Evasins: Tick Salivary Proteins that Inhibit Mammalian Chemokines.

Ticks are hematophagous arachnids that parasitize mammals and other hosts, feeding on their blood. Ticks secrete numerous salivary factors that enhance host blood flow or suppress the host inflammatory response. The recruitment of leukocytes, a hallmark of inflammation, is regulated by chemokines, which activate chemokine receptors on the leukocytes. Ticks target this process by secreting glycoproteins called Evasins, which bind to chemokines and prevent leukocyte recruitment. This review describes the recent discovery of numerous Evasins produced by ticks, their classification into two structural and functional classes, and the efficacy of Evasins in animal models of inflammatory diseases. The review also proposes a standard nomenclature system for Evasins and discusses the potential of repurposing or engineering Evasins as therapeutic anti-inflammatory agents.

A knottin scaffold directs the CXC-chemokine-binding specificity of tick evasins.

Tick evasins (EVAs) bind either CC- or CXC-chemokines by a poorly understood promiscuous or "one-to-many" mechanism to neutralize inflammation. Because EVAs potently inhibit inflammation in many preclinical models, highlighting their potential as biological therapeutics for inflammatory diseases, we sought to further unravel the CXC-chemokine-EVA interactions. Using yeast surface display, we identified and characterized 27 novel CXC-chemokine-binding evasins homologous to EVA3 and defined two functional classes. The first, which included EVA3, exclusively bound ELR+ CXC-chemokines, whereas the second class bound both ELR+ and ELR- CXC-chemokines, in several cases including CXC-motif chemokine ligand 10 (CXCL10) but, surprisingly, not CXCL8. The X-ray crystal structure of EVA3 at a resolution of 1.79 Å revealed a single antiparallel ß-sheet with six conserved cysteine residues forming a disulfide-bonded knottin scaffold that creates a contiguous solvent-accessible surface. Swapping analyses identified distinct knottin scaffold segments necessary for different CXC-chemokine-binding activities, implying that differential ligand positioning, at least in part, plays a role in promiscuous binding. Swapping segments also transferred chemokine-binding activity, resulting in a hybrid EVA with dual CXCL10- and CXCL8-binding activities. The solvent-accessible surfaces of the knottin scaffold segments have distinctive shape and charge, which we suggest drives chemokine-binding specificity. These studies provide structural and mechanistic insight into how CXC-chemokine-binding tick EVAs achieve class specificity but also engage in promiscuous binding.

Leukocyte Adhesion: Reconceptualizing Chemokine Presentation by Glycosaminoglycans.

Recruitment of immune cells from the vasculature relies on the presentation of glycosaminoglycan-bound chemokines on the luminal side of vascular endothelial cells. However, the current model of chemokine-glycosaminoglycan interactions, and its implications for receptor interactions, remains poorly developed. We propose a refined 'Chemokine Cloud' model, arguing that chemokines are not presented to leukocytes bound to glycosaminoglycans, but rather, in solution while sequestered within the hydrated glycocalyx. We posit that glycosaminoglycans provide an immobilized chemokine depot maintaining a 'cloud' of 'solution-phase' chemokines within the glycocalyx, and that it is this soluble form of any given chemokine that interacts with leukocyte-bound receptors. Our proposition clarifies certain anomalies associated with the current model of chemokine-glycosaminoglycan interactions, with implications for the design of blockers of chemokine function.

Relevance of CCL3/CCR5 axis in oral carcinogenesis.

