is controversial data regarding the localization of Kaposin in cells, we examined the subcellular localization of Kaposin using confocal microscopy. We observed that Kaposin and Kaposin-Flag showed an intense staining surrounding the nucleus. Although there was no staining at the cell membrane of transfected cells, FACS analysis using kap-2 or Flag antibodies, under nonpermeable conditions, showed positivity. Cell fractionation studies further showed that the majority of Kaposin was detected in the nuclear fraction by Western blot analysis. The cytoplasmic and detergent soluble membrane fractions did not show Kaposin protein; however, a small amount was detected in the detergent insoluble membrane fraction. Taken together, these results suggest that Kaposin exhibits multicompartmental localization in cells.

HIV-1 entry inhibitors: closing the front door

Highly active antiretroviral therapy (HAART) has led to major declines in morbidity and mortality of HIV-1-infected individuals, but the increasing prevalence of drug-resistant viral isolates, combined with the toxicity and other limitations of current treatments, make the development of new therapies a high priority. As knowledge of viral entry has expanded, this step of the viral life cycle has become a target for novel therapeutic strategies. An emerging group of antiretrovirals, known collectively as entry inhibitors, targets several distinct steps in viral entry including CD4 binding, chemokine receptor engagement and the structural changes in the viral envelope required for fusion between viral and cellular membranes. Many entry inhibitors are in various stages of clinical development, with one already licensed for use. This review will provide an overview of the mechanisms involved in the entry process, highlight promising entry blockers under development and discuss several considerations related to treatment that are unique to this class of antiretroviral drugs.