Richard Horuk

Neuronal apoptosis induced by HIV-1 gp120 and the chemokine SDF-1α is mediated by the chemokine receptor CXCR4

CXCR4, a seven transmembrane domain G-protein-coupled receptor for the Cys-X-Cys class of chemokines, is one of several chemokine receptors that can act as a co-receptor with CD4 for the human immunodeficiency virus (HIV-1) glycoprotein gp120 [1-3]. CXCR4 can mediate the entry of HIV-1 strains that specifically infect T cells, such as the IIB strain (see [4] for review). Recent reports indicate that gp120 can signal through CXCR4 [5] and it has been suggested that signal transduction, mediated by the viral envelope, might influence viral-associated cytopathicity or apoptosis [6]. Neuronal apoptosis is a feature of HIV-1 infection in the brain [7,8], although the exact mechanism is unknown. Here, we address the possible role of CXCR4 in inducing apoptosis using cells of the hNT human neuronal cell line; these cells resemble immature post-mitotic cholinergic neurons and have a number of neuronal characteristics [9-15]. We have previously shown that gp120 from the HIV-1 IIIB strain binds with high affinity to CXCR4 expressed on hNT neurons [15]. We now find that both IIIB gp120 and the Cys-X-Cys chemokine SDF-1 alpha can directly induce apoptosis in hNT neurons in the absence of CD4 and in a dose-dependent manner. To our knowledge, this is the first report of a chemokine and an HIV-1 envelope glycoprotein eliciting apoptotic responses through a chemokine receptor.

International union of pharmacology. LXXXIX. Update on the extended family of chemokine receptors and introducing a new nomenclature for atypical chemokine receptors

Sixteen years ago, the Nomenclature Committee of the International Union of Pharmacology approved a system for naming human seven-transmembrane (7TM) G protein-coupled chemokine receptors, the large family of leukocyte chemoattractant receptors that regulates immune system development and function, in large part by mediating leukocyte trafficking. This was announced in Pharmacological Reviews in a major overview of the first decade of research in this field [Murphy PM, Baggiolini M, Charo IF, Hébert CA, Horuk R, Matsushima K, Miller LH, Oppenheim JJ, and Power CA (2000) Pharmacol Rev 52:145–176]. Since then, several new receptors have been discovered, and major advances have been made for the others in many areas, including structural biology, signal transduction mechanisms, biology, and pharmacology. New and diverse roles have been identified in infection, immunity, inflammation, development, cancer, and other areas. The first two drugs acting at chemokine receptors have been approved by the U.S. Food and Drug Administration (FDA), maraviroc targeting CCR5 in human immunodeficiency virus (HIV)/AIDS, and plerixafor targeting CXCR4 for stem cell mobilization for transplantation in cancer, and other candidates are now undergoing pivotal clinical trials for diverse disease indications. In addition, a subfamily of atypical chemokine receptors has emerged that may signal through arrestins instead of G proteins to act as chemokine scavengers, and many microbial and invertebrate G protein-coupled chemokine receptors and soluble chemokine-binding proteins have been described. Here, we review this extended family of chemokine receptors and chemokine-binding proteins at the basic, translational, and clinical levels, including an update on drug development. We also introduce a new nomenclature for atypical chemokine receptors with the stem ACKR (atypical chemokine receptor) approved by the Nomenclature Committee of the International Union of Pharmacology and the Human Genome Nomenclature Committee.

CD4-independent association between HIV-1 gp120 and CXCR4: functional chemokine receptors are expressed in human neurons

BACKGROUND Chemokines are a family of proteins that chemoattract and activate immune cells by interacting with specific receptors on the surface of their targets. We have shown previously that chemokine receptors including the interleukin-8 receptor B (CXCR2) and the Duffy blood group antigen are expressed on subsets of neurons in various regions of the adult nervous system. RESULTS Using a combination of immunohistochemical staining and receptor binding studies, we show that hNT cells, which are differentiated human neurons derived from the cell line NTera2, express functional chemokine receptors of the C-X-X and C-C types. These chemokine receptors include CXCR2, CXCR4, CCR1 and CCR5. We demonstrate high-affinity binding of both types of chemokines to hNT neurons and dose-dependent chemotactic responses to these chemokines in differentiated, but no t undifferentiated, NTera 2 cells. In addition, we show that the envelop glycoprotein from the T-cell-tropic human immunodeficiency virus 1 (HIV-1) strain IIIB is a CD4-independent, dose-dependent inhibitor of the binding of stromal cell-derived factor 1 to its receptor, CXCR4. CONCLUSIONS These data support recent findings that members of the chemokine family, including CCR5 and LESTR/Fusin (CXCR4), function as coreceptors in combination with CD4 for HIV-1 invasion. This is the first report of functional expression of chemokine receptors on human neurons. Furthermore, our studies provide for direct CD4-independent association of the viral envelope protein of the HIV-1 strain III with the chemokine receptor CXCR4.

