Amanda E. I. Proudfoot

Chemokine receptors: multifaceted therapeutic targets

Chemokines and their receptors are involved in the pathogenesis of diseases ranging from asthma to AIDS. Chemokine receptors are G-protein-coupled serpentine receptors that present attractive tractable targets for the pharmaceutical industry. It is only ten years since the first chemokine receptor was discovered, and the rapidly expanding number of antagonists holds promise for new medicines to combat diseases that are currently incurable. Here, I focus on the rationale for developing antagonists of chemokine receptors for inflammatory disorders and AIDS, and the accumulating evidence that favours this strategy despite the apparent redundancy in the chemokine system.

Overcoming hurdles in developing successful drugs targeting chemokine receptors

Chemokines and their receptors are central to the inflammatory process and are attractive therapeutic targets. Drugs that inhibit chemokine receptors are approved for the treatment of HIV infection and for stem cell mobilization, but none have been approved yet for the treatment of inflammatory and/or autoimmune diseases. We analyse the challenges of developing chemokine receptor antagonists, and propose that inappropriate target selection and ineffective dosing, not the redundancy of the chemokine system, are the main barriers to their use as anti-inflammatory therapies. We highlight evidence suggesting that chemokine receptor inhibition will prove to be an effective therapy in inflammatory diseases.

Neutrophil-Derived CCL3 Is Essential for the Rapid Recruitment of Dendritic Cells to the Site of Leishmania major Inoculation in Resistant Mice

Neutrophils are rapidly and massively recruited to sites of microbial infection, where they can influence the recruitment of dendritic cells. Here, we have analyzed the role of neutrophil released chemokines in the early recruitment of dendritic cells (DCs) in an experimental model of Leishmania major infection. We show in vitro, as well as during infection, that the parasite induced the expression of CCL3 selectively in neutrophils from L. major resistant mice. Neutrophil-secreted CCL3 was critical in chemotaxis of immature DCs, an effect lost upon CCL3 neutralisation. Depletion of neutrophils prior to infection, as well as pharmacological or genetic inhibition of CCL3, resulted in a significant decrease in DC recruitment at the site of parasite inoculation. Decreased DC recruitment in CCL3−/− mice was corrected by the transfer of wild type neutrophils at the time of infection. The early release of CCL3 by neutrophils was further shown to have a transient impact on the development of a protective immune response. Altogether, we identified a novel role for neutrophil-secreted CCL3 in the first wave of DC recruitment to the site of infection with L. major, suggesting that the selective release of neutrophil-secreted chemokines may regulate the development of immune response to pathogens.

Chemokine CCL5/RANTES inhibition reduces myocardial reperfusion injury in atherosclerotic mice

Although beneficial for cardiomyocyte salvage and to limit myocardial damage and cardiac dysfunction, restoration of blood flow after prolonged ischemia exacerbates myocardial injuries. Several deleterious processes that contribute to cardiomyocyte death have been proposed, including massive release of reactive oxygen species, calcium overload and hypercontracture development or leukocyte infiltration within the damaged myocardium. Chemokines are known to enhance leukocyte diapedesis at inflammatory sites. The aim of the present study was to investigate the effect of chemokine CCL5/RANTES antagonism in an in vivo mouse model of ischemia and reperfusion. ApoE(-/-) mice were submitted to 30 min ischemia, by ligature of the left coronary artery, followed by 24 h reperfusion. Intraperitoneal injection of 10 mug of CCL5/RANTES antagonist [(44)AANA(47)]-RANTES, 5 min prior to reperfusion, reduced infarct size as well as Troponin I serum levels compared to PBS-treated mice. This beneficial effect of [(44)AANA(47)]-RANTES treatment was associated with reduced leukocyte infiltration into the reperfused myocardium, as well as decreased chemokines Ccl2/Mcp-1 and Ccl3/Mip-1alpha expression, oxidative stress, and apoptosis. However, mice deficient for the CCL5/RANTES receptor Ccr5 did not exhibit myocardium salvage in our model of ischemia-reperfusion. Furthermore, [(44)AANA(47)]-RANTES did not mediate cardioprotection in these ApoE(-/-) Ccr5(-/-) deficient mice, probably due to enhanced expression of compensatory chemokines. This study provides the first evidence that inhibition of CCL5/RANTES exerts cardioprotective effects during early myocardial reperfusion, through its anti-inflammatory properties. Our findings indicate that blocking chemokine receptor/ligand interactions might become a novel therapeutic strategy to reduce reperfusion injuries in patients during acute coronary syndromes.

