Novel Anti-Inflammatory Peptides Based on Chemokine–Glycosaminoglycan Interactions Reduce Leukocyte Migration and Disease Severity in a Model of Rheumatoid Arthritis

Inflammation is a necessary process to prevent infection and the effects of injury on the body, and its conclusion requires a controlled self-limiting process. When it becomes unresolved and chronic, it becomes destructive, leading to inflammatory disease. Leukocyte extravasation is a characteristic of inflammation and the recruitment of leukocytes from the circulation into the inflamed tissue is a multistep process (1). First, leukocytes are loosely tethered to the endothelium of blood vessel walls where they roll and interact with endothelial cell (EC)–bound chemokines and adhesion molecules such as selectins. The interaction with chemokines activates leukocyte integrins, enabling the leukocyte to mediate firm adhesion to the endothelium. Chemokines also stimulate leukocyte crawling on the endothelial surface and migration across the ECs, the basement membrane, and into the extracellular matrix of the tissue (2). Once within the tissue, leukocytes normally perform various beneficial immune duties such as tissue repair and pathogen elimination. Due to the potentially destructive nature of leukocytes, tight regulation is required otherwise inflammatory disease can ensue. Chemokines play a role in the regulation of the inflammatory process by interactions with two binding molecules: G-protein coupled receptors (GPCRs) and glycosaminoglycans (GAGs).

Chemokines are small (8–10 kDa) chemoattractants involved in numerous physiological processes such as angiogenesis, but mainly their role lies in the chemoattraction of migratory cells (3). Thus far, around 45 chemokines have been identified and are classified into four groups (C, CXC, CX3C, and CC) based on the pattern of cysteine residues in the ligands (4). Despite the range and diversity of chemokines with regards to their homeostatic or inflammatory function, they have a remarkably similar tertiary structure consisting of a conformationally disordered N terminus, a long N-loop followed by a single turn 3₁₀ helix, a three-stranded antiparallel β-sheet, and a C-terminal α-helix (5). The flexible N-terminal region is central to receptor activation because studies have shown that mutations or truncation of this region results in altered leukocyte activity (6, 7). In vitro studies have shown that more than one chemokine can bind to a given receptor and that several receptors are able to bind a given chemokine, hinting that a large amount of redundancy takes place. However, in vivo, the interaction seems to be more specific, and it has been suggested that chemokine redundancy is in fact more likely finely tuned (8). Different ligands can activate distinct signaling pathways following binding to the same receptor. For example, both CCL19 and CCL21 induce chemotaxis of CCR7-expressing cells, yet only CCL19 is able to induce receptor downregulation (9). As we learn more about chemokines, they may prove to be attractive therapeutic targets in the clinic.

The second and less well-characterized interaction of chemokines is with carbohydrates, namely GAGs. GAGs are long, linear, and heterogenous structures consisting of repeating disaccharide units that vary in the basic composition of the saccharide, the linkage to the core protein, acetylation, and N- and O-sulfation (10). GAGs are ubiquitously expressed although different types (heparan, dermatan, chondroitin, and keratan sulfate) may be found on cell surfaces within the glycocalyx and throughout the extracellular matrix of all mammalian tissues. GAGs can be surface bound or shed as soluble ectodomains (11); a process that dramatically changes their function (12). Their chain lengths can range from 1 to 25,000 disaccharide units thus together with structural variation in sulfation patterns, this presents an immense level of diversity. This amount of diversity suggests an element of control and specificity when binding their biological ligands. GAGs are usually covalently attached to a protein core (with the exception of hyaluronic acid and heparin) to form a proteoglycan (PG). The most ubiquitous of these are heparan sulfate PGs (HSPGs), which are classified into five groups of core proteins that range in size from 32 to 500 kDa. The most abundant form are the syndecans, which contain a transmembrane region anchoring them to the cell surface. In contrast, the glypicans are anchored to the cell surface via glycosyl-phosphatidylinositol groups and the other three (perlecan, agrin, and collagen XVIII) do not have direct links to the cell membrane but remain closely associated with it (10). The composition and spacing of the GAG chains on the protein is dependent on the cell type and correlates with different physiological responses of the cell (13). The differential expression of PGs on particular cells allows them to dynamically function and adapt within the local microenvironment, exhibiting an aspect of functional specificity.

GAG chains have been implicated in leukocyte transmigration and one GAG in particular is believed to have multiple roles in the extravasation process: HS (14). HS is the most abundant endothelial GAG and composes 50–90% of total endothelial PGs (15). HS has been implicated in binding to a wide range of proteins, some of which include cytokines, adhesion molecules, proteases, and growth factors. Interaction of chemokines with HS can protect them from proteolysis (16) and induce them to oligomerize; high-order oligomers are thought to be required for maximal chemokine activity (17). Furthermore, the interaction establishes chemokine gradients for migrating leukocytes by being bound and presented at the endothelial luminal surface (18) and HS is involved in the transcytosis of chemokines across the EC layer to be displayed at the luminal surface (18). Leukocyte crawling also involves a chemokine–HS interaction and is critical for the leukocyte to find optimal sites to emigrate (2). One might consider the subendothelial basement membrane represents a formidable barrier to leukocyte extravasation, often being in the range of 20–100 nm thick, yet it is also rich in extracellular HSPGs. This complex network of extracellular HSPGs such as perlecan, agrin, and type XVIII collagen can serve to bind and sequester proteins that regulate leukocyte migration such as chemokines, thereby contributing to leukocyte diapedesis (14). Hence, chemokine interaction with HS is involved in several key stages of leukocyte extravasation.

The GAG-binding motif on chemokines typically takes the form XBBXBX in CC chemokines (19) or XBBBXXBX in others, where B represents a basic and X represents any nonbasic amino acid (20). In general, these motifs are found in a separate location from the specific receptor-binding domain, and often within the C terminus. Although positively charged chemokines have a favorable charge interaction with negatively charged GAGs, several lines of evidence suggest that more than nonspecific electrostatic forces drive this interaction. For example, acidic chemokines such as CCL3 and CCL4 also bind GAGs (21), and it is thought that specificity of the GAG-chemokine interaction is introduced by Van der Waals and hydrogen-bond forces (10). The GAG-binding function of chemokines has been shown to be essential for optimal chemokine activity in vivo (19, 22–25). For example, when CXCL8 is truncated of its HS-binding C-terminal helix, it fails to bind heparin and has impaired leukocyte activation and receptor-binding properties (26). The truncation also shows reduced transcytosis across ECs and luminal surface presentation to blood leukocytes, resulting in reduced leukocyte transmigration (18, 27).

