Christine A. Power

CD4-independent infection by HIV-2 is mediated by Fusin/CXCR4

Several members of the chemokine receptor family have been shown to function in association with CD4 to permit HIV-1 entry and infection. However, the mechanism by which these molecules serve as CD4-associated cofactors is unclear. In the present report, we show that one member of this family, termed Fusin/ CXCR4, is able to function as an alternative receptor for some isolates of HIV-2 in the absence of CD4. This conclusion is supported by the finding that (1) CD4-independent infection by these viruses is inhibited by an anti-Fusin monoclonal antibody, (2) Fusin expression renders human and nonhuman CD4-negative cell lines sensitive to HIV-2-induced syncytium induction and/or infection, and (3) Fusin is selectively down-regulated from the cell surface following HIV-2 infection. The finding that one chemokine receptor can function as a primary viral receptor strongly suggests that the HIV envelope glycoprotein contains a binding site for these proteins and that differences in the affinity and/or the availability of this site can extend the host range of these viruses to include a number of CD4-negative cell types.

Extension of Recombinant Human RANTES by the Retention of the Initiating Methionine Produces a Potent Antagonist

Extension of recombinant human RANTES by a single residue at the amino terminus is sufficient to produce a potent and selective antagonist. RANTES is a proinflammatory cytokine that promotes cell accumulation and activation in chronic inflammatory diseases. When mature RANTES was expressed heterologously in Escherichia coli, the amino-terminal initiating methionine was not removed by the endogenous amino peptidases. This methionylated protein was fully folded but completely inactive in RANTES bioassays of calcium mobilization and chemotaxis of the promonocytic cell line THP-1. However, when assayed as an antagonist of both RANTES and macrophage inflammatory polypeptide-1α (MIP-1α) in these assays, the methionylated RANTES (Met-RANTES) inhibited the actions of both chemokines. T cell chemotaxis was similarly inhibited. The antagonistic effect was selective since Met-RANTES had no effect on interleukin-8- or monocyte chemotractant protein-1-induced responses in these cells. Met-RANTES can compete with both [I]RANTES and [I]MIP-1α binding to THP-1 cells or to stably transfected HEK cells recombinantly expressing their common receptor, CC-CKR-1. These data show that the integrity of the amino terminus of RANTES is crucial to receptor binding and cellular activation.

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.

Effect of a CC chemokine receptor antagonist on collagen induced arthritis in DBA/1 mice

Chemokines are small proteins that selectively activate and recruit leukocytes to sites of inflammation. Several of them, including the CC chemokines RANTES, MIP-1 alpha, MIP-1 beta, MCP-1, and the CXC chemokines IL-8, GRO-alpha, ENA-78 have been identified in rheumatoid synovium, implicating a potential role for these molecules in rheumatoid arthritis. We have investigated the expression patterns of CC chemokine receptors in the joints of mice with collagen-induced arthritis, a model for human rheumatoid arthritis. In addition, we have investigated the incidence and severity of arthritis in mice receiving administration of MetRANTES, a modified chemokine which is a nanomolar antagonist of certain CC chemokine receptors. The mRNA expression pattern of the chemokines and their receptors in the joints of arthritic mice was investigated using reverse transcriptase-PCR and in situ hybridization. An upregulation of the CC chemokine receptors mCCR1, mCCR2; mCCR3 and mCCR5 was found in the joints from arthritic mice, compared to control animals. In addition, injections of MetRANTES reduced the incidence of disease in a dose dependent manner. Furthermore, in MetRANTES-treated mice that did develop arthritis a significantly lower severity of disease was observed compared with control animals. Our data clearly demonstrate a role for CC chemokines and their receptors in inflammatory joint destruction and support the use of chemokine receptor antagonists as potential tools to control inflammatory diseases such as rheumatoid arthritis.

Selectivity and antagonism of chemokine receptors.

