The global portfolio of new antimalarial medicines under development.

With the elimination of malaria now considered a realistic goal, it would be useful for scientists and policy makers to have an inventory of the arsenal of antimalarials, current and prospective, that could help make this goal a reality. In order to provide an overview of antimalarial projects in recent clinical development, we review here the global portfolio of antimalarial drugs in clinical phases of development complemented by projects in the preclinical and early discovery phases. The portfolio is discussed in terms of the novelty of the new molecules and their potential health impact in terms of addressing the requirements for the control and eventual eradication of malaria.

Chemokine inhibition--why, when, where, which and how?

Chemokines are small chemoattractant cytokines that control a wide variety of biological and pathological processes, ranging from immunosurveillance to inflammation, and from viral infection to cancer. Genetic and pharmacological studies have shown that chemokines are responsible for the excessive recruitment of leucocytes to inflammatory sites and damaged tissue. In the present paper, we discuss the rationale behind interfering with the chemokine system and introduce various points for therapeutic intervention using either protein-based or small-molecule inhibitors. Unlike other cytokines, chemokines signal via seven-transmembrane GPCRs (G-protein-coupled receptors), which are favoured targets by the pharmaceutical industry, and, as such, they are the first cytokines for which small-molecule-receptor antagonists have been developed. In addition to the high-affinity receptor interaction, chemokines have an in vivo requirement to bind to GAGs (glycosaminoglycans) in order to mediate directional cell migration. Prevention of the GAG interaction has been shown to be a viable therapeutic strategy. Targeting chemokine intracellular signalling pathways offers an alternative small-molecule approach. One of the key signalling targets downstream of a variety of chemokine receptors identified to date is PI3Kgamma (phosphoinositide 3-kinase gamma), a member of the class I PI3K family. Thus the chemokine system offers many potential entry points for innovative anti-inflammatory therapies for autoimmune diseases, such as multiple sclerosis, rheumatoid arthritis and allergic contact dermatitis.

Treatment with Met-RANTES reduces lung injury in caerulein-induced pancreatitis.

BACKGROUND: Severe acute pancreatitis leads to a systemic inflammatory response characterized by widespread leucocyte activation and, as a consequence, distant lung injury. In CC chemokines the first two cysteine residues are adjacent to each other. The aim of this study was to evaluate the effect of Met-RANTES, a CC chemokine receptor antagonist, on pancreatic inflammation and lung injury in caerulein-induced acute pancreatitis in mice. METHODS: Acute pancreatitis was induced in mice by hourly intraperitoneal injection of caerulein. Met-RANTES was administered either 30 min before or 1 h after starting caerulein injections, and pancreatic inflammation and lung injury were assessed. There were five groups of eight mice each including controls. RESULTS: Treatment with Met-RANTES had little effect on caerulein-induced pancreatic damage. Met-RANTES, however, reduced lung injury when given either before administration of caerulein (mean(s.e.m.) lung myeloperoxidase (MPO) 1.47(0.19) versus 3.70(0.86)-fold increase over control, P = 0.024; mean(s.e.m.) microvascular permeability 1.15(0.05) versus 3.57(0.63) lavage to plasma fluorescein isothiocyanate-labelled albumin fluorescence ratio (L/P) per cent, P = 0.002) or after caerulein administration (lung MPO 1.96(0.27) versus 3.65(0.63)-fold increase over control, P = 0.029; microvascular permeability 0.94(0.04) versus 2.85(0.34) L/P per cent, P < 0.001). CONCLUSION: Treatment with Met-RANTES reduces lung damage associated with caerulein-induced pancreatitis in mice. Chemokine receptor antagonists may be of use for the treatment of the systemic complications of acute pancreatitis.

Regulation of dendritic cell recruitment into resting and inflamed airway epithelium: use of alternative chemokine receptors as a function of inducing stimulus.

Dendritic cells (DC) were purified by flow cytometry from rat tracheal mucosa; they exhibited the phenotypic characteristics of immature DC including high endocytic activity, low CD80/86 expression, and in vitro responsiveness to a broad range of CC chemokines. Daily treatment of adult rats with the selective CCR1 and CCR5 antagonist Met-RANTES reduced baseline numbers of tracheal intraepithelial DC by 50-60%, and pretreatment of animals with Met-RANTES before inhalation of aerosol containing heat-killed bacteria abolished the rapid DC influx into the epithelium that occurred in untreated controls, implicating CCR1 and CCR5 and their ligands in recruitment of immature DC precursors into resting airway tissues and during acute bacterial-induced inflammation. Comparable levels of DC recruitment were observed during airway mucosal Sendai virus infection and after aerosol challenge of sensitized animals with the soluble recall Ag OVA. However, Met-RANTES did not affect these latter responses, indicating the use of alternative chemokine receptors/ligands for DC recruitment, or possibly attraction of different DC subsets, depending on the nature of the eliciting stimulus.