Chronic inflammation is also characterized by changes in GAG patterns (28). Changes in cell-surface and secreted GAGs in cancer have been shown to strongly influence the phenotype of the tumor, allowing them to control growth rates, invasiveness, and metastatic potential (29). Moreover, a CXCL8-binding site on endothelial syndecan-3 is induced in the synovium of rheumatoid arthritis (RA) patients (30). Furthermore, in an animal model of RA, syndecan-3 functions in endothelial chemokine presentation and leukocyte recruitment, suggesting a role for this HSPG in leukocyte trafficking into the inflamed tissue (31). Altered expression of endothelial GAGs has been observed in numerous other chronic inflammatory disorders including atherosclerosis (32) and inflammatory bowel disease (33). This could promote the binding and presentation of chemokines that are selective for the particular GAG sequences expressed, therefore contributing to a site-specific localization of chemokines (34). This suggests an element of control over the function of chemokines by HS.

Clearly, the GAG-binding capability of chemokines is of importance in the multitude of steps involved in leukocyte extravasation. The potentially specific and unique interactions that chemokines have with discrete GAG sequences are, as yet, largely unexplored. Due to the number of diseases in which chemokines have been implicated, blockade of chemokine function seems an attractive strategy. This study aims to obtain more insight into the GAG-binding regions of particular chemokines, namely CCL5, CXCL8, and CXCL12γ, and their functional roles. All of these chemokines are known to interact with HS and the HS-binding domains are well established (20, 35, 36). These chemokines were chosen based on the cell types they interact with, i.e., CCL5 is largely chemotactic for monocytes, CXCL8 for neutrophils, and the relatively newly discovered CXCL12γ has a uniquely rich HS-binding C terminus and appears to stimulate migration of lymphocytes. Using a novel approach, we chemically synthesized peptide chains based only on the GAG-binding regions of each of these chemokines and tested their abilities to bind to HS and compete with chemokines in transendothelial migration assays. Sequences where the known HS-binding residues including lysine and arginine were altered to noncharged residues such as glycine, serine, and alanine were synthesized to act as controls. The lead peptide based on CXCL8 was assessed for binding to GAGs in EC lines and human RA synovium. It was then tested in a murine Ag-induced arthritis (AIA) model. This strategy could improve future prospects for exploiting GAGs as therapeutic targets and other strategies for targeting chemokine–GAG interactions.

The crystal structures of each chemokine were taken from the Protein Data Bank (PDB): CXCL8 (PDB code: 3IL8), CCL5 (PDB code: P_1U4L), and CXCL12 (PDB code: 2NWG). Using the biopolymer module of molecular modeling program, Insight II (version 2005 by Accelrys), each of the chemokines was displayed and the peptides based on chemokines were structurally defined. The chemokines were rendered using a space-filling model (cpk representation) to represent the atoms present, and are modeled as the biologically relevant dimers. Peptides based on chemokines were modeled to show the amino acid sequences of the peptide highlighted on the particular region of the chemokine where they are located. The structure of peptide CXCL12-1 (pCXCL12-1) required the construction of C-terminal α helices found in the CXCL12γ isoform, which were modeled onto residue 67 and extended to 98 residues. The design of pCXCL8-4 was based on the C-terminal α helices spliced together with a linker sequence GSGSG. The GNU image manipulation program version 2.8 was used to manipulate images and perform format conversion.

All reagents and solvents for peptide synthesis were purchased commercially from Sigma-Aldrich (Gillingham, U.K.), and peptides were synthesized at Peptide Protein Research Ltd. (Bishop's Waltham, U.K.). Solid-phase peptide synthesis was carried out on an automated peptide synthesizer Symphony employing standard Fmoc chemistry. Peptides were cleaved from the solid support and purified using reverse-phase HPLC. The crude peptide product was loaded onto a preparative C-18 Axia silica column typically running a linear gradient of water and acetonitrile buffers, each containing 0.1% trifluoroacetic acid at a gradient routinely run from 20 to 60% acetonitrile (depending on the peptide) for 1 h at a flow rate of 20 ml/min while monitoring the eluent at 225 nm. The collected fractions were lyophilized before analytical techniques such as liquid chromatography–mass spectrometry were employed to give an overall purity read out. Peptides were >90% pure. Peptide sequences can be seen in Table I.

Some peptides were N-terminally modified by the addition of tags such as biotin and FITC. These were coupled manually to the last amino acid of the N terminus using an aminohexanoic acid linker overnight, and the resulting peptide was cleaved from the resin as normal. The lead pCXCL8-1 was capped at the C terminus by using a resin functionalized with a modified Rink Amide linker and at the N terminus with 50% acetic anhydride/pyridine to be used for intra-articular administration in vivo.

The titration experiments were performed on a Fluoromax-4 spectrofluorometer (Horiba, Kyoto, Japan) coupled to an external water bath to ensure constant temperature during the measurements. Protein fluorescence emission spectra were recorded over the range of 500–600 nm upon excitation at 495 nm. The slit widths were set at 3 nm for excitation and emission, scan speed at 500 nm/min, and the temperature was set to 20°C. The use of concentrated GAG oligosaccharide stock solutions ensured a dilution of the protein sample <5%. Prior to collection of the initial (unliganded) protein emission spectra, 100 nM protein solutions were prepared from stock solutions and were equilibrated for 30 min. Following equilibration, respective GAG ligands [HS (catalog number: GAG-HS01), dermatan sulfate (DS) (catalog number: GAG-DS01); Iduron, Manchester, U.K.] were added in concentrations ranging from 5 to 50 nM. The protein/GAG solutions were then equilibrated for 1 min and fluorescence emission spectra were collected. For background correction, the emission spectra of the respective GAG concentrations were collected in PBS buffer only. They were subsequently subtracted from protein emission spectra and the resulting curves were then integrated. The mean values resulting from three independent measurements were plotted against the concentration of the added ligand. The resulting binding isotherms were analyzed by nonlinear regression using the program Origin (Microcal, Northampton, MA) to the following equation describing a bimolecular association reaction, where Fi is the initial and Fmax is the maximum fluorescence value, K_d is the dissociation constant, and [protein] and [ligand] are the total concentrations of the chemokine/peptide and the GAG ligand:

Immortalized human bone marrow ECs (HBMECs), and human cerebral microvascular ECs (HCMEC/D3s) were both donated by Prof B.B. Weksler, Cornell University, New York, and support leukocyte transendothelial migration studies (37–39). HBMECs were maintained in DMEM-F12 (Lonza, Wokingham, U.K.) supplemented with 10% FBS (Invitrogen, Paisley, U.K.) and HCMEC/D3s in rat tail collagen type 1 (R&D Systems, Abingdon, U.K.)–coated flasks (150 mg/ml in dH₂O) using EBM-2 (Lonza) containing 1% penicillin-streptomycin (Invitrogen), 1% chemically defined lipid concentrate (Invitrogen), 1 ng/ml basic fibroblast growth factor (Sigma-Aldrich), 1.4 μM hydrocortisone (Sigma-Aldrich), 5 μg/ml ascorbic acid (Sigma-Aldrich), 10 mM HEPES (PAA Laboratories, Yeovil, U.K.), 5% FBS, and 10 mM lithium chloride (Merck, Feltham, U.K.). All ECs were incubated at 37°C in a humidified incubator containing 5% CO_2. Cells were grown to 70% confluence before being used in the following experiments.