Kinetics of chemokine-glycosaminoglycan interactions control neutrophil migration into the airspaces of the lungs.

Chemokine–glycosaminoglycan (GAG) interactions are thought to result in the formation of tissue-bound chemokine gradients. We hypothesized that the binding of chemokines to GAGs would increase neutrophil migration toward CXC chemokines instilled into lungs of mice. To test this hypothesis we compared neutrophil migration toward recombinant human CXCL8 (rhCXCL8) and two mutant forms of CXCL8, which do not bind to heparin immobilized on a sensor chip. Unexpectedly, when instilled into the lungs of mice the CXCL8 mutants recruited more neutrophils than rhCXCL8. The CXCL8 mutants appeared in plasma at significantly higher concentrations and diffused more rapidly across an extracellular matrix in vitro. A comparison of the murine CXC chemokines, KC and MIP-2, revealed that KC was more effective in recruiting neutrophils into the lungs than MIP-2. KC appeared in plasma at significantly higher concentrations and diffused more rapidly across an extracellular matrix in vitro than MIP-2. In kinetic binding studies, KC, MIP-2, and rhCXCL8 bound heparin differently, with KC associating and dissociating more rapidly from immobilized heparin than the other chemokines. These data suggest that the kinetics of chemokine–GAG interactions contributes to chemokine function in tissues. In the lungs, it appears that chemokines, such as CXCL8 or MIP-2, which associate and disassociate slowly from GAGs, form gradients relatively slowly compared with chemokines that either bind GAGs poorly or interact with rapid kinetics. Thus, different types of chemokine gradients may form during an inflammatory response. This suggests a new model, whereby GAGs control the spatiotemporal formation of chemokine gradients and neutrophil migration in tissue.

TSG-6 Inhibits Neutrophil Migration via Direct Interaction with the Chemokine CXCL8

TNF-stimulated gene/protein-6 (TSG-6) is expressed by many different cell types in response to proinflammatory cytokines and plays an important role in the protection of tissues from the damaging consequences of acute inflammation. Recently, TSG-6 was identified as being largely responsible for the beneficial effects of multipotent mesenchymal stem cells, for example in the treatment of animal models of myocardial infarction and corneal injury/allogenic transplant. The protective effect of TSG-6 is due in part to its inhibition of neutrophil migration, but the mechanisms underlying this activity remain unknown. In this study, we have shown that TSG-6 inhibits chemokine-stimulated transendothelial migration of neutrophils via a direct interaction (KD, ∼25 nM) between TSG-6 and the glycosaminoglycan binding site of CXCL8, which antagonizes the association of CXCL8 with heparin. Furthermore, we found that TSG-6 impairs the binding of CXCL8 to cell surface glycosaminoglycans and the transport of CXCL8 across an endothelial cell monolayer. In vivo this could limit the formation of haptotactic gradients on endothelial heparan sulfate proteoglycans and, hence, integrin-mediated tight adhesion and migration. We further observed that TSG-6 suppresses CXCL8-mediated chemotaxis of neutrophils; this lower potency effect might be important at sites where there is high local expression of TSG-6. Thus, we have identified TSG-6 as a CXCL8-binding protein, making it, to our knowledge, the first soluble mammalian chemokine-binding protein to be described to date. We have also revealed a potential mechanism whereby TSG-6 mediates its anti-inflammatory and protective effects. This could inform the development of new treatments for inflammation in the context of disease or following transplantation.

An engineered monomer of CCL2 has anti-inflammatory properties emphasizing the importance of oligomerization for chemokine activity in vivo

We demonstrated recently that P8A‐CCL2, a monomeric variant of the chemokine CCL2/MCP‐1, is unable to induce cellular recruitment in vivo, despite full activity in vitro. Here, we show that this variant is able to inhibit CCL2 and thioglycollate‐mediated recruitment of leukocytes into the peritoneal cavity and recruitment of cells into lungs of OVA‐sensitized mice. This anti‐inflammatory activity translated into a reduction of clinical score in the more complex inflammatory model of murine experimental autoimmune encephalomyelitis. Several hypotheses for the mechanism of action of P8A‐CCL2 were tested. Plasma exposure following s.c. injection is similar for P8A‐CCL2 and wild‐type (WT) CCL2, ruling out the hypothesis that P8A‐CCL2 disrupts the chemokine gradient through systemic exposure. P8A‐CCL2 and WT induce CCR2 internalization in vitro and in vivo; CCR2 then recycles to the cell surface, but the cells remain refractory to chemotaxis in vitro for several hours. Although the response to P8A‐CCL2 is similar to WT, this finding is novel and suggests that despite the presence of the receptor on the cell surface, coupling to the signaling machinery is retarded. In contrast to CCL2, P8A‐CCL2 does not oligomerize on glycosaminoglycans (GAGs). However, it retains the ability to bind GAGs and displaces endogenous JE (murine MCP‐1) from endothelial surfaces. Intravital microscopy studies indicate that P8A‐CCL2 prevents leukocyte adhesion, while CCL2 has no effect, and this phenomenon may be related to the mechanism. These results suggest that oligomerization‐deficient chemokines can exhibit anti‐inflammatory properties in vivo and may represent new therapeutic modalities.

