GPCRs with seven transmembrane domains mediate the recognition of chemokine-encoded messages (7). These GPCRs comprise a polypeptide chain with three intracellular and three extracellular loops, a serine/threonine-rich intracellular COOH-terminal and an acidic NH₂-terminal extracellular domain. Receptor signaling and internalization is mediated by the transmembrane domains, cytoplasmic loops and COOH-terminal domain. The NH₂-terminal domain and a pocket created by the transmembrane domains and extracellular loops are involved in ligand recognition (1). A unique structural feature of the chemokine receptors is the DRYLAIV amino acid sequence present in the second intracellular loop domain, which is required for efficient coupling with G proteins of the G_αi class (5, 12). The chemokine receptors are classified into four subfamilies in accordance with the cysteine motifs of their main ligands: CXCR, CCR, CX3CR, and XCR (1, 12). Upon binding of chemokines, chemokine receptors undergo conformational changes giving rise to the activation of intracellular effectors via G proteins and/or β-arrestins, initiating signal transduction pathways and cellular responses (17–23).

In addition, several atypical chemokine receptors (ACKRs) have been identified (6, 24). These atypical receptors are characterized by a modified or lacking DRYLAIV motif, resulting in the inability of eliciting conventional G protein-coupled signaling processes. The ACKRs influence the internalization and function of chemokines through interaction with β-arrestin signaling pathways. They regulate inflammatory and immune responses by functioning as scavenger or decoy receptors or chemokine transporters (1, 3, 12).

Most inflammatory chemokines bind to several receptors and most chemokine receptors recognize multiple ligands. This binding promiscuity is characteristic for the chemokine network (3, 5). Thus, the chemokine/chemokine receptor network seems highly redundant (25). However, this functional redundancy is not absolute (26, 27). It has been suggested that chemokines are under temporal and spatial control in vivo, and that the localization and timing determine a different biological outcome in different tissues. To ensure appropriate inflammatory responses and to avoid undesirable inflammation, this complex system must be tightly controlled, thereby enabling fine-tuning of leukocyte responses to different inflammatory stimuli. The mechanisms which regulate the interactions between chemokines and chemokine receptors, including down-regulation of chemokine activity by atypical receptors, alternative signaling responses and posttranslational modifications (PTMs), have recently been reviewed (28–30). In contrast to the originally expected redundancy of the chemokine network, recent work demonstrates extreme specificity of the chemokine/chemokine receptor system. Girbl et al., identified distinct and non-redundant roles for two murine CXCR2 ligands CXCL1 and CXCL2 in neutrophil transendothelial migration (31). In addition, Coombs et al. revealed that differential trafficking of the chemokine receptors CXCR1 and CXCR2 regulates neutrophil clustering and dispersal at sites of tissue damage in zebrafish (32). Furthermore, Dyer et al. provide evidence for both redundancy and specificity of the chemokine receptors CCR1, CCR2, CCR3 and CCR5 dependent on the context (33).

The interactions of chemokines and chemokine receptors were traditionally described by a two-step/two-site mechanism (34–36). In the spatial formulation (i.e., two-site), the NH₂-terminus of the receptor recognizes the chemokine globular core (site 1 interaction), followed by the insertion of the unstructured chemokine NH₂-terminus into the receptor transmembrane bundle (site 2 interaction). In the functional formulation (i.e., two-step), site 1 provides affinity and specificity, whereas site 2 elicits receptor activation. With this knowledge, it is not surprising that minor modifications at the NH₂-terminus of chemokines may have profound effects on their activity. However, more and more evidence supports a more complex model (multiple steps/multiple binding sites in the interaction of chemokines and their receptors) to mediate increasingly diverse outcomes (37). The new paradigms in chemokine receptor signal transduction have recently been reviewed by Kleist et al. (38). These authors indicate that we should move beyond the two-site model, since chemokine receptor signaling is influenced by PTMs of chemokine receptors, chemokine, and chemokine receptor dimerization and endogenous non-chemokine ligands.

GAGs are negatively charged, linear polysaccharides comprising repeated disaccharide units, varying in basic composition of the saccharide, linkage, and patterns of acetylation and N- and O-sulphation. The structures of GAGs are highly variable in composition and length, ranging from 1 to 25,000 disaccharide units. Therefore, these polysaccharides exhibit the largest diversity among biological macromolecules (8, 39). GAGs interact with a wide variety of proteins, including proteases, growth factors, cytokines, chemokines and adhesion molecules, enabling them to participate in physiological processes, such as protein function, cellular adhesion and signaling (9, 40). GAGs can be classified into six groups: heparan sulfate (HS), heparin, chondroitin sulfate (CS), dermatan sulfate (DS), keratan sulfate (KS), and hyaluronic acid (HA) (39). The structures and disaccharide composition of GAGs are shown in Figure 1. The disaccharide subunits are composed of an amino sugar residue [N-acetyl-D-galactosamine (GalNAc) or N-acetyl-D-glucosamine (GlcNAc)] and an uronic acid residue [D-glucuronic acid (GlcA) or L-iduronic acid (IdoA)] or D-galactose (Gal) (41, 42). Interestingly, HS has a multidomain structure with sulphated IdoA-containing domains or NS-domains (usually 5-10 disaccharides) separated by flexible spacers of low sulphation that have an acetylated GlcA-GlcNAc sequence (43). HS proteoglycans (HSPGs) account for 50 to 90% of total endothelial proteoglycans (PGs) (44). GAGs, including heparin and HA, can be present in plasma as soluble molecules. Alternatively, GAGs are encountered in surface-bound forms as PGs (8, 39). GAGs, other than heparin and HA, are frequently found covalently attached to protein cores, thereby forming PGs (9). These structures are ubiquitously present on cell surfaces as well as in the extracellular matrix (ECM). There, the PGs serve as a macromolecular coating, also known as glycocalyx, which can interact with proteins such as chemokines (8).

Chemokine binding to GAGs is required for chemokine-induced leukocyte migration in vivo. Mutants demonstrating impaired GAG-binding capacity retained the ability to induce chemotaxis in vitro, but failed to elicit cell migration in vivo (45–49). Chemokines interact with GAGs of the ECM and endothelial cell surfaces (39, 45, 50, 51). Immobilization of chemokines enables the formation of a chemokine gradient, which is indispensable for leukocyte recruitment. This tethering mechanism prevents the diffusion of the chemokines in the blood stream and facilitates localized high concentrations of chemokines that are produced (39, 45). Furthermore, GAGs may play a role in the abluminal-to-luminal transcytosis of chemokines (52, 53). In addition, GAGs may protect chemokines against proteolysis and may influence chemokine-GPCR signaling, thereby regulating chemokine function (9, 54–56).