ECs were seeded at a density of 2.5 × 10⁵ in Nunc Lab-Tek eight-well chamber slides (Sigma-Aldrich) using 500 μl serum-free medium and left to reach confluence for 24 h. Some cells were treated with heparanase I (10 U/ml) and heparanase III (2 U/ml; both from Sigma-Aldrich) as described by Whittall et al. (39) for 1.5 h at 37°C to remove the HS. Following the heparanase treatment, cells were fixed with 4% PFA for 10 min before being incubated with 2 μg/ml mouse anti-human HS Ab (10e4; Amsbio, Abingdon, U.K.) for 1 h in 2% BSA/PBS at room temperature (RT). The cells were washed 3 times in PBS. The primary Ab was detected by Alexa Fluor 488 goat anti-mouse IgM (1:200 in 2% BSA/PBS) and the cells washed before being counterstained in DAPI (2 μg/ml; DAPI) for 3 min. For a control, mouse IgM was used at an equivalent concentration in place of the primary Ab. Slides were mounted using Hydromount (Thermo Fisher Scientific, Loughborough, U.K.), visualized with a light fluorescence microscope (Leica DM LB), and analyzed with Cell^˄D software.

For all leukocyte collection, a total of 20 ml blood was collected from healthy volunteers, after informed consent, into lithium heparin-treated vacutainers. To isolate polymorphonuclear cells (PMNs), the blood was centrifuged at 500 × g for 30 min with polymorphprep (Axis-Shield, Oslo, Norway), followed by the removal of mononuclear cells (MNs) and erythrocytes. The remaining PMNs were washed with an equal volume of HBSS and dH₂O for 10 min at 400 × g followed by erythrocyte lysis using ammonium chloride solution (Stemcell Technologies, Cambridge, U.K.) for 7 min at 37°C. The PMNs were then harvested by centrifugation at 300 × g for 5 min, resuspended in 0.1% BSA in RPMI 1640 (Lonza), and held at 4°C until required. For MN isolation, the same process applied except the MNs were harvested after initial centrifugation and the PMNs and erythrocytes were discarded.

A total of 2.5 × 10⁵ ECs in 500 μl cell growth medium were cultured on 3 μm pore transwell filters (Thermo Fisher Scientific) in a 24-well flat-bottom microplate (Corning Costar, Sigma-Aldrich) with 800 μl of cell growth medium in the basal chambers until confluence was reached (24 h at 37°C). The solution in the apical chamber was replaced with 500 μl fresh serum-free medium and the solution in the basal chamber was replaced with a solution of serum-free medium containing 100 ng/ml CXCL8 or CXCL12γ; for controls, serum-free medium containing no CXCL8 or CXCL12γ was used. The samples were incubated at 37°C for 30 min for endothelial activation to occur as described by Whittall et al. (39). ECs were activated by incubating monolayers with the following chemokines: recombinant human CXCL8, CCL5 (both from PeproTech, London, U.K.), and CXCL12γ (R&D Systems) at concentrations of 100 ng/ml in serum-free medium for 30 min at 37°C (39). The negative controls contained no chemokines. Monolayers were established by optimizing seeding density of ECs and allowing them to reach confluence over 24 h. At this time point the transendothelial electrical resistance value was maximum (using an EVOM2 voltmeter) and experiments were carried out identically thereafter. A total of 5 × 10⁵ PMNs was then added to the apical chambers, followed by incubation at 37°C for a further 30 min in the continued presence of CXCL8 or CXCL12γ. PMN migration was quantified by determining the number of neutrophils that had migrated through to the basal chamber in 30 min, by manual counting using a disposable hemocytometer (Immune Systems, Torquay, U.K.) and displayed as a percentage of transendothelial migration when compared with chemokine alone.

MN cell migration was carried out in an identical manner to PMN cell migration with three notable differences. The chemokine was human recombinant CCL5 (100 ng/ml), the ECs were cultured on 5 μm pore transwell filters (Thermo Fisher Scientific), and a total of 4 × 10⁵ MNs were added to the apical chamber, followed by incubation at 37°C for 3 h following addition of MNs in the continued presence of CCL5.

To assess the ability of peptides based on CXCL8, CXCL12γ, and CCL5 to inhibit leukocyte migration, a range of concentrations (0.5, 5, 50, 500, and 5000 nM) was added to the basal chamber of the transwell system at the same time as the corresponding chemokine. Serum-free medium containing chemokine alone was used as the positive control and containing no chemokine as the negative control. The ability of the test peptide at inhibiting leukocyte migration was also compared with a peptide control (Table I; a sequence containing charged HS binding sites [i.e., Lys and Arg] to noncharged amino acids [i.e., Ala, Gly, or Ser]) using the HCMEC/D3 cell line.

To assess the role of HS in leukocyte migration, ECs in the transwell chambers were treated with heparanase I and III as described above and by others (39). Following incubation with heparanases, relevant chemokines were added and leukocyte transendothelial migration was carried out as before.

GAG-binding plates (Iduron) were coated with 25 μg/ml of HS (catalog number H7640-10MG; Sigma-Aldrich) in standard assay buffer (100 mM NaCl, 50 mM sodium acetate, 0.2% v/v Tween 20, pH 7.2) overnight at RT. After washing the plate three times with standard assay buffer, the wells were blocked with blocking solution (1% w/v BSA in PBS) for 1 h at 37°C and the wash step repeated. CXCL8 (PeproTech) was titrated for detection and used thereafter at 0.75 μg/ml in blocking solution. CXCL8 was incubated with different competitor concentrations of peptide (pCXCL8-1: 0.5, 5, 50, 500, 5000 nM) for 2 h at 37°C in triplicate. Unbound chemokine and peptide was removed by three wash steps followed by addition of a 220 ng/ml solution of biotinylated anti-human CXCL8 (PeproTech) in blocking solution for 1 h at 37°C. Following three more washes, 220 ng/ml ExtrAvidin-alkaline phosphatase (Sigma-Aldrich) in blocking solution was added to the wells for 30 min at 37°C. The plate was washed three times, and then 200 μl of a development reagent, SigmaFAST p-nitrophenyl phosphate (Sigma-Aldrich), was added and left to develop for 40 min at RT. The OD was then read at 405 nm using a spectrophotometer. Percentage inhibition of chemokine binding to HS was calculated as follows, with the control being standard assay buffer only:

ECs were detached from T75 flasks and incubated with labeled chemokine or peptide in FACs buffer (1% BSA/PBS) for 1 h on ice. The peptide was titrated and used at 5 μg/ml of FITC-labeled (f)pCXCL8-1 or control pCXCL8-1c (fpCXCL8-1c).

To assess CXCL8 binding to ECs, CXCL8 was labeled with Atto 425 (Bio-Techne, Abingdon, U.K.) according to the manufacturer’s instructions. The fluorescently labeled CXCL8 was titrated and subsequently used at 50 μg/ml (5.3 nM) in FACs buffer. For a control, cells were incubated with FACs buffer only. Binding to ECs was carried out as before, for 1 h on ice. To assess whether pCXCL8-1 could compete with CXCL8 for binding sites, 0.5, 5, and 50 nM of unlabeled peptide was added at the same time as fluorescent CXCL8.