Chemokine and chemokine receptor expression in the central nervous system

A decade ago several new cytokines were described that orchestrated the activation and migration of immune cells. These newly described cytokines, of which interleukin-8 (IL-8) was a representative member, defined a novel group of molecules called chemokines (chemotactic cytokines). Chemokines are low molecular weight, 8-12 kDa, basic proteins that have been classified into four distinct families, CXC, CC, C and CX3C, based on the position of their first two conserved cysteine residues. The expression and biological function of chemokines along with their cognate receptors have been well described on various subsets of leukocytes. Only more recently have these molecules been described on various cells within the central nervous system. These pro-inflammatory proteins have been implicated in a variety of diseases within the central nervous system from Multiple Sclerosis to AIDS dementia. While chemokines are likely to enhance the evolution of central nervous system inflammatory disorders they also have other roles in normal brain function and development. This review summarizes the role of chemokines and their receptors in the normal and pathophysiological brain.

CCR1+/CCR5+ Mononuclear Phagocytes Accumulate in the Central Nervous System of Patients with Multiple Sclerosis

Mononuclear phagocytes (monocytes, macrophages, and microglia) are considered central to multiple sclerosis (MS) pathogenesis. Molecular cues that mediate mononuclear phagocyte accumulation and activation in the central nervous system (CNS) of MS patients may include chemokines RANTES/CCL5 and macrophage inflammatory protein-1alpha/CCL3. We analyzed expression of CCR1 and CCR5, the monocyte receptors for these chemokines, on circulating and cerebrospinal fluid CD14+ cells, and in MS brain lesions. Approximately 70% of cerebrospinal fluid monocytes were CCR1+/CCR5+, regardless of the presence of CNS pathology, compared to less than 20% of circulating monocytes. In active MS lesions CCR1+/CCR5+ monocytes were found in perivascular cell cuffs and at the demyelinating edges of evolving lesions. Mononuclear phagocytes in early demyelinating stages comprised CCR1+/CCR5+ hematogenous monocytes and CCR1-/CCR5- resident microglial cells. In later stages, phagocytic macrophages were uniformly CCR1-/CCR5+. Cultured in vitro, adherent monocytes/macrophages up-regulated CCR5 and down-regulated CCR1 expression, compared to freshly-isolated monocytes. Taken together, these findings suggest that monocytes competent to enter the CNS compartment derive from a minority CCR1+/CCR5+ population in the circulating pool. In the presence of ligand, these cells will be retained in the CNS. During further activation in lesions, infiltrating monocytes down-regulate CCR1 but not CCR5, whereas microglia up-regulate CCR5.

The CC chemokine I-309 inhibits CCR8-dependent infection by diverse HIV-1 strains.

Using a chemokine receptor model based on known receptor sequences, we identified several members of the seven transmembrane domain G-protein superfamily as potential chemokine receptors. The orphan receptor ChemR1, which has recently been shown to be a receptor for the CC chemokine I-309, scored very high in our model. We have confirmed that I-309, but not a number of other chemokines, can induce a transient Ca2+ flux in cells expressing CCR8. In addition, the human erythroleukemic cell line K562 responded chemotactically in a dose-responsive manner to this chemokine. Since several chemokine receptors have been shown to be required as coreceptors for HIV-1 infection, we asked whether human immunodeficiency virus type 1 (HIV-1) could efficiently utilize CCR8. Here we show that the CCR8 receptor can serve as a coreceptor for diverse T-cell tropic, dual-tropic, and macrophage-tropic HIV-1 strains and that I-309 was a potent inhibitor of HIV-1 envelope-mediated cell-cell fusion and virus infection. Furthermore, we show by flow cytometry and immunohistochemistry that antibodies generated against the CCR8 receptor amino-terminal peptide cross-reacted with U-87 MG cells stably expressing CCR8, THP-1 cells, HL-60 cells, and human monocytes, a target cell for HIV-1 infectivity in vivo.

Beneficial role of the GPR30 agonist G-1 in an animal model of multiple sclerosis.

The beneficial effects of estrogens in multiple sclerosis are thought to be mediated exclusively by the classical nuclear estrogen receptors ERalpha and ERbeta. However, recently many reports revealed that estrogens are able to mediate rapid signals through a G protein-coupled receptor (GPCR), known as GPR30. In the present study, we set out to explore whether effects mediated through this receptor were anti-inflammatory and could account for some of the beneficial effects of estrogen. We demonstrate that GPR30 is expressed in both human and mouse immune cells. Furthermore a GPR30-selective agonist, G-1, previously described by us, inhibits the production of lipopolysaccharide (LPS)-induced cytokines such as TNF-alpha and IL-6 in a dose-dependent manner in human primary macrophages and in a murine macrophage cell line. These effects are likely mediated solely through the estrogen-specific receptor GPR30 since the agonist G-1 displayed an IC(50) far greater than 10 microM on the classical nuclear estrogen receptors as well as a panel of 25 other GPCRs. Finally, we show that the agonist G-1 is able to reduce the severity of disease in both active and passive EAE models of multiple sclerosis in SJL mice and that this effect is concomitant with a G-1-mediated decrease in proinflammatory cytokines, including IFN-gamma and IL-17, in immune cells harvested from these mice. The effect of G-1 appears indirect, as the GPR30 agonist did not directly influence IFN-gamma or IL-17 production by purified T cells. These data indicate that G-1 may represent a novel therapeutic agent for the treatment of chronic autoimmune, inflammatory diseases.