A hallmark of immune cell trafficking at sites of inflammation and in normal immune surveillance is the migration of leukocytes from the circulation across the endothelium. Therefore, leukocytes need to adhere to the luminal surface of the endothelium. As an inflammatory response develops, cytokines and other inflammatory mediators stimulate the local expression of cell adhesion molecules. First, leukocytes attach to the endothelium by a low-affinity interaction between selectins on the endothelium and their carbohydrate counter-ligands mediating leukocyte tethering and rolling (52, 57–60). In this way, chemokines are able to bind to their leukocyte-specific chemokine receptor(s) resulting in the activation of integrins on the leukocyte. The interaction between the leukocyte integrins and their ligands, such as immunoglobulin-like intercellular adhesion molecules, mediates firm adhesion to the endothelium, enabling the leukocyte to force its way between endothelial cells (52). During this transendothelial migration, the leukocyte squeezes in between two neighboring endothelial cells without disrupting the integrity of the endothelial barrier (61). For neutrophils, this is accomplished by homotypic binding of platelet endothelial cell adhesion molecule-1 (PECAM-1) on the neutrophil with PECAM-1 within the endothelial junction (62). Moreover, it has been shown that PECAM-1 is able to bind heparin and HS by a site that is distinct from that required for haemophilic binding (63). In addition, leukocytes were shown to migrate through endothelial cells (64–66). This process of transcellular migration involves many of the same molecules and mechanisms that regulate paracellular migration.

To ensure the directional guidance of leukocytes across the endothelium and through the ECM into the tissue, a chemoattractant gradient is necessary. However, soluble chemokine gradients cannot persist on the luminal endothelial surface, since they are disturbed by the blood flow (67–69). In addition, soluble chemoattractant gradients would activate leukocytes in the circulation prior to their selectin-mediated adhesive interaction with the endothelium, resulting in the loss of leukocytes' ability to initiate adhesion and emigration (70). Therefore, it has been proposed that chemokines that are bound or immobilized on the luminal endothelial surface more effectively promote leukocyte adhesion to the endothelium and subsequent migration. A first proof of this haptotaxis was the in situ binding of CXCL8 to endothelial cells of venules and veins in human skin and the ability of immobilized CXCL8 to induce in vitro neutrophil migration (71, 72). In addition, chemokines undergo transcytosis through the endothelium and are presented at the luminal surface to adherent leukocytes. Both CXCL8 and CCL5 were bound at the abluminal surface of the endothelium, internalized into caveolae and transported transcellularly to the luminal surface (57). It has even been shown that a COOH-terminally truncated CXCL8 analog with impaired heparin binding and impaired immobilization on endothelial HS was unable to be transcytosed and lost its capacity to induce neutrophil migration in vitro and in vivo (57). Because many of the cell types that produce chemokines are extravascular, chemokine transcytosis and presentation by the endothelium provides a mechanism where through chemokines can stimulate leukocyte emigration (73). A similar mechanism has been shown for CCL19 in the high endothelial venules of lymphoid tissues where it mediates T cell recruitment and suggests a role for this mechanism in normal immune surveillance (74). Noteworthy, endothelial cells also produce chemokines themselves, which are stored in Weibel-Palade bodies and do not need to be transcytosed (73, 75).

In addition, ACKR1 or the duffy antigen receptor for chemokines (DARC), expressed on red blood cells and endothelial cells of postcapillary venules, was shown to bind chemokines, such as CXCL1, CXCL8, CCL2, and CCL5, in inflamed and normal human tissues (76, 77). Mice with targeted disruption of the ACKR1 gene show no developmental abnormalities, but show increased inflammatory infiltrates in lung, liver and/or peritoneum when challenged with lipopolysaccharide (LPS) and/or thioglycolate (78, 79). These data suggest that the intensity of inflammatory reactions is modulated by ACKR1 and that ACKR1 acts as a sink for chemokines. Endothelial ACKR1 may also play a role in chemokine transcytosis in endothelial cells, since it is localized to endothelial caveolae and binds and internalizes chemokines (80, 81). Moreover, it has been suggested that ACKR1 acts as a chemokine-presenting molecule on the endothelium (82). Girbl et al. revealed a self-guided migration response of transmigrating neutrophils. More specifically, neutrophil-derived CXCL2 was presented on ACKR1 at endothelial junctions, thereby enabling unidirectional, paracellular transendothelial migration of neutrophils in vivo (31). However, chemokines bound to ACKR1 on red blood cells do not activate neutrophils anymore (82). Therefore, GAGs may be more important in chemokine presentation.

Although very difficult to prove in vivo, chemoattractant gradient formation has been reported in tissues and venules. Recently, chemokines were shown to localize within postcapillary venules in a GAG-dependent way. For example, localized extravascular release of CXCL2 induced directed migration of neutrophils along a haptotactic gradient on the endothelium toward the tissue as visualized by intravital microscopy (50). This sequestration of chemokines occurred only in venules and was HS-dependent. Transgenic mice overexpressing heparanase showed altered and random crawling of neutrophils in response to CXCL2, which was translated into a decreased number of emigrated neutrophils. In addition, fluorescently labeled CXCL8 formed an extracellular gradient in zebrafish tissue that decays within a distance of 50–100 μm from the producing cells and that was immobilized on HSPGs on the local venous vasculature (51). Inhibition of this interaction compromised both directional guidance and restriction of neutrophil motility. This suggests that leukocytes, once in the tissue, can migrate to the site of inflammation through the gradient of local GAG-bound chemokines. Analogously, endogenous HS-dependent gradients of CCL21 were detected within mouse skin, guiding dendritic cells toward lymphatic vessels (83). These data support the hypothesis that chemokine production at sites of inflammation results in the generation of GAG-mediated chemokine gradients and chemokine presentation by GAGs on the endothelial cell surface, thereby preventing their diffusion and degradation and retaining a high local concentration of the chemokines (52). Finally, blood vessels pattern HS gradients between the apical and basolateral axis (84). Resting and inflamed postcapillary skin venules, as well as high endothelial venules of lymph nodes, show higher HS densities in the basal lamina. Furthermore, the luminal glycocalyx of skin vessels and microvascular dermal cells contained much lower HS densities than their basolateral ECM. Noteworthy, progressive skin inflammation by intradermal injections of complete Freund's adjuvant resulted in massive ECM deposition and in further enrichment of the HS content nearby inflamed vessels. Recently, silencing of exostosin-1, a key enzyme in the biosynthesis of HS, was shown to reduce the directional guidance of neutrophils across inflamed endothelial barriers (85). This again suggesting an important role for basolateral HS. Strikingly, however, effector T cell transendothelial migration is not altered upon silencing of exostosin-1, suggesting that chemotactic signals from intra-endothelial chemokine stores are sufficient to induce the migration of effector T cells.