Stem cell factor-induced leukotriene B4 production cooperates with eotaxin to mediate the recruitment of eosinophils during allergic pleurisy in mice.

The understanding of the mechanisms underlying eosinophil recruitment in vivo may aid in the development of novel strategies for the treatment of allergic disorders. In this study, we investigated the role of chemokines in the cascade of events leading to eosinophil recruitment in a stem cell factor (SCF)- and leukotriene B(4) (LTB(4))-dependent allergic pleurisy model in mice. The intrapleural administration of the eosinophil-active chemokines eotaxin, RANTES, and macrophage-inflammatory protein 1alpha (MIP-1alpha) induced a time- and dose-dependent eosinophil recruitment. Pretreatment with anti-eotaxin, but not anti-RANTES or anti-MIP-1alpha, blocked the recruitment of eosinophils following Ag challenge of sensitized animals, and significant eotaxin immunoreactivity was detected in the pleural cavity of these animals. Similarly, only the anti-eotaxin inhibited the eosinophil recruitment induced by injection of SCF in naive animals. However, blockade of SCF did not inhibit the release of eotaxin after Ag challenge of sensitized mice. Akin to its effects on SCF and in the allergic reaction, eotaxin-induced eosinophil recruitment was blocked by the LTB(4) receptor antagonist CP105696. Nevertheless, SCF, but not eotaxin, appeared to regulate the endogenous release of LTB(4) after Ag challenge. Finally, we show that low doses of eotaxin synergized with LTB(4) to induce eosinophil recruitment in the pleural cavity. Overall, the present results show that eotaxin and SCF-induced LTB(4) cooperate to induce eosinophil recruitment into sites of allergic inflammation. Cooperation between inflammatory mediators must be an important phenomenon in vivo, explaining both the ability of lower concentrations of mediators to induce a full-blown functional response and the effectiveness of different strategies at inhibiting these responses.

Aminooxypentane-RANTES induces CCR3 activation and internalization of CCR3 from the surface of human eosinophils.

Eosinophils are predominant effector cells in allergic diseases attracted by several CC chemokines into the inflammatory tissue. According to their important role in attracting leukocytes, several kinds of chemokine receptor antagonists have been developed. Therefore, the aim of this study was to investigate the effect of aminooxypentane (AOP)-RANTES on the activation of the CC chemokine receptor 3, CCR3, exemplary on human eosinophils, because they represent the dominant CCR3+ cell type. AOP-RANTES dose-dependently induced an increase of intracellular calcium concentration ([Ca(2+)](i)) and a release of reactive oxygen species, which could be inhibited by pertussis toxin, in human eosinophils from normal nonatopic donors. AOP-RANTES was as effective as RANTES but less effective than eotaxin and eotaxin-2 in the activation of the respiratory burst. Flow-cytometric analyses revealed that eosinophils constitutively expressed the CC chemokine receptors CCR1 and CCR3, whereas CCR5 was not expressed. AOP-RANTES, RANTES, eotaxin and eotaxin-2, but not Met-RANTES, induced a downregulation of CCR3 at 37 degrees C. Reexpression of CCR3 on eosinophils was observed within 120 min. Whereas no differences of CCR3 downregulation and recycling after stimulation with AOP-RANTES, RANTES, eotaxin and eotaxin-2 were found there exists a distinct profile of activity with respect to the activation of the respiratory burst in human eosinophils.

Aggretin, a heterodimeric C-type lectin from Calloselasma rhodostoma (Malayan pit viper), stimulates platelets by binding to α2ß1 integrin and glycoprotein Ib, activating Syk and phospholipase Cγ 2, but does not involve the glycoprotein VI/Fc receptor γ chain collagen receptor.