To confirm whether CXCL8 and pCXCL8-1 were binding to HS, cells were pretreated with heparanase I and III as described previously (39), before the addition of fluorescently labeled CXCL8 or peptide.

Following the incubations, cells were washed by spinning down with FACs buffer twice at 500 × g for 5 min. Cells were then analyzed for binding using a NovoCyte flow cytometer and analyzed using NovoExpress software. Cells were gated on forward scatter, side scatter on all viable cells and then a second gate was placed on singlets. To assess binding, forward scatter area and FITC area were used to gate on positive and negative cells of peptide binding, and BV510 area was used to detect Atto425 emission of CXCL8 binding.

Synovial tissue was obtained from Keele University with full ethical approval from the Birmingham, East, North, and Solihull Research Ethics Committee (study ID: 11/WM/0035). Sections were cut at 10 μm using a cryostat and mounted onto polylysine slides (Thermo Fisher Scientific) and stored at −20°C until use.

Sections were equilibrated at RT for 30 min prior to use and then with ice-cold binding buffer (0.1% BSA in HBSS containing 10 mM HEPES) for 5 min. Labeled peptide and chemokine were added for 1 h in binding buffer and these were biotinylated pCXCL8-1 (bpCXCL8-1; 5 μg/ml), which was detected with streptavidin Alexa Fluor 488 (1:200; BioLegend, London, U.K.) and Atto 425 (Bio-Techne)–labeled CXCL8 [(5 μg/ml/0.53 nM); PeproTech]. In each case a double stain was performed with von Willebrand factor Ab (15.5 μg/ml; DAKO, Cambridge, U.K.) followed by a goat anti-rabbit Alexa Fluor 594 (1:400; Thermo Fisher Scientific). For controls, binding buffer in the absence of bpCXCL8-1 and Atto 425–labeled CXCL8 were used and rabbit IgG (15.5 μg/ml; DAKO) in the place of anti–von Willebrand factor.

To assess whether pCXCL8-1 could compete with CXCL8 for binding sites, an equimolar amount of unlabeled pCXCL8-1 (0.5 nM) was added at the same time as Atto 425–labeled CXCL8. To confirm whether CXCL8 was binding to HS, tissue was pretreated with heparanase I and III, as described previously (40), before the addition of labeled CXCL8. For a control, tissue was treated with binding buffer only.

Finally, sections were washed in binding buffer and then counterstained with DAPI (2 μg/ml in dH₂O) for 5 min. Sections were rinsed, mounted in Hydromount (Thermo Fisher Scientific), and visualized with a fluorescence light microscope (Leica DM LB), and analyzed with Cell^˄D software.

Experiments were conducted in 7–10 wk old inbred male C57BL/6 wild-type (WT) mice. Animals were housed and maintained under conventional management at animal facilities at Liverpool John Moores University, U.K. Procedures were performed with ethical approval from the Home Office, U.K., and were carried out under the project license PPL 40/3047.

Murine AIA was induced as described previously (31, 41). All reagents used for AIA induction were from Sigma-Aldrich. In short, mice were immunized s.c. with 1 mg/ml of methylated BSA (mBSA) emulsified with an equal volume of Freund’s complete adjuvant and injected i.p. with 100 μl heat-inactivated Bordetella pertussis toxin, the subsequent immune response was boosted 1 wk later. Then 3 wk after the initial immunization, AIA was induced by intra-articular injection of 10 mg/ml mBSA into the right knee joint and PBS into the left knee joint to serve as a control.

Briefly, 6 h post–arthritis induction, 10 μl of peptide (pCXCL8-1_aa) (5 μg) and control peptide (pCXCL8-1c_aa) were injected intra-articularly [0.5 ml monoject (29G) insulin syringe, BD Micro-Fine, Franklyn Lakes, NJ] into the right knee of 10 animals. At the end of experiments (day 3 and day 7 postinduction of AIA, n = 5) animals were sacrificed and joints were collected for histology.

Joints were fixed in neutral buffered formal saline and decalcified with 10% formic acid for 72 h before embedding in paraffin. Midsagittal sections (2 μm thickness) were cut and stained with H&E for scoring of degree of infiltration (normal = 0 to severe = 5), synovial hyperplasia in the lining layer (0–3), and cellular exudate in the joint space (0–3) as described previously (31, 41). Sections were stained with toluidine blue for scoring of degree of cartilage depletion (0–3) based on PG loss. Blind scoring was undertaken and all parameters were pooled to give an arthritic index (mean ± SEM).

Neutrophil infiltration was quantitated by counting the number of neutrophils in the synovium in H&E sections in five random fields of view at ×1000 magnification as described previously (31). Means and SEs were then calculated.

Using a mouse TNF-α ELISA Ready-SET-Go! Kit (eBioscience, Altrincham, U.K.) the serum concentration of TNF-α was measured according to the manufacturer’s instructions.

Statistical analysis was carried out on GraphPad Prism 5.0. A one-way ANOVA followed by Dunnett post hoc test was used for multiple comparisons and a two-tailed Student t test for comparisons of two variables. A p value <0.05 was deemed as significant.

Chemokines were chosen based on the cells they interact with and their roles in acute or chronic phases of inflammatory diseases. CXCL8 (Fig. 1A) is a potent chemoattractant of neutrophils, CCL5 (Fig. 1E) is largely associated with the chemotaxis of monocytes and T cells, and CXCL12γ (Fig. 1G) for lymphocytes; the dysregulation of each results in upregulated leukocyte migration. The residues of the chemokines implicated in GAG binding have been shown to be contained within the C-terminal α helix of CXCL8 (35) and CXCL12γ (36), and within the 40s loop of CCL5 (20). Therefore, our peptides were modeled on these particular regions without the inclusion of the tertiary chemokine structure. In addition, control peptides were synthesized whereby the positively charged amino acids such as lysine and arginine were substituted with noncharged amino acids such as alanine, glycine, and/or serine. The sequences can be seen in Table I.

A number of CXCL8 peptides were modeled to include varying amounts of HS-binding domains. A 10-aa peptide (pCXCL8-1) and a 15-aa peptide (pCXCL8-2) were synthesized based on the C-terminal α helix and included Lys⁶⁴, Lys⁶⁷, and Arg⁶⁸ (pCXCL8-1) plus the addition of Arg⁶⁰ in pCXCL8-2 as seen in Fig. 1B. CXCL8 also has another HS-binding domain located within the proximal loop, Lys²⁰. Therefore, a longer 54-aa peptide was synthesized to include all of these HS-binding sites (pCXCL8-3; Fig. 1C). As CXCL8 exists in both a monomeric and dimeric form, a further dimeric peptide was modeled to include both α helices held in place with a linker molecule as seen in Fig. 1D, comprising 41 aa in total (pCXCL8-4). Hence, this peptide therefore had a total of eight HS binding sites. The lead peptide pCXCL8-1 was modified for in vivo work (pCXCL8-1_aa) to protect it from proteolytic degradation and reduced half-life. Due to the direct intra-articular administration of the peptide, it was decided that the peptide would not need increased plasma half-life so instead it was opted to protect the peptide at the terminal regions. To do this, the peptide was amidated and acetylated, thereby altering the peptide ends into an uncharged state (Table I). This more closely mimics the native protein and these modifications would increase the metabolic stability of the peptide as well as its ability to resist enzymatic degradation.