The binding of chemokines to GAGs and oligomerization have been proven to be indispensable for chemokine activity in vivo (45, 48, 86, 87). Proudfoot et al. demonstrated that mutations in the GAG-binding sites of CCL2, CCL4 and CCL5 result in abrogated GAG binding and a compromised recruitment of cells in vivo when injected intraperitoneally, although receptor binding and in vitro chemotactic activity in Boyden chemotaxis chambers were seldom affected. Even at a dose 10,000-fold higher than the active dose of the wild-type chemokines, the mutants with reduced affinity for GAGs showed no activity in vivo. Noteworthy, the losses in potency in vitro can be attributed to the small losses of receptor affinity and to the impaired interaction with GAGs on the recruited cells, because GAGs can enhance the localization of chemokines to these cells in vitro (88). This also indicates that in general chemokines do not need to be immobilized on GAGs to induce chemotaxis in vitro. However, recently cis presentation of CXCL4 on GAGs, expressed on leukocytes, was reported to affect in vitro cell migration (89).

In addition, inactivation of bifunctional HS N-deacetylase sulphotransferase (NDST-1) in endothelial cells, which is required for sulphation of HS chains, results in impaired neutrophil infiltration in various inflammation models, although these mutant mice develop normally (53). The neutrophil adhesion and migration were reduced because of impaired chemokine transcytosis across the endothelium and reduced chemokine presentation on the endothelial surface. In addition, neutrophil infiltration was decreased to a certain extent due to altered rolling velocity and weaker binding of L-selectin to endothelial cells. In summary, endothelial HS has an important function during inflammation: acting as a ligand for L-selectin during neutrophil rolling, playing a role in chemokine transcytosis and being responsible for the binding and presentation of chemokines at the luminal surface of the endothelium.

Most chemokines are highly basic proteins and therefore it was stated that chemokine-GAG binding largely depends on non-specific electrostatic interactions. However, a certain degree of specificity mediated by van der Waals and hydrogen bonds has been ascribed to this interaction. This was exemplified by binding of the acidic chemokines CCL3 and CCL4 to GAGs (8, 45). In addition, the electrostatic interactions do not necessarily reflect the binding capacity of chemokines to heparin. Although CXCL11 and CXCL12 bound with higher affinity to a non-specific cation exchange resin than CCL5, CCL5 bound stronger to the heparin Sepharose column (9). Before, Kuschert et al. described that GAGs interact with chemokines in a selective manner, providing evidence for GAG sequence specificity. At first, chemokines were shown to exhibit a wide variation in the affinity for heparin and endothelial cells, with, for example, higher affinity binding of CCL5 compared to CCL3 (90, 91). Second, chemokines were shown to possess selectivity in the strength of interaction with GAGs, suggesting that chemokines can discriminate between them. For CCL5, the order is heparin, DS, HS, CS, whereas for CXCL8 and CCL2 the order is heparin, HS, CS and DS. Further, CXCL8 and CXCL1 were shown to bind preferentially to a subset of heparin molecules, whereas CXCL4 and CXCL7 did not show this preference (92). It is important to realize that almost all studies rely on the use of natural GAGs, which are heterogeneous in length and carboxylation and sulphation patterns. Detailed knowledge on interaction of proteins with specific GAG structures depends on the availability of well-described, homogeneous GAG structures. However, chemical synthesis of such specific GAG structures is far more complex than the synthesis of oligonucleotides or peptides.

Several groups have determined the GAG-binding sites in chemokines by using mutagenesis studies, nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. These studies revealed that typically basic amino acids (Arg, Lys, and His) are involved in GAG binding and that the main GAG-binding motifs on chemokines frequently take the form BBXB or BBBXXBX, in which B and X represent a basic and any amino acid, respectively (93). First, it was stated that the GAG-binding domains are located at a site distant from the specific receptor-binding domain, often within the COOH-terminus of the chemokines. However, the GAG-binding domains were located sometimes in the 40 s loop or in the 20 s loop of chemokines. On the other hand, the GAG-binding motif of CXCL10 is a more widely distributed non-BBXB pattern. Thus, for some chemokines the GAG-binding site is not restricted to the COOH-terminus and has an overlap with receptor-binding sites. Therefore, the question whether chemokines simultaneously bind to GAGs on the endothelium and to their receptor on leukocytes remains unanswered and may be chemokine-dependent. Since chemokines show distinctly different GAG-binding epitopes, these data provide a strong indication for specificity of chemokine-GAG binding.

In addition to the GAG-binding motifs of chemokines, specific chemokine-binding epitopes on GAGs have been identified. Although, for example, N-sulphated groups on HS were not necessary, 2-O-sulphated groups on the iduronic acid units were required for the formation of a GAG-dependent CXCL4 tetramer (94). In addition, a binding site for CXCL8 and CCL3 was identified on HS (95, 96).

Many chemokines form dimers or higher-order oligomers, thereby adding more complexity to the structural biology of the chemokine system (97). In addition, CXCL12 dimerization was reported to depend on the presence of heparin (98). CXC chemokines dimerize through the interaction of residues in their β1-strands, thereby forming a six-stranded β-sheet structure topped by two α-helices. Importantly, this dimer structure leaves the NH₂-terminus, N-loop and β3-strand exposed on the surface of the dimer. In this way, CXC chemokine dimers still bind and activate chemokine receptors. In contrast, many CC chemokines dimerize into elongated structures by the formation of an antiparallel β-sheet between the NH₂-terminal regions. Therefore, it was stated that CC chemokine dimers are inactive. In addition to dimers, several chemokines form higher-order oligomers. For example, CXCL4 and CCL3 form tetramers and polymers, respectively (97).