Aggretin, a potent platelet activator, was isolated from Calloselasma rhodostoma venom, and 30-amino acid N-terminal sequences of both subunits were determined. Aggretin belongs to the heterodimeric snake C-type lectin family and is thought to activate platelets by binding to platelet glycoprotein alpha(2)beta(1). We now show that binding to glycoprotein (GP) Ib is also required. Aggretin-induced platelet activation was inhibited by a monoclonal antibody to GPIb as well as by antibodies to alpha(2)beta(1). Binding of both of these platelet receptors to aggretin was confirmed by affinity chromatography. No binding of other major platelet membrane glycoproteins, in particular GPVI, to aggretin was detected. Aggretin also activates platelets from Fc receptor gamma chain (Fcgamma)-deficient mice to a greater extent than those from normal control mice, showing that it does not use the GPVI/Fcgamma pathway. Platelets from Fcgamma-deficient mice expressed fibrinogen receptors normally in response to collagen, although they did not aggregate, indicating that these platelets may partly compensate via other receptors including alpha(2)beta(1) or GPIb for the lack of the Fcgamma pathway. Signaling by aggretin involves a dose-dependent lag phase followed by rapid tyrosine phosphorylation of a number of proteins. Among these are p72(SYK), p125(FAK), and PLCgamma2, whereas, in comparison with collagen and convulxin, the Fcgamma subunit neither is phosphorylated nor coprecipitates with p72(SYK). This supports an independent, GPIb- and integrin-based pathway for activation of p72(SYK) not involving the Fcgamma receptor.

The BBXB motif of RANTES is the principal site for heparin binding and controls receptor selectivity.

The chemokine RANTES (regulated on activation normal T cell expressed and secreted; CCL5) binds selectively to glycosaminoglycans (GAGs) such as heparin, chondroitin sulfate, and dermatan sulfate. The primary sequence of RANTES contains two clusters of basic residues, (44)RKNR(47) and (55)KKWVR(59). The first is a BBXB motif common in heparin-binding proteins, and the second is located in the loop directly preceding the C-terminal helix. We have mutated these residues to alanine, both as point mutations as well as triple mutations of the 40s and 50s clusters. Using a binding assay to heparin beads with radiolabeled proteins, the (44)AANA(47) mutant demonstrated an 80% reduction in its capacity to bind heparin, whereas the (55)AAWVA(59) mutant retained full binding capacity. Mutation of the (44)RKNR(47) site reduced the selectivity of RANTES binding to different GAGs. The mutants were tested for their integrity by receptor binding assays on CCR1 and CCR5 as well as their ability to induce chemotaxis in vitro. In all assays the single point mutations and the triple 50s cluster mutation caused no significant difference in activity compared with the wild type sequence. However, the triple 40s mutant showed a 80-fold reduction in affinity for CCR1, despite normal binding to CCR5. It was only able to induce monocyte chemotaxis at micromolar concentrations. The triple 40s mutant was also able to inhibit HIV-1 infectivity, but consistent with its abrogated GAG binding capacity, it no longer induced enhanced infectivity at high concentrations.

Chemokine receptors--the next therapeutic target for HIV?

To date, the available therapies for the treatment of HIV infection are targeted against proteins encoded by the virus itself. Thus, combination drug therapies for HIV with reverse transciptase and protease inhibitors have resulted in spectacular reductions of viraemia, often leading to a remarkable improvement in symptoms and recovery from disease in infected people. There is still however, a great need for improved therapies since many patients are unable to take these drugs, either for reasons of intolerance, strain resistance, complexity of regimen or prohibitive cost. Multiple therapies aimed at different events in the HIV life cycle will ensure switching of treatments to combat resistant viruses, and also allow treatment flexibility if patients are unable to tolerate particular therapies. One event that could provide a key to reducing or even eliminating viral infection would be to prevent the virus from entering the host cell. Intense efforts are now underway to produce drugs that target chemokine receptors, one of the essential components for HIV cell entry. HIV needs two receptors on the host cell surface for efficient attachment and infection. The first is CD4 and the second, identified in 1996, is a member of the family of chemokine receptors, members of the G-protein coupled 7TM superfamily, which are involved in the trafficking of leukocytes in immune surveillance and inflammation. Many small, orally bioavailable molecules that block various 7TM receptors are used to treat a panoply of diseases including ulcers, allergies, migraines, and schizophrenia. These molecules are the cornerstone of the pharmaceutical industry's contribution to the fight against so many diseases. Small molecule inhibitors of the HIV-coreceptors are now entering the first stages of clinical trials as new therapeutics for the fight against AIDS.