Predictions of CCR1 Chemokine Receptor Structure and BX 471 Antagonist Binding Followed by Experimental Validation

A major challenge in the application of structure-based drug design methods to proteins belonging to the superfamily of G protein-coupled receptors (GPCRs) is the paucity of structural information (1). The 19 chemokine receptors, belonging to the Class A family of GPCRs, are important drug targets not only for autoimmune diseases like multiple sclerosis but also for the blockade of human immunodeficiency virus type 1 entry (2). Using the MembStruk computational method (3), we predicted the three-dimensional structure of the human CCR1 receptor. In addition, we predicted the binding site of the small molecule CCR1 antagonist BX 471, which is currently in Phase II clinical trials (4). Based on the predicted antagonist binding site we designed 17 point mutants of CCR1 to validate the predictions. Subsequent competitive ligand binding and chemotaxis experiments with these mutants gave an excellent correlation to these predictions. In particular, we find that Tyr-113 and Tyr-114 on transmembrane domain 3 and Ile-259 on transmembrane 6 contribute significantly to the binding of BX 471. Finally, we used the predicted and validated structure of CCR1 in a virtual screening validation of the Maybridge data base, seeded with selective CCR1 antagonists. The screen identified 63% of CCR1 antagonists in the top 5% of the hits. Our results indicate that rational drug design for GPCR targets is a feasible approach.

CCR1-specific non-peptide antagonist: efficacy in a rabbit allograft rejection model

The classic signs of acute cellular rejection during organ transplantation include the infiltration of mononuclear cells into the interstitium. This recruitment of leukocytes into the transplanted tissue is promoted by chemokines like RANTES. Since RANTES is a potent agonist for the CC chemokine receptor CCR1, we examined whether the CCR1 antagonist BX 471 was efficacious in a rabbit kidney transplant rejection model. BX 471 was able to compete with high affinity with the CCR1 ligands MIP-1alpha and RANTES for binding to HEK 293 cells expressing rabbit CCR1. BX 471 was a competitive antagonist of rabbit CCR1 in Ca(2+) flux studies. Two separate studies in which animals were subcutaneously implanted with slow release pellets of BX 471 demonstrated that animals implanted with BX 471 had increased survival compared with untreated controls or animals implanted with placebo. The mean survival time for the placebo group was 12.33+/-1.7 days. The animals in the BX 471 treated group had mean survival times of 16.9+/-2.1 and 16.0+/-1.7 days, respectively, for the two studies. Analysis of the combined data by Student t-test gave a P value of 0.03 that is significant at the 0.05 level. In addition, there was a marked reduction in the urea and creatinine levels in the BX 471 treated animals compared with the control and placebo groups in both studies. Finally, pathologic analysis of the kidneys in the rabbit renal transplantation model from animals in the different groups showed that BX 471 was similar to cyclosporin in its ability to prevent extensive infarction of transplanted kidneys. Based on the data from these studies, BX 471 shows clear efficacy at the single dose tested compared with animals treated with placebo.

The Promiscuous Chemokine Binding Profile of the Duffy Antigen/Receptor for Chemokines Is Primarily Localized to Sequences in the Amino-terminal Domain

The Duffy antigen (DARC) is a promiscuous chemokine receptor that also binds Plasmodium vivax. DARC belongs to a family of heptahelical chemokine receptors that includes specific (IL-8RA) and shared (IL-8RB) IL-8 receptors. Ligand binding specificity of IL-8 receptors was localized to the amino-terminal extracellular (E1) domain. To determine the basis for promiscuous chemokine binding by DARC, a chimeric receptor composed of the E1 domain of DARC and hydrophobic helices and loops from IL-8RB (DARC/IL-8RB) was constructed. Scatchard analysis of stable transfectants demonstrated that the DARC/IL-8RB chimeric receptor bound IL-8 and melanoma growth stimulating activity (MGSA) with Kvalues almost identical to the native receptors. The hybrid receptor also bound RANTES, MCP-1, and MGSA-EA (which binds DARC, but not IL-8RB), but not MIP-1α, similarly to DARC. Ligand binding to DARC transfectants was unaltered by anti-Fy3, but inhibited by Fy6, which binds an epitope in the E1 domain. The epitope recognized by Fy3 was localized to the third extracellular loop by analysis of insect cells expressing chimeric receptors composed of complementary portions of DARC and IL-8RB. These findings implicate the E1 domain of DARC in multispecific chemokine binding.