As discussed before, chemokine-GAG binding is important for the localization and the presentation of chemokines on cell surfaces as haptotactic gradients. Moreover, many chemokines oligomerize on GAGs and are stabilized by GAG binding. This chemokine oligomerization and stabilization is essential for chemokine activity in vivo (45, 86, 87, 91, 99). For example, monomeric P8A-CCL2 was incapable of recruiting leukocytes in two in vivo models of inflammation. Surprisingly, in vitro, the monomeric variants are fully active. Also in other studies, monomeric forms of CXCL8, CCL5, CCL4 and the non-oligomerizing chemokine CCL7 have been shown to bind their receptor and to induce chemotaxis in vitro (100–103). Therefore, it can be stated that the monomeric form is sufficient for receptor binding and induction of the directed migration of cells. However, some steps in the process of in vivo migration may involve oligomers. Sometimes the monomeric and dimeric forms of the chemokine show different receptor binding and GAG interactions. Both interactions are essential for in vivo activity, as exemplified by CXCL8 (104). Moreover, the steepness of the chemokine gradient determined by reversible oligomerization is an important factor in the chemotactic response (104, 105).

It was even stated that oligomerization of chemokines increases their affinity for GAGs by providing a more extensive binding surface. In the presence of GAGs, CCL2 formed a tetramer, whereas normally only a dimer is formed (106). In contrast, the CXCL4 tetramer is stable in the absence of GAGs. Dyer et al. showed that oligomerization-deficient mutants of CCL5 and CXCL4 have reduced affinity for heparin, HS and CS compared with their wild-type counterparts (99). In addition, oligomerization may be required for chemokines to simultaneously bind the receptor and the GAG. Certainly, when the chemokine has overlapping GAG- and receptor-binding sites. Alternatively, Graham et al. suggested a “chemokine cloud” model in which chemokines are presented as molecules sequestered in “solution” in a hydrated glycocalyx (107).

In summary, chemokine oligomerization may be important for the local concentration of the chemokine, thereby preventing their diffusion and degradation. Indeed, GAGs protected chemokines from degradation. CCL11 binding to heparin protected the chemokine from proteolysis by plasmin, cathepsin G and elastase (55). In addition, heparin and HS specifically prevented the processing of CXCL12 by CD26/dipeptidyl peptidase IV (DPP IV) (54, 56). Since cleavage of chemokines by proteases can affect their activity, this protection can serve as an additional degree of regulation prolonging the duration of the chemokine signal (108).

The two lymphocyte attractants XCL1 and XCL2 bound GAGs (222, 223). Both convert between a canonical chemokine folded monomer and a unique dimer. Interestingly, the monomer forms were responsible for receptor binding and activation, whereas the dimer forms were involved in GAG binding. Recently, a major GAG-binding site of XCL1 was determined as mutations of the amino acids Arg23 and Arg43 greatly diminished GAG binding (224). Despite their structural similarity, XCL2 displayed a higher affinity for heparin than XCL1. In addition, the XCL1 dimer was responsible for inhibiting HIV-1 activity.

In summary, it can be stated that the interaction between chemokines and GAGs on the cell surface is not essential for GPCR binding and signaling. However, GAG binding enhances the activity of low chemokine concentrations by sequestration of chemokines on the cell surface, inducing polymerization of chemokines and increasing their local concentration. Therefore, cell surface GAGs enhance the effect of chemokines on high-affinity receptors within the local microenvironment.

The chemokine network exerts an indispensable role in the antiviral immune response. Accordingly, some viruses have developed strategies to modulate chemokine activity, thereby affecting leukocyte migration and aiming at evasion or manipulation of the host immune response. These viral mechanisms are highly sophisticated as they possibly have been selected during evolution over millions of years. Large DNA viruses, poxviruses and herpesviruses in particular, use a substantial part of their genome to neutralize the antiviral activity of the immune system of the host. One of their strategies involves the expression of proteins that have the ability to modulate chemokine activity: viral chemokine homologs, viral chemokine receptor homologs and viral chemokine-binding proteins (vCKBPs). The latter group includes secreted proteins that display no sequence similarity with mammalian proteins. These vCKBPs can interfere with chemokine function via binding to either the GAG-binding epitope of chemokines or the chemokine receptor-binding epitope of chemokines, resulting in disruption of the chemokine gradient or abrogated interaction of the chemokine with its chemokine receptor, respectively (234–237). In contrast to the observed inhibitory activity of vCKBPs on chemokine function, a vCKBP that does not inhibit, but potentiates chemokine activity has been detected in both herpes simplex virus type 1 (HSV-1) and HSV-2 (238). The fact that viruses produce proteins that disrupt the chemokine gradient emphasizes the importance of the chemokine-GAG interaction (234). In this paragraph, we will focus on vCKBPs that inhibit chemokine activity by interfering with the chemokine-GAG interaction.

Ticks are bloodsucking parasites that, like many pathogens, have developed certain mechanisms to evade the immune response of their host. Tick saliva contains a wide range of immunomodulatory proteins including a class of CKBPs, termed Evasins. These proteins bound and neutralized chemokines, thereby preventing recruitment of cells of the innate immune system and allowing ticks to remain undetected by their host (255). Until now, the class of Evasins comprises three family members: Evasin-1, Evasin-3 and Evasin-4. Since Evasin-1 and Evasin-4 are structurally related, they constitute the subclass C8 fold, whereas Evasin-3 belongs to the subclass C6 fold containing 8 and 6 cysteines, respectively. The C8 fold Evasins bound to CC chemokines: CCL3, CCL4, CCL18 (Evasin-1) and CCL5, CCL11 (Evasin-4). In contrast, the C6 fold Evasin-3 had high affinity for the CXC chemokines CXCL1 and CXCL8 (and their murine related proteins: CXCL1 and CXCL2). Potent anti-inflammatory activity of Evasins has been demonstrated in several in vivo animal models of disease (255, 256).