The chemokine CCL3 is a chemotactic cytokine crucial for inflammatory cell recruitment in homeostatic and pathological conditions. CCL3 might stimulate cancer progression by promoting leukocyte accumulation, angiogenesis and tumour growth. The expression of CCL3 and its receptors CCR1 and CCR5 was demonstrated in oral squamous cell carcinoma (OSCC), but their role was not defined. Here, the functions of CCL3 were assessed using a model of chemically induced tongue carcinogenesis with 4-nitroquinoline-1-oxide (4NQO). Lineages of OSCC were used to analyse the effects of CCL3 in vitro. The 4NQO-induced lesions exhibited increased expression of CCL3, CCR1 and CCR5. CCL3-/- and CCR5-/- mice presented reduced incidence of tongue tumours compared to wild-type (WT) and CCR1-/- mice. Consistently, attenuated cytomorphological atypia and reduced cell proliferation were observed in lesions of CCL3-/- and CCR5-/- mice. OSCC from CCL3-/- mice exhibited lower infiltration of eosinophils and reduced expression of Egf, Fgf1, Tgf-ß1, Vegfa, Vegfb, Itga-4, Vtn, Mmp-1a, Mmp-2 and Mmp-9 than WT mice. In vitro, CCL3 induced invasion and production of CCL5, IL-6, MMP -2, -8, -9. Blockage of CCL3 in vitro using α-CCL3 or Evasin-1 (a CCL3-binding protein) impaired tumour cell invasion. In conclusion, CCL3/CCR5 axis has pro-tumourigenic effects in oral carcinogenesis. The induction of inflammatory and angiogenic pathways and eosinophils recruitment appear to be the underlying mechanism explaining these effects. These data reveal potential protective effects of CCL3 blockade in oral cancer.

Glycosaminoglycan Interactions with Chemokines Add Complexity to a Complex System.

Chemokines have two types of interactions that function cooperatively to control cell migration. Chemokine receptors on migrating cells integrate signals initiated upon chemokine binding to promote cell movement. Interactions with glycosaminoglycans (GAGs) localize chemokines on and near cell surfaces and the extracellular matrix to provide direction to the cell movement. The matrix of interacting chemokine-receptor partners has been known for some time, precise signaling and trafficking properties of many chemokine-receptor pairs have been characterized, and recent structural information has revealed atomic level detail on chemokine-receptor recognition and activation. However, precise knowledge of the interactions of chemokines with GAGs has lagged far behind such that a single paradigm of GAG presentation on surfaces is generally applied to all chemokines. This review summarizes accumulating evidence which suggests that there is a great deal of diversity and specificity in these interactions, that GAG interactions help fine-tune the function of chemokines, and that GAGs have other roles in chemokine biology beyond localization and surface presentation. This suggests that chemokine-GAG interactions add complexity to the already complex functions of the receptors and ligands.

Varicella zoster virus glycoprotein C increases chemokine-mediated leukocyte migration.

Varicella zoster virus (VZV) is a highly prevalent human pathogen that establishes latency in neurons of the peripheral nervous system. Primary infection causes varicella whereas reactivation results in zoster, which is often followed by chronic pain in adults. Following infection of epithelial cells in the respiratory tract, VZV spreads within the host by hijacking leukocytes, including T cells, in the tonsils and other regional lymph nodes, and modifying their activity. In spite of its importance in pathogenesis, the mechanism of dissemination remains poorly understood. Here we addressed the influence of VZV on leukocyte migration and found that the purified recombinant soluble ectodomain of VZV glycoprotein C (rSgC) binds chemokines with high affinity. Functional experiments show that VZV rSgC potentiates chemokine activity, enhancing the migration of monocyte and T cell lines and, most importantly, human tonsillar leukocytes at low chemokine concentrations. Binding and potentiation of chemokine activity occurs through the C-terminal part of gC ectodomain, containing predicted immunoglobulin-like domains. The mechanism of action of VZV rSgC requires interaction with the chemokine and signalling through the chemokine receptor. Finally, we show that VZV viral particles enhance chemokine-dependent T cell migration and that gC is partially required for this activity. We propose that VZV gC activity facilitates the recruitment and subsequent infection of leukocytes and thereby enhances VZV systemic dissemination in humans.

Evasins: Therapeutic Potential of a New Family of Chemokine-Binding Proteins from Ticks.