The chemokine superfamily can be sub‐divided into two groups based on their amino terminal cysteine spacing. The CXC chemokines are primarily involved in neutrophil‐mediated inflammation and, so far, two human receptors have been cloned. The CC chemokines tend to be involved in chronic inflammation, and recently we have cloned a fourth leukocyte receptor for this group of ligands. Understanding what makes one receptor bind its range of agonists is important if we are to develop potent selective antagonists. We have started to investigate the molecular basis of this receptor selectivity by looking at why CC chemokines do not bind to the CXC receptors in several ways. First, we looked at the role of the three‐dimensional structure of the ligand, and have solved the three‐dimensional structure of RANTES using nuclear magnetic resonance spectroscopy. The structure is similar to that already determined for the CC chemokine macrophage inflammatory protein‐1β, and it has a completely different dimer interface to that of the CXC chemokine interleukin‐8 (IL‐8). However, the monomer structures of all the chemokines are very similar, and at physiological concentrations the proteins are likely to be monomeric. Second, by examining all the known CC and CXC chemokines, we have found a region that differs between the two subfamilies. Mutations of one of the residues in this region, Leu‐25 in IL‐8, to tyrosine (which is conserved at this position in CC chemokines) enables the mutant IL‐8 to bind CC‐chemokine receptor‐1 (CC‐CKR‐1) and introduces monocyte chemoattractant activity. Using other mutations in this region, we can show a direct interaction with the N‐terminus of CC‐CKR‐1. Third, we have found that modification of the amino terminus of RANTES by addition of one amino acid makes it into an antagonist with nanomolar potency. Taken together, this data suggests a two‐site model for receptor activation and for selectivity between CC and CXC chemokines, with an initial receptor contact provided by the main body of the chemokine, and activation provided by the amino terminal region.

Ticks produce highly selective chemokine binding proteins with antiinflammatory activity

Bloodsucking parasites such as ticks have evolved a wide variety of immunomodulatory proteins that are secreted in their saliva, allowing them to feed for long periods of time without being detected by the host immune system. One possible strategy used by ticks to evade the host immune response is to produce proteins that selectively bind and neutralize the chemokines that normally recruit cells of the innate immune system that protect the host from parasites. We have identified distinct cDNAs encoding novel chemokine binding proteins (CHPBs), which we have termed Evasins, using an expression cloning approach. These CHBPs have unusually stringent chemokine selectivity, differentiating them from broader spectrum viral CHBPs. Evasin-1 binds to CCL3, CCL4, and CCL18; Evasin-3 binds to CXCL8 and CXCL1; and Evasin-4 binds to CCL5 and CCL11. We report the characterization of Evasin-1 and -3, which are unrelated in primary sequence and tertiary structure, and reveal novel folds. Administration of recombinant Evasin-1 and -3 in animal models of disease demonstrates that they have potent antiinflammatory properties. These novel CHBPs designed by nature are even smaller than the recently described single-domain antibodies (Hollinger, P., and P.J. Hudson. 2005. Nat. Biotechnol. 23:1126–1136), and may be therapeutically useful as novel antiinflammatory agents in the future.

Airway hyperresponsiveness, but not airway remodeling, is attenuated during chronic pulmonary allergic responses to Aspergillus in CCR4−/− mice

The role of CC chemokine receptor 4 (CCR4) during the development and maintenance of Th2type allergic airway disease is controversial. In this study, we examined the role of CCR4 in the chronic allergic airway response to live Aspergillus fumigatus spores, or conidia, in A. fumigatussensitized mice. After the conidia challenge, mice lacking CCR4 (CCR4‐/‐ mice) exhibited significantly increased numbers of airway neutrophils and macrophages, and conidia were more rapidly eliminated from these mice compared with control CCR4 wild‐type (CCR4+/+) mice. Significant airway hyperresponsiveness to intravenous methacholine was observed at day 3 in CCR4‐/‐ mice, whereas at days 7 and 30, airway hyperresponsiveness was attenuated in these mice compared with control mice. A major reduction in peribronchial and airway eosinophilia was observed in CCR4‐/‐ mice at all times after conidia challenge in contrast to CCR4+/+ mice. Further, whole lung levels of interleukin (IL) 4 and IL‐5 were significantly increased in CCR4‐/‐ mice at day 3, whereas these Th2 cytokines and IL‐13 were significantly decreased at day 30 in CCR4‐/‐ mice compared with their wild‐type counterparts. Peribronchial fibrosis and goblet cell hyperplasia were similar in both groups of mice throughout the course of this model. In summary, CCR4 modulates both innate and acquired immune responses associated with chronic fungal asthma.