Peptides based on CCL5 were chosen to include varying amounts of the 40s loop. Three peptides in total were designed. pCCL5-1 comprised 4 aa, ⁴⁴RKNR^47, and this was extended in the N-terminal direction to increase the overall mass to include Thr⁴³ (pCCL5-2; 5 aa) and Val⁴² (pCCL5-3; six residues) as seen in Fig. 1F and Table I.

The peptide based on CXCL12γ (pCXCL12-1) takes advantage of its enriched C-terminal α helix. The 30-aa helix contains 18 basic residues, 9 of which are clustered into four acknowledged HS-binding domains, and therefore the design for the peptide was simply to incorporate the 30 aa of this unique C terminus (Fig. 1H, Table I).

The affinity of the peptide interaction with HS and DS was determined by isothermal fluorescent titration. Peptides or intact chemokine were labeled on the N terminus with FITC (f) and fluorescence quenching was measured upon interaction with HS or DS. This was repeated for control peptides where the putative HS-binding sites had been altered to noncharged amino acids such as glycine, serine, and alanine (Table I). The resulting binding isotherms (Fig. 2) were analyzed by nonlinear regression giving the K_d values shown in Table II.

All measured fCXCL8 peptides gave a higher affinity (lower K_d) than WT fCXCL8. WT fCXCL8 bound to HS with a K_d of 128 nM whereas fpCXCL8-1 and -2 gave values of 15 and 61 nM respectively. The modified version of fpCXCL8-1 (fpCXCL8-1_aa) bound tighter than WT fCXCL8 although weaker than fpCXCL8-1 (by ∼4-fold), which is attributed to the modifications at the N and C termini. Interestingly, fpCXCL8-1, with three HS-binding domains, had a 4-fold higher affinity for HS than fpCXCL8-2, with four HS-binding domains. fpCXCL8-1 also exhibited some selectivity for HS over DS (15 nM compared with 44 nM), similar behavior to WT fCXCL8, whereas fpCXCL8-2 binds with similar affinity to both (61 nM compared with 52 nM). Control peptides for fpCXCL8-1, fpCXCL8-1_aa, and fpCXCL8-2 showed no significant interaction with HS (Fig. 2B–D), and therefore no K_d values could be calculated. Quenching around 10% and late saturation of FITC fluorescence is indicative of a very weak and/or a nonspecific interaction, thus highlighting the importance of the positively charged amino acids for GAG interaction.

The peptide based on CCL5, pCCL5-3, exhibited a strong affinity for HS with a K_d value of 8 nM. This peptide showed selectivity for HS over DS (8 nM compared with 148 nM). As can be seen in Fig. 2E, the control peptide showed <10% FITC quenching, again highlighting the importance of the positively charged amino acids associated with GAG binding.

Due to the unique C terminus of CXCL12γ, one would assume strong affinity for GAGs as confirmed by isothermal fluorescent titration (IFT) measurements. pCXCL12-1 gave K_d values of 2 and 3 nM to HS and DS, respectively (Fig. 2F). Interestingly, the control peptide for pCXCL12-1 showed some interaction with HS as indicated by the initial increase of the curve. However, this interaction appeared to be nonspecific due to the lack of saturation of FITC fluorescence. The control peptide did contain positively charged amino acids that are reported to be uninvolved with GAG binding; however, these could be giving rise to some interaction with HS seen in this study.

Before assessing the ability of peptides to bind to cellular HS and prevent leukocyte migration, it was necessary to determine the distribution of HS on ECs and elucidate the importance of this GAG during leukocyte transendothelial migration. The distribution of HS was examined by immunofluorescence as seen in Fig. 3A and 3B, which shows a mesh-work–like pattern of HS on the cellular surface. When the cells were pretreated with the enzymes heparanase I and III to degrade HS, the mesh-work–like pattern was lost, indicating a loss of HS on the cell surface (Fig. 3C, 3D). As a control, cells were stained with an IgM isotype.

To examine the function of HS in leukocyte transendothelial migration, ECs were pretreated as before with HS-degrading enzymes and leukocytes were assessed for chemotaxis in response to chemokines in the basal chamber. Chemokine concentrations were titrated (0–500 ng/ml) and used at 100 ng/ml. As can be seen in Fig. 3E, neutrophil migration was significantly reduced when treated with heparanase I and III compared with CXCL8 alone (p < 0.0001). The amount of neutrophil migration was equivalent to baseline in the absence of CXCL8 (data not shown). However, when assessing the effect of heparanase treatment on the migration of monocytes to CCL5 (100 ng/ml), a lesser role for HS was observed, although it was still significant (p = 0.0017). From Fig. 3F it can be seen that monocyte migration was reduced when HS was removed; however, this reduction was ∼40% compared with 80% for neutrophils.

It is well established that GAGs are important in the binding and presentation of chemokines on the luminal surface of ECs (18, 27). To assess whether this is the case for our EC transwell system, we pretreated ECs with heparanases and then tested the binding of fluorescently labeled CXCL8 or pCXCL8-1 by flow cytometry. As can be seen in Fig. 3G, CXCL8 bound to HCMEC/D3 cells significantly more than the control (FACs buffer only), exhibiting a total of ∼40% positive cells. When the HCMEC/D3s were pretreated with heparanase I and III, the number of CXCL8-positive cells was significantly reduced (p < 0.001). These data agree with findings in Fig. 3E, showing that HS is crucial for the binding and presentation of CXCL8. After confirming this was the case, we then wanted to see if the same was true for pCXCL8-1. This peptide exhibited almost 100% binding to HCMEC/D3 cells whereas the control pCXCL8-1c showed no binding (Fig. 3H); this again highlights the importance of the positively charged HS-binding domains in the peptide for EC interaction. This also agrees with IFT data that pCXCL8-1 has a higher affinity for HS than CXCL8, by the fact that pCXCL8-1 binds with a 2.5-fold increase in the percent of EC binding compared with CXCL8 (Fig. 3G, 3H). When the cells were treated with heparanases, the percentage positivity of pCXCL8-1 binding was significantly reduced (p < 0.001) (Fig. 3H).

To characterize the antichemotactic ability of peptides based on chemokines, they were tested in an in vitro model of inflammation. Using a transendothelial cell assay in two cell lines, the ability of the peptide to prevent leukocyte migration toward a chemokine stimulus was measured (Fig. 4). pCXCL8-1 significantly reduced neutrophil migration in the HCMEC/D3 cell line compared with the corresponding control peptide (Table I) at all concentrations of peptide apart from 5000 nM. The optimum concentration appeared to be 0.5 nM of peptide with a reduction of neutrophil migration up to 60% (p < 0.001) compared with the absence of peptide. The same peptide and concentration in the HBMEC cell line showed similar effects (p < 0.05) although more variability was observed.