Blood-sucking parasites, such as ticks, remain attached to their hosts for relatively long periods of time in order to obtain their blood meal without eliciting an immune response. One mechanism used to avoid rejection is the inhibition of the recruitment of immune cells, which can be achieved by a class of chemokine-binding proteins (CKBPs) known as Evasins. We have identified three distinct Evasins produced by the salivary glands of the common brown dog tick, Rhipicephalus sanguineus. They display different selectivities for chemokines, the first two identified show a narrow selectivity profile, while the third has a broader binding spectrum. The Evasins showed efficacy in animal models of inflammatory disease. Here, we will discuss the potential of their development for therapeutic use, addressing both the advantages and disadvantages that this entails.

Modulation of Chemokine Responses: Synergy and Cooperativity.

Chemokine biology is mediated by more complex interactions than simple monomolecular ligand-receptor interactions, as chemokines can form higher order quaternary structures, which can also be formed after binding to glycosaminoglycans (GAGs) on endothelial cells, and their receptors are found as dimers and/or oligomers at the cell surface. Due to the complexity of the chemokine binding and signaling system, several mechanisms have been proposed to provide an explanation for the synergy observed between chemokines in leukocyte migration. Pioneering studies on interactions between different chemokines have revealed that they can act as antagonists, or synergize with other chemokines. The synergism can occur at different levels, involving either two chemokine receptors triggered simultaneously or sequentially exposed to their agonists, or the activation of one type of chemokine receptor triggered by chemokine heterocomplexes. In addition to the several chemokines that, by forming a heterocomplex with chemokine receptor agonists, act as enhancers of molecules of the same family, we have recently identified HMGB1, an endogenous damage-associated molecular patterns (DAMPs) molecule, as an enhancer of the activity of CXCL12. It is now evident that synergism between chemokines is crucial at the very early stage of inflammation. In addition, the low-affinity interaction with GAGs has recently been shown to induce cooperativity allowing synergy or inhibition of activity by displacement of other ligands.

The Interaction of Heparin Tetrasaccharides with Chemokine CCL5 Is Modulated by Sulfation Pattern and pH.

Interactions between chemokines such as CCL5 and glycosaminoglycans (GAGs) are essential for creating haptotactic gradients to guide the migration of leukocytes into inflammatory sites, and the GAGs that interact with CCL5 with the highest affinity are heparan sulfates/heparin. The interaction between CCL5 and its receptor on monocytes, CCR1, is mediated through residues Arg-17 and -47 in CCL5, which overlap with the GAG-binding (44)RKNR(47) "BBXB" motifs. Here we report that heparin and tetrasaccharide fragments of heparin are able to inhibit CCL5-CCR1 binding, with IC50 values showing strong dependence on the pattern and extent of sulfation. Modeling of the CCL5-tetrasaccharide complexes suggested that interactions between specific sulfate and carboxylate groups of heparin and residues Arg-17 and -47 of the protein are essential for strong inhibition; tetrasaccharides lacking the specific sulfation pattern were found to preferentially bind CCL5 in positions less favorable for inhibition of the interaction with CCR1. Simulations of a 12-mer heparin fragment bound to CCL5 indicated that the oligosaccharide preferred to interact simultaneously with both (44)RKNR(47) motifs in the CCL5 homodimer and engaged residues Arg-47 and -17 from both chains. Direct engagement of these residues by the longer heparin oligosaccharide provides a rationalization for its effectiveness as an inhibitor of CCL5-CCR1 interaction. In this mode, histidine (His-23) may contribute to CCL5-GAG interactions when the pH drops just below neutral, as occurs during inflammation. Additionally, an examination of the contribution of pH to modulating CCL5-heparin interactions suggested a need for careful interpretation of experimental results when experiments are performed under non-physiological conditions.

Targeting chemokines: Pathogens can, why can't we?