Recently, Denisov et al. explored the three-dimensional structures of Evasin-3 and the CXCL8 – Evasin-3 complex. Evasin-3 bound to the CXCL8 monomer and disrupted the interaction of CXCL8 with GAGs and with its receptor CXCR2. When Met-Evasin-3 (a variant of Evasin-3 with a methionine residue at the NH₂-terminus) was added to the CXCL8-GAG complex in in vitro experiments, GAG binding was abrogated. Evasin-3 disrupted the continuous stretch of positively charged amino acids of the GAG-binding domain of CXCL8, thereby preventing binding of the chemokine to GAGs. Consequently, Evasin-3 competed with GAG binding and replaced the GAGs from the CXCL8-GAG complex. Furthermore, two novel CXCL8-binding truncated Evasin-3 variants: linear tEv3 17-56 and cyclic tcEv3 16-56 dPG were synthesized. These variants demonstrated high proteolytic stability in human plasma and inhibited CXCL8-induced neutrophil migration in vitro to a similar extent as native Evasin-3. Both synthetic Evasin-3 variants showed high affinity for CXCL8, although lower than the affinity of native Evasin-3 for CXCL8. The long NH₂- and COOH-termini of native Evasin-3 apparently affect the internal dynamics of its structure, resulting in lower K_D values (256).

Potent in vivo anti-inflammatory activity of Evasin-3 has been shown in several animal models. Evasin-3 inhibited CXCL1-induced neutrophil recruitment to the peritoneal as well as the knee cavity. In addition, Evasin-3 treatment in a murine model of antigen-induced arthritis led to an overall reduction of leukocyte recruitment to the joint and periarticular tissues. Especially neutrophil influx in the knee joint was decreased (by 70%). Moreover, treatment diminished inflammatory hypernociception and the local production of TNF-α. Evasin-3 inhibited the adhesion of leukocytes to the synovial endothelium as observed in intravital microscopy experiments. In addition, Evasin-3 reduced lethality in a murine model of intestinal ischemia-reperfusion injury (255). Evasin-3 treatment was evaluated in a murine model of myocardial ischemia-reperfusion injury as well. A single administration of Evasin-3 during myocardial ischemia resulted in reduced infarct size. This beneficial effect could be allocated to the inhibition of neutrophil influx and the decrease in ROS production (257). Another study demonstrated the positive effect of Evasin-3 on atherosclerotic vulnerability for ischemic stroke. In a mouse model of carotid atherosclerosis, treatment with Evasin-3 resulted in decreased intraplaque neutrophilic inflammation and matrix metalloproteinase-9 content. However, in a murine model of ischemic stroke, no poststroke clinical outcomes were ameliorated by treatment with Evasin-3, suggesting that this treatment is not useful to prevent ischemic brain injury (258). In addition, Evasin-3 reduced neutrophilic inflammation in both lung and pancreas in a murine model of acute pancreatitis. Macrophage recruitment to the pancreas was reduced as well. Evasin-3 treatment reduced ROS release in the lung. Furthermore, treatment with Evasin-3 was associated with reduced lung and pancreas apoptosis and pancreas necrosis (259).

TSG-6 is an inflammation-associated protein with tissue-protective and anti-inflammatory characteristics. Its therapeutic effects have been studied in a wide range of disease models. TSG-6 interacted with various ligands including GAGs and its ability to bind to chemokines has been revealed recently. TSG-6 is the first soluble CKBP discovered in mammals (260, 261). This CKBP bound chemokines of the CC and CXC subfamilies (CCL2, CCL5, CCL7, CCL19, CCL21, CCL27, CXCL4, CXCL8, CXCL11, and CXCL12) and associated with their GAG-binding site, interfering with their interaction with GAGs (190, 262).

GAG-binding peptides can be used as another strategy to target the chemokine-GAG interaction. The design of several GAG-binding peptides derived from different chemokines has been reported. We synthesized a CXCL9-derived GAG-binding peptide, namely CXCL9(74–103). In contrast to most other chemokines, the COOH-terminal region of CXCL9 is exceptionally long, highly positively charged and conserved among species. CXCL9 is a CXCR3 ligand and recruits activated T lymphocytes and NK cells. Moreover, this chemokine has angiostatic properties (16, 231, 263). Remarkably, when natural CXCL9 was purified, the highly charged COOH-terminus (a peptide of up to 30 amino acids) was almost always cleaved from the intact chemokine (231). In a next step, the potential role of this natural COOH-terminal peptide was evaluated. COOH-terminal CXCL9-derived peptides with different length were synthesized. These CXCL9-derived peptides do neither activate, nor recruit leukocytes through CXCR3 (231).

CXCL9(74–103) bound with high affinity to soluble and cellular GAGs. The longest 30 amino acid peptide, CXCL9(74–103), was the most potent competitor and competed with CXCL8, muCXCL1, muCXCL6, CXCL11, CCL2, and CCL3 for binding to GAGs (174, 231, 264). This indicated that CXCL9(74–103) may compete with a wide variety of chemokines that belong to different subclasses. Furthermore, the importance of amino acids 74–78 was emphasized as CXCL9(74–103) was the most potent GAG-binding peptide. Although these amino acids were required, they were not sufficient for GAG binding. CXCL9(74–103) included two typical GAG-binding motifs (BBXB: ⁷⁵KKQK⁷⁸ and BBBXXB: ⁸⁵KKKVLK⁹⁰) (231). A shorter peptide, CXCL9(74–93) showed similar affinity for HS and LMWH in comparison with CXCL9(74–103). However, the affinity of the shorter peptide for binding to CS was lower compared with CXCL9(74–103), indicating that shortening of CXCL9(74–103) resulted in a narrowing of the GAG-binding spectrum (174).

Since CXCL9(74–103) competed with intact chemokines for binding to GAGs in vitro and showed abrogated binding to and signaling through CXCR3, it was hypothesized that CXCL9(74–103) would compete with active chemokines for GAG binding in vivo, thereby inhibiting leukocyte migration. In addition, as CXCL9(74–103) is derived from a chemokine, it might show specificity for certain GAG sequences expressed on the endothelium in an inflammatory situation (231). The potential anti-inflammatory activity of this peptide was first assessed in two murine acute inflammation models characterized by neutrophil infiltration. The CXCL9(74–103) peptide competed with the most potent human neutrophil-attracting chemokine, i.e., CXCL8 for GAG binding and blocked neutrophil migration in both a gout model and an inflammation model that involved intra-articular injection with CXCL8 (231). Furthermore, treatment with CXCL9(74–103) in a murine model of CXCL8-induced neutrophil recruitment to the peritoneal cavity reduced the potency of CXCL8 to induce neutrophil infiltration. In an intravital microscopy experiment, binding of CXCL9(74–103) to the endothelium was visualized in the murine cremaster muscle model. Administration of CXCL8 resulted in neutrophil recruitment. However, treatment with CXCL9(74–103) led to decreased adherence of neutrophils to the endothelial cells (174). In a murine model of antigen-induced arthritis the peptide reduced the recruitment of leukocytes, especially neutrophils, to the synovial cavity and prevented articular and cartilage damage. Moreover, CXCL9(74–103) reduced neutrophil infiltration and neutrophil-dependent inflammation in the ears of the mice in a murine contact hypersensitivity model (265).