To characterize the modified version of pCXCL8-1 (pCXCL8-1_aa), it was tested in the transwell system before being used in vivo. As indicated by the graphs, there was a significant reduction of neutrophil migration in both cell lines again between the 0.5–500 nM range of peptide. These results display a similarity to pCXCL8-1 data, confirming that the modifications have not affected the biological activity of the peptide.

The larger pCXCL8-2 was able to reduce neutrophil migration in both cell lines; however, this was less than observed for pCXCL8-1. Using HCMEC/D3 cells at 5 nM of pCXCL8-1, neutrophil migration was reduced by ∼60%, yet at the same concentration of pCXCL8-2 this reduction was only ∼30% compared with the no-peptide control; pCXCL8-2 was able to reduce neutrophil migration to the same extent as pCXCL8-1, although at a 10-fold higher concentration. The control peptide had no effect on neutrophil migration and showed significant differences compared with the test peptide. There was some variability between cell lines for this peptide, with significance being reached at 0.5 nM of peptide in the HBMEC cell line compared with a 10-fold higher concentration in the HCMEC/D3 cell line.

pCXCL8-3 significantly reduced neutrophil migration by ∼50% at 5, 50, and 500 nM (p < 0.001) in the HCMEC/D3 cell line, although less than pCXCL8-1 (Supplemental Fig. 1A). However, the pCXCL8-4 dimer was unable to inhibit CXCL8-induced chemotaxis in the HCMEC/D3 cell line (Supplemental Fig. 1B). Due to the complications associated with longer peptides during synthesis, the shorter pCXCL8-1 was chosen in preference as a lead peptide due to the simplicity of its synthesis making it advantageous therapeutically.

The peptide based on the unique C-terminal α helix of CXCL12γ showed a stark decrease of neutrophil migration when compared with its no-peptide control in the HCMEC/D3 cell line. This peptide reduced neutrophil migration back to baseline levels of ∼20% in the HCMEC/D3 cell line at 0.5 nM (p < 0.01). It demonstrated potency beyond that of others, requiring as little as 0.0005 nM to significantly reduce neutrophil migration by up to 40%. The peptide also inhibited neutrophil migration significantly more than using the corresponding control peptide at 0.5–50 nM. Similar reduction occurred in the HBMEC cell line although the pattern of inhibition with increasing peptide concentration differed between the two cell types. pCXCL12-1 was assessed for its ability to reduce CXCL8-mediated neutrophil migration across the HCMEC/D3 cell line. The peptide did not significantly reduce neutrophil migration in response to CXCL8, demonstrating some specificity.

pCCL5-3 was unable to reduce monocyte migration across the HCMEC/D3 cell line in response to CCL5 and did not differ compared with its corresponding control peptide. In agreement, the peptide also showed a similar trend in the HBMEC cell line although it did reach significance when compared with CCL5 alone at 5000 nM of peptide (p = 0.0039).

To examine if the CXCL8 peptide bound to ECs in situ, fluorescently labeled pCXCL8-1 was incubated with human RA synovium, and its binding compared with intact CXCL8 labeled with Atto 425. It has been shown previously that CXCL8 binding to HS is upregulated in the ECs of human RA synovium (30). To confirm where the positive staining was observed, a double stain was carried out using von Willebrand factor to highlight ECs (in red). pCXCL8-1 (Fig. 5A) and CXCL8 (Fig. 5D) were found to associate with blood vessels, and with the extracellular matrix (Fig. 5B, 5E). Staining was absent in the negative control sections in the absence of primary Ab, fluorescent CXCL8, or pCXCL8-1 (Fig. 5G, 5H).

Having confirmed that both the peptide pCXCL8-1 and CXCL8 bind to RA synovium, the question remained whether the peptide competes with CXCL8 for HS binding. Serial sections of human RA synovia were used to assess fluorescent CXCL8 binding (Fig. 6A), heparanase treatment prior to CXCL8 binding (Fig. 6D), and an equimolar concentration of pCXCL8-1 (0.5 nM) together with CXCL8 (Fig. 6G). The representative images seen in Fig. 6A and 6B show that CXCL8 (in green) is associated with ECs stained using von Willebrand Ab (in red). However, when the synovial sections were pretreated with heparanase I and III prior to the addition of CXCL8, there was loss of CXCL8 binding (Fig. 6D). Additionally, when equimolar concentrations of labeled CXCL8 and unlabeled pCXCL8-1 were added to the sections, a similar loss of CXCL8 binding was observed (Fig. 6G). This behavior is consistent with competitive binding of pCXCL8-1 and CXCL8 for HS.

The ability of pCXCL8-1 to inhibit binding of CXCL8 to HS was also evaluated using GAG-binding plates in an ELISA-like competition assay (Fig. 6M). As expected, given its highly positively charged nature, pCXCL8-1 clearly competed with CXCL8. The amount of CXCL8 binding was reduced by all concentrations of pCXCL8-1 and this reached significance at 50 nM (p < 0.05), reducing the amount of CXCL8 binding by at least 40%. To examine this competitive effect on ECs, fluorescently labeled CXCL8 and a range of concentrations of unlabeled pCXCL8-1 were added to HCMEC/D3 cells and the amount of CXCL8 binding was quantified by flow cytometry. As can be seen in Fig. 6N, the amount of CXCL8 binding to HCMEC/D3s was reduced in a dose-dependent manner over 0.5–50 nM (p < 0.001).

Based on its success at reducing neutrophil migration in vitro and its higher affinity for HS than WT CXCL8, pCXCL8-1 could possess anti-inflammatory properties. By competing with chemokines for GAG binding, this peptide could diminish the ability of chemokines to recruit leukocytes to a site of inflammation. Consequently, the anti-inflammatory and therapeutic capability of pCXCL8-1_aa was tested in an AIA mouse model. The AIA model shares many histopathological and clinical similarities to human RA (41, 42), making it a useful and relevant model to investigate our peptide. AIA was induced in the right stifle joint of mice and pCXCL8-1_aa and corresponding control peptide was injected 6 h later; when swelling routinely appears in AIA mice and so mimics the early condition in humans.