Current status of chemokine receptor inhibitors in development

The chemokine network plays pivotal role in a large number of inflammatory, allergic and autoimmune diseases, as well as in the promotion of tumor growth and metastasis. Considerable effort has been put in the pharmaceutical industry to identify therapeutic agents that specifically target chemokine receptors. Despite the fact that several promising programs have proven unsuccessful in Phase II trials the research activity both in academia and industry is still highly intense, whereas for some of the chemokine receptors the progress is still at the preclinical stage. In this review the authors discuss possible reasons beyond successes and failures of early clinical development programs and discuss the most relevant and recent pharmacological approaches with the aim to point out new theories, open issues and expectations in this research field.

Chapter 4. Interactions of chemokines with glycosaminoglycans.

Many proteins require interactions with cell surface glycosaminoglycans (GAGs) to exert their biologic activity. The effect of GAG binding on protein function ranges from essential roles in development, organogenesis, cell growth, cell adhesion, inflammation, tumorigenesis, and interactions with pathogens. A classic example is the role of GAGs in the interaction of fibroblast growth factors with their receptors, where GAGs play a role in specificity determination and control of receptor-ligand engagement. The other well-studied example involves the binding of antithrombin to heparin/heparan sulfate, which results in the inactivation of the coagulation cascade. In view of their specialized activity in cellular recruitment, chemokines interact with GAGs, minimally as a mechanism for localization of chemokines to specific anatomical spaces enabling them to act as directional signals for migrating cells. The biological relevance of these interactions has been recently demonstrated by functional characterization of mutants that are deficient in GAG binding. These mutants bind receptor normally in vitro but are unable to recruit cells in vivo. Observations like this have motivated investigations to identify GAG-binding epitopes on chemokines, the specificity and affinity of chemokines for different GAGs, the oligomerization of chemokines on GAGs, and the efficacy of GAG-binding mutants in the context of in vivo cell recruitment and animal models of disease. To this end, several techniques have been developed to measure the interactions of chemokines with GAGs. In this chapter we describe these various assays with particular reference to those that have been used to assess the binding of chemokines to GAGs and to define their epitopes. In the end, we believe both in vitro and in vivo characterization are absolutely necessary for understanding these interactions and their biologic relevance in the context of the whole organism.

Treatment with Evasin-3 reduces atherosclerotic vulnerability for ischemic stroke, but not brain injury in mice

Neutrophilic inflammation might have a pathophysiological role in both carotid plaque rupture and ischemic stroke injury. Here, we investigated the potential benefits of the CXC chemokine-binding protein Evasin-3, which potently inhibits chemokine bioactivity and related neutrophilic inflammation in two mouse models of carotid atherosclerosis and ischemic stroke, respectively. In the first model, the chronic treatment with Evasin-3 as compared with Vehicle (phosphate-buffered saline (PBS)) was investigated in apolipoprotein E-deficient mice implanted of a cast’ carotid device. In the second model, acute Evasin-3 treatment (5 minutes after cerebral ischemia onset) was assessed in mice subjected to transient left middle cerebral artery occlusion. Although CXCL1 and CXCL2 were upregulated in both atherosclerotic plaques and infarcted brain, only CXCL1 was detectable in serum. In carotid atherosclerosis, treatment with Evasin-3 was associated with reduction in intraplaque neutrophil and matrix metalloproteinase-9 content and weak increase in collagen as compared with Vehicle. In ischemic stroke, treatment with Evasin-3 was associated with reduction in ischemic brain neutrophil infiltration and protective oxidants. No other effects in clinical and histological outcomes were observed. We concluded that Evasin-3 treatment was associated with reduction in neutrophilic inflammation in both mouse models. However, Evasin-3 administration after cerebral ischemia onset failed to improve poststroke outcomes.