Bilinexin, a snake C-type lectin from Agkistrodon bilineatus venom agglutinates platelets via GPIb and alpha2beta1.

A new snake protein, named bilinexin, has been purified from Agkistrodon bilineatus venom by ion-exchange chromatography and gel filtration chromatography. Under non-reducing conditions it has a mass of 110 kDa protein on SDS-PAGE. On reduction, it can be separated into five subunits with masses in the range 13-25 kDa. The N-terminal sequences of these subunits are very similar to those of convulxin or the alboaggregins, identifying bilinexin as a new member of the snake C-type lectin family, unusual in having multiple subunits. Bilinexin agglutinates fixed platelets. washed platelets and platelet rich plasma (PRP) without obvious activation (shape change) as confirmed by light microscope examination. Both inhibitory and binding studies indicate that antibodies against alpha2beta1 inhibit not only platelet agglutination induced by bilinexin, but also bilinexin binding to platelets. VM16d, a monoclonal anti-GPIbalpha antibody, completely inhibits platelet agglutination induced by bilinexin, and polyclonal antibodies against GPIbalpha prevent its binding to platelets. However, neither convulxin, polyclonal anti-GPVI antibodies, nor GPIIb/IIIa inhibitors affect its binding to and agglutination of platelets. Bilinexin neither activates GPIIb/IIIa integrin on platelets nor induces tyrosine phosphorylation of platelet proteins, nor increases intracellular Ca2+ in platelets. Like alboaggregin B, bilinexin agglutinates platelets, which makes it a good tool to investigate the differences in mechanism between snake C-type lectins causing platelet agglutination and those that induce full activation.

Functional expression of CCR1, CCR3, CCR4, and CXCR4 chemokine receptors on human platelets.

Platelets are known to contain platelet factor 4 and beta-thromboglobulin, alpha-chemokines containing the CXC motif, but recent studies extended the range to the beta-family characterized by the CC motif, including RANTES and Gro-alpha. There is also evidence for expression of chemokine receptors CCR4 and CXCR4 in platelets. This study shows that platelets have functional CCR1, CCR3, CCR4, and CXCR4 chemokine receptors. Polymerase chain reaction detected chemokine receptor messenger RNA in platelet RNA. CCR1, CCR3, and especially CCR4 gave strong signals; CXCR1 and CXCR4 were weakly positive. Flow cytometry with specific antibodies showed the presence of a clear signal for CXCR4 and weak signals for CCR1 and CCR3, whereas CXCR1, CXCR2, CXCR3, and CCR5 were all negative. Immunoprecipitation and Western blotting with polyclonal antibodies to cytoplasmic peptides clearly showed the presence of CCR1 and CCR4 in platelets in amounts comparable to monocytes and CCR4 transfected cells, respectively. Chemokines specific for these receptors, including monocyte chemotactic protein 1, macrophage inflammatory peptide 1alpha, eotaxin, RANTES, TARC, macrophage-derived chemokine, and stromal cell-derived factor 1, activate platelets to give Ca(++) signals, aggregation, and release of granule contents. Platelet aggregation was dependent on release of adenosine diphosphate (ADP) and its interaction with platelet ADP receptors. Part, but not all, of the Ca(++) signal was due to ADP release feeding back to its receptors. Platelet activation also involved heparan or chondroitin sulfate associated with the platelet surface and was inhibited by cleavage of these glycosaminoglycans or by heparin or low molecular weight heparin. These platelet receptors may be involved in inflammatory or allergic responses or in platelet activation in human immunodeficiency virus infection.

A key role for CC chemokine receptor 4 in lipopolysaccharide-induced endotoxic shock.