Histologically, AIA was characterized by infiltration of the synovial sublining by leukocytes including neutrophils and exudate containing leukocytes in the joint cavity, hyperplasia of the synovial lining, and loss of PGs from the articular cartilage (Table III; images not shown) as observed in H&E-stained sections (Fig. 7A, 7D, 7G, respectively). These changes did not occur in contralateral knee joints that were injected with PBS instead of mBSA and appeared histologically normal (Fig. 7C, 7F, 7I). The inflammation, exudate, and hyperplasia appeared less severe in pCXCL8-1_aa–treated mice when compared with the corresponding control peptide (Fig. 7B, 7E, 7H). To quantitate these changes, the parameters were scored as a measure of disease severity and differences between peptide- and control peptide–treated mice were evident. At day 7 postinjection, leukocyte infiltration (p = 0.03), cellular exudate (p = 0.012), and hyperplasia (p = 0.009) were reduced with pCXCL8-1_aa. When all histological parameters were pooled to give an overall arthritic index as a measure of overall disease severity, pCXCL8-1_aa significantly improved the arthritic score when compared with the control peptide at day 7 postinjection of mBSA (p = 0.008). Differences were not observed at day 3. A similar trend was observed for serum levels of TNF-α in AIA mice (Fig. 7J). Concentrations of TNF-α were reduced at both time points yet reached significance at day 7 postinjection of mBSA.

To determine if pCXCL8-1_aa could alter neutrophil infiltration into the synovium, the numbers of these cells were counted in synovial sections (Fig. 7K). At 7 d after arthritis induction, pCXCL8-1_aa reduced the numbers of neutrophils in the synovium from 5.6 ± 1.1 to 3.1 ± 0.8 with control peptide (p < 0.05). At 3 d after arthritis induction there was no significant effect.

A recent shift in the interest of the chemokine system as a therapeutic target has focused on GAGs. Their strong involvement in many disease areas shows potential for the development of glycan-targeting therapeutics. In this study, we have contributed to this development by showing that peptides based on chemokines can act as potential anti-inflammatories. Due to the presence of GAG-binding domains within the peptides and the undoubted interaction with negatively charged GAGs, two central questions were formed. Do the peptides based on chemokines actively compete with chemokines for GAG binding, thereby reducing the amount of chemokine being bound and presented; and, therefore, do they inhibit chemokine-induced leukocyte migration and reduce disease severity? Based on the necessity of chemokine binding to GAGs for exerting biological functions in vivo, there is a clear rationale for exploiting the chemokine–GAG interaction. Our peptide design approach takes advantage of the enriched HS-binding domains of chemokines and uses the discrete pocket of highly charged residues as novel anti-inflammatory peptides.

As our approach is intended to keep close to the natural WT protein, no new GAG-binding sites at different locations of the chemokine have been introduced, and the peptides exhibit sequence identity with each of the chemokines they are based upon. We intend that this would prevent interference with hydrogen bonding and Van der Waals forces to maintain the chemokines’ in-built specificity for the GAG. If maintenance of hydrogen bonding and hydrophobic forces can be achieved, this approach could be a novel and effective way to create efficient and selective GAG antagonists as this is what creates the specificity between chemokines and GAGs (43). Utilizing the HS-binding domain of chemokines as small chain peptides (lead peptide is 10 aa) is a new approach to combating the chemokine–GAG interaction. Previous approaches have incorporated much more of the chemokine structure with knocked-out GPCR activation and have included an unnatural sequence to increase the binding affinity of the peptide to GAGs, yet offer increased potential for off-target effects (44). A further advantage of the current novel therapeutic approach is the predictable mechanism by which the peptide works. The peptides based on chemokines interact with GAGs and compete with the WT chemokine for GAG binding. The peptide based on CXCL8 could compete with CXCL8 for binding to HS on binding plates, EC lines, and ECs in RA tissue. The binding was associated with EC GAGs as when either ECs or RA tissue was treated with an enzyme to degrade the HS, the binding was visibly reduced. Furthermore, control peptides lacking amino acids that interact with GAGs, such as lysine and arginine, bound significantly less to ECs, which agreed with binding isotherm data where the control peptides showed little interaction with HS. This suggests that the interaction of the peptides that do have the lysine and arginine amino acids are in fact interacting with the GAGs present on the EC, and these amino acids are important in the interaction. As GAG expression differs between tissues, especially in inflamed tissue where chemokine-binding sites on ECs are upregulated (40), the peptide could bind to GAGs preferentially in the inflamed tissue and prevent further chemokine-induced leukocyte migration.

As a control for our experiments, all peptides were subject to chemotaxis assays whereby the peptide alone was placed in the basal chamber and leukocytes were allowed to migrate across an endothelial layer. These peptides did not induce chemotaxis of leukocytes and leukocyte migration remained at baseline (data not shown). This suggests no functional role of the peptides other than to act as GAG-binding antagonists of WT chemokines. Our peptide design approach is clearly dependent upon the knowledge of the GAG-binding site in the target protein, which was based on published data. Using these data, we synthesized control peptides that lack the reported HS-binding residues and were replaced with a noncharged amino acid. These control peptides were fundamental in distinguishing anti-inflammatory properties of peptides in vitro and in vivo.

Our peptides were subjected to experiments to assess binding to soluble GAGs. As an initial assessment of peptide interaction with soluble GAGs, they were tested for their binding affinities to HS and DS by IFT. These two GAGs were chosen to show any selectivity by the peptide for particular GAGs. A binding isotherm of WT CXCL8 was measured and peptides based on CXCL8 were compared. In agreement with the literature, WT CXCL8 binds with higher affinity to HS than DS (21, 30). This preference is also apparent for the CCL5 peptide whereas the CXCL12 peptide shows equal preference for HS and DS. In all cases of peptides based on CXCL8, the peptides had a higher affinity than WT CXCL8 for HS and DS. These increased binding affinities of CXCL8 peptides compared with WT CXCL8 show that the simple peptide design using only identical sequences within chemokines is sufficient to induce a competitive antagonist, perhaps even posing as safe biological molecules based on their natural design. The high fluorescence quenching value (as indicated on the y-axis in Fig. 2) refers to more efficient fluorescence deactivation and thus to tighter binding of the GAG ligand. This is especially true of pCXCL12-1 where quenching occurs in the first additions of GAG ligand and reaches a quenching value up to 80%. The interaction could be electrostatic in nature; however, when the salt level of the solution is increased from physiological levels (137–400 mM), the affinity of this peptide for HS remains similar (data not shown). This is indicative of a specific peptide-HS interaction. Taken together, these data show a strong affinity of the peptides for GAGs (in the low nanomolar range) making them viable antagonistic molecules. Furthermore, different peptides show selectivity in targeting particular GAGs.

Using an Ab to an HS epitope, we found HS to be present on EC cell lines used in this study, occurring as a mesh-like structure. Confirmation of this expression was achieved with heparanases; when the ECs were incubated with both heparanase I and III, the expression of HS was reduced to background. HS on ECs has been widely reported to bind, concentrate, and present chemokines to leukocytes during extravasation (24, 27, 34, 45). Indeed, we have shown that CXCL8 binding to ECs is reduced when the cells are treated with heparanases alongside the fact that our control peptide, pCXCL8-1c, shows little interaction with ECs corroborates HS as a key player in chemokine activity. The implications of this are clearly shown in Fig. 3E, where neutrophil transmigration through an endothelial monolayer that has been pretreated with heparanases is vastly reduced.