Chemoattractant cytokines, or chemokines, are the largest sub-family of cytokines. About 50 distinct chemokines have been identified in humans. Their principal role is to stimulate the directional migration of leukocytes, which they achieve through activation of their receptors, following immobilization on cell surface glycosaminoglycans (GAGs). Chemokine receptors belong to the G protein-coupled 7-transmembrane receptor family, and hence their identification brought great promise to the pharmaceutical industry, since this receptor class is the target for a large percentage of marketed drugs. Unfortunately, the development of potent and efficacious inhibitors of chemokine receptors has not lived up to the early expectations. Several approaches to targeting this system will be described here, which have been instrumental in establishing paradigms in chemokine biology. Whilst drug discovery programs have not yet elucidated how to make successful drugs targeting the chemokine system, it is now known that certain parasites have evolved anti-chemokine strategies in order to remain undetected by their hosts. What can we learn from them?

Identification of the pharmacophore of the CC chemokine-binding proteins Evasin-1 and -4 using phage display.

To elucidate the ligand-binding surface of the CC chemokine-binding proteins Evasin-1 and Evasin-4, produced by the tick Rhipicephalus sanguineus, we sought to identify the key determinants responsible for their different chemokine selectivities by expressing Evasin mutants using phage display. We first designed alanine mutants based on the Evasin-1·CCL3 complex structure and an in silico model of Evasin-4 bound to CCL3. The mutants were displayed on M13 phage particles, and binding to chemokine was assessed by ELISA. Selected variants were then produced as purified proteins and characterized by surface plasmon resonance analysis and inhibition of chemotaxis. The method was validated by confirming the importance of Phe-14 and Trp-89 to the inhibitory properties of Evasin-1 and led to the identification of a third crucial residue, Asn-88. Two amino acids, Glu-16 and Tyr-19, were identified as key residues for binding and inhibition of Evasin-4. In a parallel approach, we identified one clone (Y28Q/N60D) that showed a clear reduction in binding to CCL3, CCL5, and CCL8. It therefore appears that Evasin-1 and -4 use different pharmacophores to bind CC chemokines, with the principal binding occurring through the C terminus of Evasin-1, but through the N-terminal region of Evasin-4. However, both proteins appear to target chemokine N termini, presumably because these domains are key to receptor signaling. The results also suggest that phage display may offer a useful approach for rapid investigation of the pharmacophores of small inhibitory binding proteins.

Chemokine cooperativity is caused by competitive glycosaminoglycan binding.

Chemokines comprise a family of secreted proteins that activate G protein-coupled chemokine receptors and thereby control the migration of leukocytes during inflammation or immune surveillance. The positional information required for such migratory behavior is governed by the binding of chemokines to membrane-tethered glycosaminoglycans (GAGs), which establishes a chemokine concentration gradient. An often observed but incompletely understood behavior of chemokines is the ability of unrelated chemokines to enhance the potency with which another chemokine subtype can activate its cognate receptor. This phenomenon has been demonstrated to occur between many chemokine combinations and across several model systems and has been dubbed chemokine cooperativity. In this study, we have used GAG binding-deficient chemokine mutants and cell-based functional (migration) assays to demonstrate that chemokine cooperativity is caused by competitive binding of chemokines to GAGs. This mechanistic explanation of chemokine cooperativity provides insight into chemokine gradient formation in the context of inflammation, in which multiple chemokines are secreted simultaneously.

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.

International Union of Basic and Clinical Pharmacology. [corrected]. 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.

Evasin-4, a tick-derived chemokine-binding protein with broad selectivity can be modified for use in preclinical disease models.