CC chemokine receptor (CCR)4, a high affinity receptor for the CC chemokines thymus and activation-regulated chemokine (TARC) and macrophage-derived chemokine (MDC), is expressed in the thymus and spleen, and also by peripheral blood T cells, macrophages, platelets, and basophils. Recent studies have shown that CCR4 is the major chemokine receptor expressed by T helper type 2 (Th2) polarized cells. To study the in vivo role of CCR4, we have generated CCR4-deficient (CCR4(-/-)) mice by gene targeting. CCR4(-/-) mice developed normally. Splenocytes and thymocytes isolated from the CCR4(-/-) mice failed to respond to the CCR4 ligands TARC and MDC, as expected, but also surprisingly did not undergo chemotaxis in vitro in response to macrophage inflammatory protein (MIP)-1alpha. The CCR4 deletion had no effect on Th2 differentiation in vitro or in a Th2-dependent model of allergic airway inflammation. However, CCR4(-/-) mice exhibited significantly decreased mortality on administration of high or low dose bacterial lipopolysaccharide (LPS) compared with CCR4(+/+) mice. After high dose LPS treatment, serum levels of tumor necrosis factor alpha, interleukin 1beta, and MIP-1alpha were reduced in CCR4(-/-) mice, and decreased expression of MDC and MIP-2 mRNA was detected in peritoneal exudate cells. Analysis of peritoneal lavage cells from CCR4(-/)- mice by flow cytometry also revealed a significant decrease in the F4/80(+) cell population. This may reflect a defect in the ability of the CCR4(-/-) macrophages to be retained in the peritoneal cavity. Taken together, our data reveal an unexpected role for CCR4 in the inflammatory response leading to LPS-induced lethality.

Differential activation of CC chemokine receptors by AOP-RANTES.

RANTES (regulated on activation normal T cell expressed) has been found at elevated levels in biological fluids from patients with a wide range of allergic and autoimmune diseases and is able to attract several subtypes of leukocytes including eosinophils and monocytes into inflamed tissue. Amino-terminal modifications of RANTES produce receptor antagonists which are candidates for blocking this cellular recruitment. Met-RANTES has been shown to modulate inflammation in vivo, while AOP-RANTES is a potent inhibitor of R5 human immunodeficiency virus type 1 (HIV-1) strains and has been shown to down-modulate CCR5 and prevent recycling of the receptor. We have studied the effect of AOP-RANTES in eosinophil activation and have found that it is able to efficiently elicit eosinophil effector functions through CCR3, as measured by the release of reactive oxygen species and calcium mobilization, whereas Met-RANTES is inactive in these assays. AOP-RANTES is found to inhibit CCR3-mediated HIV-1 infection with moderate potency, in contrast to its potent inhibition of CCR5-mediated HIV-1 infection. Furthermore, we have investigated the abilities of these modified proteins to down-modulate CCR1 and CCR3 from the surface of monocytes and eosinophils. We show here that AOP-RANTES is much less effective than RANTES in down-modulation of CCR1. Surprisingly, recycling of CCR1 was minimal after incubation with RANTES while there was complete recycling with AOP-RANTES. In the case of CCR3, no significant difference was found between RANTES and AOP-RANTES in down-modulation and recycling. It therefore appears that trafficking of RANTES receptors follows different patterns, which opens up potential new targets for therapeutic intervention.

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.

Amino-terminally modified RANTES analogues demonstrate differential effects on RANTES receptors.

Modification of the amino terminus of regulated on activated normal T-cell expressed (RANTES) has been shown to have a significant effect on biological activity and produces proteins with antagonist properties. Two amino-terminally modified RANTES proteins, Met-RANTES and aminooxypentane-RANTES (AOP-RANTES), exhibit differential inhibitory properties on both monocyte and eosinophil chemotaxis. We have investigated their binding properties as well as their ability to activate the RANTES receptors CCR1, CCR3, and CCR5 in cell lines overexpressing these receptors. We show that Met-RANTES has weak activity in eliciting a calcium response in Chinese hamster ovary cells expressing CCR1, CCR3, and CCR5, whereas AOP-RANTES has full agonist activity on CCR5 but is less effective on CCR3 and CCR1. Their ability to induce chemotaxis of the murine pre-B lymphoma cell line, L1.2, transfected with the same receptors, consolidates these results. Monocytes have detectable mRNA for CCR1, CCR2, CCR3, CCR4, and CCR5, and they respond to the ligands for these receptors in chemotaxis but not always in calcium mobilization. AOP-RANTES does not induce calcium mobilization in circulating monocytes but is able to do so as these cells acquire the macrophage phenotype, which coincides with a concomitant up-regulation of CCR5. We have also tested the ability of both modified proteins to induce chemotaxis of freshly isolated monocytes and eosinophils. Cells from most donors do not respond, but occasionally cells from a particular donor do respond, particularly to AOP-RANTES. We therefore hypothesize that the occasional activity of AOP-RANTES to induce leukocyte chemotaxis is due to donor to donor variation of receptor expression.

The platelet collagen receptor glycoprotein VI is a member of the immunoglobulin superfamily closely related to FcalphaR and the natural killer receptors.