DEFINITION, FUNCTION AND PATHOPHYSIOLOGICAL SIGNIFICANCE OF CHEMOKINE RECEPTORS

Chemokines and their receptors are at the core of many processes in biology, from routine immunosurveillance and the inflammatory process, through to the infection of cells by HIV. In the past two years, various bioinformatic and cloning strategies have led to an explosion in the number of chemokines and receptors that have been identified. Although the picture is far from complete, several themes are emerging. In particular, there are important differences between observations in vitro, where there appears to be much redundancy, and studies in vivo, where distinct roles are clearer. In this review, Timothy Wells, Christine Power and Amanda Proudfoot discuss the chemokines and their receptors and recent data from immunological and virology studies, and speculate on the potential of interfering with the chemokine network as a useful approach to ameliorating disease.

The strategy of blocking the chemokine system to combat disease

One of the key characteristics of inflammation is the recruitment of leukocytes to the site of inflammation. Most anti-inflammatory strategies act intracellularly on the target cells, but after the cells have migrated to the site. We therefore propose that the prevention of cellular recruitment by blockade of the relevant chemokine receptor/ligand pair would present a novel therapy in that it would act upstream of the therapies currently in use. The chemokine system is a complex family of over 40 ligands and 18 receptors and as such may appear difficult to inhibit selectively. In the first part of the article we discuss the specificity mechanisms that are beginning to be unraveled which we believe occur at multiple levels. These levels of control of specificity include the temporal regulation of both the ligands and their receptors, which are under the control of pro-inflammatory cytokines; the localization of chemokines on cell surfaces through their interactions with glycosaminoglycans; differential receptor/ligand interactions; and different patterns of receptor trafficking, to name but a few. The chemokine system has been validated as providing good therapeutic targets by several approaches. In our laboratory, we have used a chemokine receptor antagonist in models of inflammation in vivo to demonstrate that this approach is successful in reducing inflammation. Chemokine receptors belong to the class of seven transmembrane spanning receptors, which have proven to be excellent targets by the pharmaceutical industry for many diseases. The number of small molecule inhibitors of chemokine receptors is rapidly growing in the patent literature, and reports both in the literature as well as conferences in the field have shown them to be effective in inflammatory disease models, as well as inhibiting HIV-1 infection. Since clinical trials will begin this year with some of these molecules, hopefully we will fairly soon have the answer of the efficacy of this therapeutic approach.

Strategies for chemokine antagonists as therapeutics

Chemokines are responsible for specific recruitment of leukocytes that are involved both in homing as well as in inflammation. Dysregulation of the system results in excessive recruitment to inflammatory sites and thus prevention of this recruitment is an effective anti-inflammatory strategy. Chemokine receptors are not limited only to cellular recruitment but are also the essential co-factor along with CD4 that enable HIV-1 viruses to infect cells. In this review we discuss the various points of intervention that can be addressed to provide anti-inflammatory and anti-HIV infectivity therapeutics. These include prevention of the receptor-ligand interaction, prevention of the chemokine-glycosaminoglycan interaction, interfering with the signaling pathways that are induced upon receptor activation, and modification of receptor trafficking pathways. We summarize the status of the approaches that have been undertaken to produce therapeutics that block chemokine action.