In contrast to other strategies that interfere with the chemokine-GAG interaction, the CXCL9-derived peptide, CXCL9(74–103), was not synthesized with the intention to specifically target the action of CXCL9, but rather focuses on the inhibition of the activity of a broader range of chemokines. Recently, two other groups reported the development of chemokine-derived GAG-binding peptides as well. McNaughton et al. synthesized CCL5-, CXCL8-, and CXCL12γ-derived peptides based on the knowledge of their GAG-binding regions (232). The peptides display sequence identity with the chemokines they are derived from. This approach aims at preserving the intrinsic specificity of the chemokine for the GAG by not interfering with hydrogen binding and Van der Waals interactions. The lead peptide pCXCL8-1, consisting of ten amino acids, was modeled based on the COOH-terminal α-helix of CXCL8, a region that mediates an important role in GAG binding. This peptide showed increased affinity for HS and DS in comparison with intact CXCL8 and displayed selectivity for HS over DS. Moreover, pCXCL8-1 competed with CXCL8 for binding to HS. In contrast to pCXCL8-1, a CXCL12γ-derived peptide failed to inhibit CXCL8-induced neutrophil migration, despite its high affinity for HS. This indicates that specificity plays a role in the interaction between the peptide and HS. Accordingly, the binding sites for the CXCL8- and the CXCL12γ-derived peptides on HS may differ. The peptide pCXCL8-1 was modified (NH₂-terminal acetylation and COOH-terminal amidation) in order to protect it from proteolytic cleavage by exopeptidases upon administration in vivo. The resulting peptide pCXCL8-1_aa was tested in an in vivo murine model of antigen-induced arthritis. The number of neutrophils in the synovium was reduced. Furthermore, the inflammation, cellular exudate and hyperplasia were decreased upon treatment with pCXCL8-1_aa. The peptide improved the arthritic score that is a measure of severity of disease in this mouse model (232). Martinez-Burgo et al. synthesized three different peptides derived from CXCL8: a COOH-terminal peptide (54–72) (wild-type peptide), a peptide (54–72) where the glutamic acid residue at position 70 was replaced with a lysine residue (E70K peptide) and a scrambled peptide consisting of the same amino acids as peptide 1 in a random order. Although only detectable at much higher concentrations compared to intact CXCL8, the three peptides, and the E70K peptide in particular, showed binding to heparin. The observed low-affinity binding of the peptides appeared to be dependent on charge. Furthermore, the peptides did not affect chemokine-GPCR binding. Only the E70K peptide showed the ability to inhibit CXCL8-mediated transendothelial migration of neutrophils (233).

ProtAffin Biotechnologie AG developed a protein-based technology platform, also known as the CellJammer technology platform, in order to interfere with protein-GAG interactions via protein engineering. This approach enabled the generation of GAG-binding decoy proteins, namely dominant-negative mutant proteins. On the one hand, these proteins are characterized by increased GAG-binding affinity (= dominant mutations). On the other hand, dominant-negative mutant proteins display impaired receptor binding/activation (= negative mutations) (266–268). In order to improve GAG-binding affinity, non-crucial amino acids in the GAG-binding domain of the wild-type protein were substituted with basic amino acids. This resulted in an increase of the electrostatic component of the protein-GAG interaction. Furthermore, to disrupt the bioactivity of the wild-type protein, amino acids responsible for natural chemokine-receptor interactions are either substituted with alanine residues or deleted (266). The strategy relies on the intrinsic selectivity of the GAG-binding protein for its specific GAG epitope. Consequently, dominant-negative mutant proteins that bind with higher affinity to the GAG have the ability to displace their wild-type counterpart protein from the specific GAG target sequence. With this mode of action, dominant-negative mutant proteins antagonized protein-GAG interactions (266, 267).

A series of CXCL8 mutants was engineered by site-directed mutagenesis. Subsequently, by using a combinatorial approach consisting of different techniques, the affinities of the different mutants for HS were assessed. The pro-inflammatory human CXCL8 was designed toward an anti-inflammatory dominant-negative CXCL8 mutant. On the one hand, four non-crucial amino acids of human CXCL8 were replaced with basic lysine residues in order to knock-in high GAG-binding affinity. On the other hand, the six NH₂-terminal amino acids of human CXCL8, including the ELR motif, were deleted in order to knock-out the binding to its two GPCRs, namely CXCR1 and CXCR2. This resulted in the mutant PA401 or CXCL8[Δ6F17KF21KE70KN71K] that was selected from the series of dominant-negative CXCL8 mutants as it showed the best interaction profile with HS (266, 269, 270). Moreover, complete knocked-out CXCR1 and CXCR2 activity was observed in this mutant (269, 271). The wild-type chemokine that is displaced by the dominant-negative chemokine mutant, may still activate leukocytes. However, since the endothelial contact with GAGs is disrupted, transmigration of the chemokine should not take place (271). In addition, other chemokines than CXCL8 can be displaced from GAGs by PA401 as well. PA401 had the ability to displace CCL2 from GAGs with a similar IC50 value in comparison with CXCL8. Furthermore, CXCL10, CXCL12, CCL11 and other chemokines were displaced by PA401 as well, although they were characterized by higher IC50 values (267, 271–273). PA401 had the ability to inhibit CXCL8-induced neutrophil migration (reduction by 75%) in a transendothelial migration assay (273).