We have cloned the platelet collagen receptor glycoprotein (GP) VI from a human bone marrow cDNA library using rapid amplification of cDNA ends with platelet mRNA to complete the 5' end sequence. GPVI was isolated from platelets using affinity chromatography on the snake C-type lectin, convulxin, as a critical step. Internal peptide sequences were obtained, and degenerate primers were designed to amplify a fragment of the GPVI cDNA, which was then used as a probe to screen the library. Purified GPVI, as well as Fab fragments of polyclonal antibodies made against the receptor, inhibited collagen-induced platelet aggregation. The GPVI receptor cDNA has an open reading frame of 1017 base pairs coding for a protein of 339 amino acids including a putative 23-amino acid signal sequence and a 19-amino acid transmembrane domain between residues 247 and 265. GPVI belongs to the immunoglobulin superfamily, and its sequence is closely related to FcalphaR and to the natural killer receptors. Its extracellular chain has two Ig-C2-like domains formed by disulfide bridges. An arginine residue is found in position 3 of the transmembrane portion, which should permit association with Fcgamma and its immunoreceptor tyrosine-based activation motif via a salt bridge. With 51 amino acids, the cytoplasmic tail is relatively long and shows little homology to the C-terminal part of the other family members. The ability of the cloned GPVI cDNA to code for a functional platelet collagen receptor was demonstrated in the megakaryocytic cell line Dami. Dami cells transfected with GPVI cDNA mobilized intracellular Ca(2+) in response to collagen, unlike the nontransfected or mock transfected Dami cells, which do not respond to collagen.

Glycosaminoglycans interact selectively with chemokines and modulate receptor binding and cellular responses.

Chemokines selectively recruit and activate a variety of cells during inflammation. Interactions between cell surface glycosaminoglycans (GAGs) and chemokines drive the formation of haptotactic or immobilized gradients of chemokines at the site of inflammation, directing this recruitment. Chemokines bind to glycosaminoglycans on human umbilical vein endothelial cells (HUVECs) with affinities in the micromolar range: RANTES > MCP-1 > IL-8 > MIP-1alpha. This binding can be competed with by soluble glycosaminoglycans: heparin, heparin sulfate, chondroitin sulfate, and dermatan sulfate. RANTES binding showed the widest discrimination between glycosaminoglycans (700-fold), whereas MIP-1alpha was the least selective. Almost identical results were obtained in an assay using heparin sulfate beads as the source of immobilized glycosaminoglycan. The binding of chemokines to glycosaminoglycan fragments has a strong length dependence, and optimally requires both N- and O-sulfation. Isothermal titration calorimetry data confirm these results; IL-8 binds heparin fragments with a K(d) of 0.39-2.63 microM, and requires five saccharide units to bind each monomer of chemokine. In membranes from cells expressing the G-protein-coupled chemokine receptors CXCR1, CXCR2, and CCR1, soluble GAGs inhibit the binding of chemokine ligands to their receptors. Consistent with this, heparin and heparin sulfate could inhibit IL-8-induced neutrophil calcium flux. Chemokines can therefore form complexes with both cell surface and soluble GAGs; these interactions have different functions. Soluble GAG chemokines complexes are unable to bind the receptor, resulting in a block of the biological activity. Previously, we have shown that cell surface GAGs present chemokines to the G-protein-coupled receptors, by increasing the local concentration of protein. A model is presented which brings together all of these data. The selectivity in the chemokine-GAG interaction suggests selective disruption of the haptotactic gradient may be an achievable therapeutic approach in inflammatory disease.

Chemokine receptors and their antagonists in allergic lung disease.

The trafficking and homing of leukocytes in normal homeostasis and in disease is under the control of a variety of cytokine and lipid mediators. One family of small cytokines particularly involved in inflammation which has been identified is the chemokine family. Their action is mediated by a large superfamily of seven transmembrane spanning G-protein coupled receptors. One of the hopes in this field has been there may be selectivity in terms of which cells are recruited to sites of inflammation by virtue of their chemokine receptor expression pattern. This means that it may be possible to find antagonists of chemokine receptors that can selectively down regulate certain cell type recruitment, without provoking a generalized immunosuppression. In this review, we discuss the current state of understanding of the chemokine receptor field. The therapeutic potential of this field can be judged from recent data on the use of protein chemokine antagonists in allergic disease. The data so far obtained in animal studies point to the potential clinical uses of this emerging class of therapeutic agents.