Rhipicephalus sanguineus, the common brown dog tick, produces several chemokine-binding proteins which are secreted into the host in its saliva to modulate the host response during feeding. Two of these demonstrate very restricted selectivity profiles. Here, we describe the characterization of the third, which we named Evasin-4. Evasin-4 was difficult to produce recombinantly using its native signal peptide in HEK cells, but expressed very well using the urokinase-type plasminogen activator signal peptide. Using SPR, Evasin-4 was shown to bind most CC chemokines. Investigation of the neutralization properties by inhibition of chemokine-induced chemotaxis showed that binding and neutralization did not correlate in all cases. Two major anomalies were observed: no binding was observed to CCL2 and CCL13, yet Evasin-4 was able to inhibit chemotaxis induced by these chemokines. Conversely, binding to CCL25 was observed, but Evasin-4 did not inhibit CCL25-induced chemotaxis. Size-exclusion chromatography confirmed that Evasin-4 forms a complex with CCL2 and CCL18. In accordance with the standard properties of unmodified small proteins, Evasin-4 was rapidly cleared following in vivo administration. To enhance the in vivo half-life and optimize its potential as a therapeutic agent, Fc fusions of Evasin-4 were created. Both the N- and C-terminal fusions were shown to retain binding activity, with the C-terminal fusion showing a modest reduction in potency.

CCL18 exhibits a regulatory role through inhibition of receptor and glycosaminoglycan binding.

CCL18 has been reported to be present constitutively at high levels in the circulation, and is further elevated during inflammatory diseases. Since it is a rather poor chemoattractant, we wondered if it may have a regulatory role. CCL18 has been reported to inhibit cellular recruitment mediated by CCR3, and we have shown that whilst it is a competitive functional antagonist as assessed by Schild plot analysis, it only binds to a subset of CCR3 receptor populations. We have extended this inhibitory activity to other receptors and have shown that CCL18 is able to inhibit CCR1, CCR2, CCR4 and CCR5 mediated chemotaxis, but has no effect on CCR7 and CCR9, nor the CXC receptors that we have tested. Whilst CCL18 is able to bind to CCR3, it does not bind to the other receptors that it inhibits. We therefore tested the hypothesis that it may displace glycosaminoglycan (GAG) chemokines bound either in cis- on the leukocyte, or in trans-presentation on the endothelial surface, thereby inhibiting the recruitment of leukocytes into the site of inflammation. We show that CCL18 selectivity displaces heparin bound chemokines, and that chemokines from all four chemokine sub-classes displace cell bound CCL18. We propose that CCL18 has regulatory properties inhibiting chemokine function when GAG-mediated presentation plays a role in receptor activation.

The Activity of CCL18 is Principally Mediated through Interaction with Glycosaminoglycans.

The CC chemokine ligand 18 (CCL18) was first identified as a chemoattractant for naïve T cells. It has been reported to recruit T and B lymphocytes, and we show here, natural killer (NK) cells, but with low efficacy. Investigation of its ability to elicit G-protein-coupled signaling showed that it does not involve extracellular signal-regulated kinase (ERK) phosphorylation, and it is not able to induce receptor internalization, as assessed on CCR3. CCL18 has recently been reported to possess activities unrelated to cellular recruitment, but it had no effect on T lymphocyte proliferation. We postulated that a more potent chemoattractant may be produced under inflammatory conditions but only minor truncations were observed, with the major form being the full-length protein. In view of the lack of potent immunomodulatory properties, we wondered if binding to CCL18 by the tick chemokine binding proteins Evasin-1 and -4 was an artifact of the methods used, but complex formation was confirmed by size exclusion chromatography, and abrogation of its binding to, and antagonism of, CCR3. Its receptor has remained elusive since its cloning in 1997, although it has been reported to induce migration of breast cancer cells by signaling through PITPNM3, but we show that this receptor is not expressed on lymphocytes. We have developed a radiolabeled equilibrium competition binding assay and demonstrated that it bound with high affinity to peripheral blood leukocytes (PBLs), but the binding was displaced similarly by both unlabelled CCL18 as well as heparin. Both heparin binding and binding to PBLs are considerably abrogated by mutation of the BBXB motif in the 40s loop suggesting an essential role of the CCL18-glycosaminoglycan interaction.