The anti-inflammatory activity of PA401 was evaluated in several disease models. Treatment with PA401 reduced renal ischemia-reperfusion injury in a rat model. More specifically, proximal tubular damage was reduced and a decreased number of infiltrating granulocytes was observed. Furthermore, PA401 reduced acute allograft damage in a rat kidney transplantation model. PA401 treatment lowered glomerular infiltration of monocytes and CD8⁺ T cells. In addition, tubular interstitial inflammation and tubulitis, which is an indication of acute allograft rejection, were diminished as well. Moreover, the highest dose of PA401 improved glomerular and vascular rejection (271). In a murine model of acute inflammation, PA401 dose-dependently inhibited neutrophil recruitment to the knee cavity after intra-articular injection with murine CXCL1. Thus, PA401 showed anti-inflammatory activity in an inflammation model that was induced by a murine functional homolog of CXCL8. Furthermore, PA401 treatment was evaluated in a murine model of antigen-induced arthritis and resulted in inhibition of leukocyte adhesion, diminished neutrophil recruitment and inflammation-related hypernociception (269). Moreover, PA401 had the ability to disrupt the CXCL8-GAG interaction in bronchoalveolar fluid samples from patients with cystic fibrosis. As a consequence, the release of CXCL8 from the GAGs rendered the chemokine susceptible to proteolytic degradation, resulting in reduced migration of neutrophils (274). PA401 was also tested as a novel therapeutic approach in two murine models characterized by neutrophilic lung inflammation. In a murine model of LPS-induced lung inflammation, the administration of PA401 reduced the total number of cells and the number of neutrophils in bronchoalveolar lavage (BAL). Furthermore, PA401 had the ability to decrease lung congestion and inflammatory cells in the lung tissue (230) and normalized plasma inflammatory markers (275). In a murine model of tobacco smoke-induced lung inflammation, PA401 treatment showed broad anti-inflammatory activities, including reduced inflammatory cells and soluble inflammatory markers. A reduction in the number of neutrophils, macrophages, lymphocytes and epithelial cells in BAL was observed (230). Furthermore, PA401 treatment was tested in a murine model of urinary tract infection and resulted in decreased recruitment of neutrophils to the urine. Normalization of the tissue architecture could be observed in mice treated with PA401 when inspecting histopathology sections of renal micro-abscesses (273). In a murine model of bleomycin-induced pulmonary fibrosis, a dose-dependent decrease in total cell counts and neutrophils was observed in BAL samples upon PA401 treatment. Moreover, decreased levels of CXCL1 were detected in lung tissue (273). In an experimental autoimmune uveitis model in rats, treatment with PA401 influenced severity and incidence of disease. The mean maximal clinical disease scores were decreased after both pre-symptomatic and symptomatic treatment with PA401. Furthermore, slightly less retinal destruction was observed after PA401 treatment (273).

A phase I first-in-human clinical trial (NCT01627002) was performed to examine the safety, tolerability, immunogenicity and pharmacokinetics of PA401 in healthy volunteers. Moreover, the effect of PA401 on lung inflammation following an LPS challenge was investigated in this study as well.

The CellJammer approach was applied to develop a second dominant-negative chemokine mutant, namely a CCL2/MCP-1-based decoy protein named PA508. Met-CCL2[Y13AS21KQ23R] was selected out of four novel CCL2 mutants as it showed both the highest affinity for HS and knocked-out CCR2 activity. The serine and glutamine residues at position 21 and 23 respectively, are characterized by solvent-exposed areas above 30% and are located close to the GAG-binding site of the chemokine. Consequently, these amino acid residues were substituted with basic amino acids in order to increase the GAG-binding affinity. Moreover, these mutations were beneficial for the disruption of receptor activation, considering the partial overlap between the GAG-binding and receptor activation sites in CCL2. Since the tyrosine residue at position 13 is a key residue for receptor signaling, it was replaced by an alanine residue, thereby abrogating CCR2 activation and signaling. The NH₂-terminal methionine residue, originating from recombinant protein synthesis in E. coli, was not removed as it turned out to increase the binding affinity to heparin and decreased binding affinity for CCR2 (276). PA508 has a remarkable specificity for CCL2, as it did not influence transendothelial migration of monocytic cells induced by chemokines such as CCL5 and CXCL1. Moreover, this specificity was confirmed in vivo as PA508 showed no antagonistic activity in CCR2^−/− mice, indicating that PA508 specifically targeted the CCL2-CCR2 axis (277).

The anti-inflammatory activity of PA508 has been demonstrated in several in vivo models. The administration of PA508 resulted in a mild ameliorating effect in rat experimental autoimmune uveitis. PA508-treated rats were protected from the development of severe inflammation of the inner eye (276). Furthermore, treatment with PA508 reduced the influx of leukocytes in a murine air pouch model of TNF-α-induced leukocyte recruitment. In a mouse model of wire-induced neointimal hyperplasia, PA508 reduced neointimal plaque area in wire-injured arteries. In addition, this reduction was associated with diminished macrophage infiltration. The smooth muscle cell content in the neointima was increased upon treatment. Thus, treatment with PA508 reduced neointima formation and resulted in a more stable, less inflammatory plaque phenotype (277). Administration of PA508 attenuated myocardial ischemia-reperfusion injury in mice. Treatment preserved heart function and reduced myocardial infarction size. The latter resulted from PA508-mediated inhibition of myocardial macrophage-related inflammation and reduction of myofibroblast and collagen content (277). In a murine model of zymosan-induced peritonitis, administration of PA508 reduced infiltration of a pro-inflammatory subset of monocytes (Gr1 and F4/80 double positive), whereas the number of peritoneal macrophages was not affected (278). PA508 treatment showed also promising results in murine experimental autoimmune encephalomyelitis. PA508-treated mice showed a delayed disease onset and an overall better clinical score. Furthermore, mice that received PA508 demonstrated decreased maximal disease severity, preserved body weight and increased survival. Treatment with PA508 resulted in a reduction of inflammatory cell infiltrates in the spinal cord and the cerebellum, as observed during histological analysis and demyelination was reduced (278).

Chemokines and their respective chemokine mutants are characterized by a short serum half-life. In order to increase the bioavailability and thus prolong the serum half-life of PA508 for chronic indications, a novel mutant CCL2-human serum albumin (HSA) fusion protein was designed. Considering the steric influence of HSA, a diminished GAG-binding affinity of this new mutant CCL2-HSA chimera was expected. Therefore, a series of novel mutants with additional basic amino acids was developed. Eventually, the fusion of the selected CCL2 mutant with HSA resulted in HSA(C34A)-(Gly)₄Ser-Met-CCL2[Y13AN17KS21KQ23KS34K] that demonstrated high and selective GAG-binding affinity and improved stability (279).

The CellJammer technology was used also to generate CCL5-based decoy proteins. Initially, ten CCL5 mutants were developed. All mutants retained their NH₂-terminal methionine residue that resulted from bacterial expression, rendering them into functional receptor antagonists. Two mutants, A22K and H23K, were selected since they demonstrated highest stability and improved GAG binding. Their GAG-binding affinity was increased by engineering an extended three-dimensional GAG-binding epitope in addition to the linear ⁴⁴RKNR⁴⁷ GAG-binding domain. Both mutants displayed completely abrogated chemotaxis on monocytes, due to their extra NH₂-terminal methionine. Treatment with the H23K mutant in rat autoimmune uveitis led to earlier recovery from ocular inflammation, whereas treatment with the A22K mutant did not demonstrate any anti-inflammatory effect. Brandner et al. hypothesized that the minor therapeutic effect of both mutants could be due to a deficiency in GAG-induced oligomerization caused by these mutations. The H23K mutant exhibited higher GAG-binding affinity and partially retained the ability to form GAG-induced oligomers, which could explain its observed therapeutic effect in comparison with the A22K mutant of CCL5 (280).

In addition, a CXCL12α-based decoy protein was developed. Deletion of the first eight amino acids of the chemokine already resulted in completely impaired chemotaxis. Furthermore, the amino acid residues at positions 29 and 39 were mutated as these positions are important for the first receptor binding step and for GAG binding. This resulted in the dominant-negative CXCL12α mutant CXCL12α[Δ8L29KV39K], which demonstrated impaired CXCR4 signaling in combination with increased and specific GAG affinity. The effect of this mutant was assessed in an in vivo murine breast cancer seeding model. Treatment with the CXCL12α mutant inhibited migration of cancer cells, resulting in a decreased number of liver metastases (281).

NOX-A12 is an aptamer inhibitor of CXCL12 that can be classified more specifically as a Spiegelmer. Mirror-image aptamers or Spiegelmers are synthetic oligonucleotides that are composed of non-natural L-nucleotides. Since naturally occurring enzymes are stereoselective for nucleic acids in the D-configuration, Spiegelmers are protected from nucleolytic cleavage, implying a native biological stability (282, 283). Spiegelmers can be selected in vitro to bind a wide variety of molecular targets with high affinity and specificity (283, 284). Binding to a specific molecular target relies on the three-dimensional structure of a Spiegelmer, which is determined by its nucleotide sequence (283, 285). This interaction via three-dimensional structures is similar to antibody–antigen binding. Moreover, aptamers are functionally comparable to antibodies regarding binding affinity and specificity for their targets. They display advantages relative to antibodies, including smaller size, higher stability, fast in vitro chemical production, wide spectrum of potential targets and non-immunogenicity (285, 286).

The Spiegelmer NOX-A12 or olaptesed pegol is a 45-nucleotide long L-RNA aptamer that interferes with chemokine-GAG interactions (228, 283, 286). NOX-A12 was developed to bind and antagonize CXCL12 in the tumor microenvironment and for cell mobilization (228, 287). CXCL12 plays a critical role in physiological and pathological processes such as embryogenesis, haematopoiesis, angiogenesis and inflammation (200, 288). This chemokine functions by interaction with its two receptors CXC chemokine receptor 4 (CXCR4) and atypical chemokine receptor 3 (ACKR3) (200). NOX-A12 bound its molecular target with subnanomolar affinity (287). Because of its small size, NOX-A12 was rapidly excreted through renal filtration (285). However, in order to extend its plasma half-life, NOX-A12 was conjugated to a branched polyethylene glycol (PEG) moiety of 40 kDa (PEGylated) (289).

CXCL12 also plays a critical role in the pathogenesis of chronic lymphocytic leukemia (CLL). This chemokine is important for migration and retention of CLL cells in tissues such as the BM. Bone marrow stromal cells (BMSCs) constitutively secrete CXCL12 and consequently attract CLL cells into the supportive microenvironment via activation of the CXCR4 receptor that is expressed on these leukemic cells. CXCL12 is presented to CXCR4 by binding to GAGs on the cell surface or ECM. In the tissue microenvironment, the CLL cells are protected from cytotoxic drugs and they receive survival signals via several factors, including CXCL12. Interfering with the cross-talk between CLL and stromal cells in order to eliminate CLL cells from the protective microenvironment and sensitize CLL cells to conventional therapy can be done by targeting CXCL12/CXCR4 signaling (228, 229).

Hoellenriegel et al. investigated the effect of NOX-A12 on the migration of CLL cells and drug sensitivity. Moreover, they more thoroughly investigated the mechanism of action of NOX-A12 and revealed its ability to compete with GAGs for CXCL12 binding. SPR measurements were performed with immobilized heparin and CXCL12 was injected together with NOX-A12. The latter competed with the immobilized heparin for binding to CXCL12, resulting in detachment of heparin-bound CXCL12. Consequently, the binding site of NOX-A12 on CXCL12 was presumably close to or overlaps with the heparin-binding site of CXCL12. Hence, the mode of action of NOX-A12 involved competition with heparin for binding to CXCL12 and explained the detachment of CXCL12 from extracellular GAGs (228).

Another Spiegelmer that binds to and neutralizes a chemokine, i.e. Emapticap pegol or NOX-E36, bound to CCL2, also known as monocyte chemoattractant protein (MCP)-1 (283, 290). However, to the best of our knowledge, it has not been explored or proven whether the mechanism of action of the latter Spiegelmer is similar to the one of NOX-A12. Accordingly, NOX-E36 will not be further discussed in this review.

NOX-A12 has been used in a variety of different disease models, ranging from chronic kidney disease to several types of cancer. An overview of studies that report treatment with NOX-A12 in different (animal) models and clinical trials can be found in Table 2. Currently, NOX-A12 is being tested alone and in combination with the immune checkpoint inhibitor Pembrolizumab in an ongoing phase I/II clinical trial of metastatic pancreatic and colorectal cancer (NCT03168139). In addition, the recruitment of patients with newly diagnosed glioblastoma/glioma has started in order to initiate a phase I/II clinical trial to evaluate combination treatment of NOX-A12 and radiotherapy¹.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.