Using our peptides based on chemokines, we were able to show a reduction of leukocyte migration in a cell-culture model of inflammation. By placing a chemokine in the basal chamber, the chemokine would be transcytosed or diffuse to the EC surface, thereby creating a chemotactic gradient for the leukocytes placed into the apical chamber (27). The peptides were tested in two separate cell lines to confirm results. Peptides based on CXCL8 (apart from pCXCL8-4) were able to reduce leukocyte transendothelial migration. Interestingly, in agreement with IFT binding affinity data, the smaller pCXCL8-1 with three HS binding sites showed increased efficacy and was most efficacious in the low nanomolar range unlike pCXCL8-2, which has four HS-binding domains and required a higher concentration to elicit similar results. pCXCL8-3 could inhibit CXCL8-induced neutrophil migration from 0.5 to 5000 nM; however, the optimum concentration was 500 nM and therefore much higher than pCXCL8-1. Conversely, pCXCL8-4 showed no antichemotactic ability in vitro. Our results suggest that the two lysine and one arginine residues in the final 10 aa of the C terminus of CXCL8 are especially important in its functional interaction with HS in terms of driving leukocyte migration. In addition, there is specificity in the interaction because adding the CXCL12γ peptide instead of CXCL8 peptide had no inhibitory effect on CXCL8-driven neutrophil migration, despite the increased affinity of this peptide for HS as shown by IFT. Therefore, the CXCL8 peptide–binding site on HS may be different from that of the CXCL12γ peptide.

Our peptide utilizing the C terminus of CXCL12γ was highly potent and showed a marked decrease in neutrophil transendothelial migration compared with a control. However, unlike some of the other peptides, a difference between cell lines was observed. In addition, pCXCL12-1 shows specificity in action as it was unable to inhibit CXCL8-induced neutrophil migration. The peptide based on CCL5 showed promising IFT data with a high affinity for HS; however, this did not translate into success in vitro. pCCL5-3 was unable to exhibit an antichemotactic effect on mononuclear cells using HCMEC/D3 cells and a very small effect using HMBECs. This could be due to a number of different reasons, one being the very small size of the peptide (773 kDa) or perhaps that mononuclear cell migration is through an alternative mechanism. As we have shown, heparanase treatment does not have as extensive a reduction of CCL5-mediated leukocyte migration as it does on CXCL8 migration. This suggests a role for perhaps another GAG or a molecule like the Duffy Ag receptor for chemokines, whose role is less clear in chemokine presentation to leukocytes.

The results show that CXCL8 binds to RA synovium, as does pCXCL8-1. The pattern of binding was similar to that of HS staining (30), being associated with blood vessels, and some was observed in the extracellular matrix. Studies have previously shown similar CXCL8 binding, which was attributable to the presence of HSPGs on ECs and the extracellular matrix (30). It was investigated if pCXCL8-1 could compete for CXCL8 binding to GAGs in cultured ECs and RA synovia. We clearly demonstrated a reduction in CXCL8 binding in the presence of peptide, moreover the binding is HS dependent as the use of heparanases also reduces CXCL8 binding. In addition, the competitive nature of pCXCL8-1 was shown in HS-binding plates in an ELISA-like assay, despite the varying concentrations used, which was necessitated by the different techniques.

Following effects in vitro and in situ, our lead peptide, pCXCL8-1, was evaluated in an AIA murine model after modifications to render it less susceptible to proteolytic degradation (pCXCL8-1_aa). Previous work has validated this model as suitable to test therapeutic agents that interfere with the CXCL8 axis (46) as the model’s pathology is driven by the mouse functional homolog of CXCL8. We have shown that pCXCL8-1_aa was able to reduce several parameters involved in arthritis, significantly reducing the overall arthritic score as a measure of disease severity. The reduction was characterized by reduced leukocyte infiltrate into the synovium and joint space, particularly including neutrophils. Neutrophils are considered influential cells in the development of inflammatory joint disease, as supported by several studies involving experimental models of arthritis. Neutrophils are found in high numbers within the human rheumatoid joint where they play a significant role in inflicting damage to the tissue, bone, and cartilage by secretion of proteases and toxic oxygen species, as well as driving further inflammation through secretion of cytokines, chemokines, and PGs (47). This suggests that targeting CXCL8-driven neutrophil extravasation with CXCL8 peptides is successful and beneficial. Furthermore, it is possible that inhibiting a mainly neutrophil-attracting chemokine (CXCL8) can have a direct or indirect effect on other cells and pathological features of RA. Hyperplasia of the synovial lining layer occurs in RA and is proposed to occur via the recruitment of monocytes in the sublining blood vessels, which then migrate and insert into the lining layer (48, 49). The monocytes are activated, differentiate into macrophages, and create hyperplasia. Monocyte adherence to the endothelium is increased by the presence of CXCL8 and so by reducing the amount of CXCL8 presentation, the peptide may be able to reduce monocyte recruitment and hence hyperplasia of the lining layer. By reducing neutrophil recruitment, it is possible that recruitment of other cell types via reduced chemokine and cytokine production such as TNF-α is also reduced. In this study we show that pCXCL8-1_aa reduces the levels of TNF-α in the circulation of AIA mice. TNF-α is a central cytokine involved in inflammation and tissue degradation in RA and blocking this cytokine is the major current therapy for the disease. The decrease in TNF-α levels suggest that administration of pCXCL8-1 can reduce systemic inflammation, which is a feature of RA, in addition to having local effects in the joint.

In summary, GAGs are an abundant class of highly sulfated polysaccharides that are known to drive and control protein activity by interacting with basic amino acids on the target protein. We have targeted this functional interaction as a potential way of antagonizing the target protein’s pathological role. Chemokines are clearly beneficial in the battle against infectious organisms and during wound healing after tissue injury, yet excessive and ongoing chemokine expression has been associated with inflammatory disorders, characterized by an inappropriate increase in leukocyte infiltration (50). The chemokine system, therefore, seems an attractive target for modulating such diseases. Interestingly, other species have already manipulated the chemokine system to their own benefit. For example, viruses and ticks have successfully used this strategy to evade the host’s immune system (51) by producing homologs of chemokines and chemokine receptors, thereby altering and controlling their activity. The chemokine system has been the subject of therapeutic interest for many years with previous strategies focusing on chemokine receptor antagonists (52). Unfortunately, the labors of this train of thought have been largely unsuccessful in clinical trials. More recent strategies have employed the use of the chemokine–GAG interaction. The recently developed CellJammer approach seeks to develop mutant chemokines with increased GAG-binding affinity and knocked-out GPCR function, thus creating an antagonist for WT chemokines. This approach has been successfully applied to create antagonists for CXCL8 and CCL2 (46, 53). CXCL8- and CCL2-based decoy molecules were shown to moderate inflammation in various mouse models such as ischemia and reperfusion, AIA, renal allograft rejection, and experimental autoimmune uveitis. Most recently, a C-terminal peptide based on CXCL9 was shown to inhibit neutrophil extravasation and monosodium urate crystal–induced gout in mice (54). The amounting evidence in support for the chemokine–GAG strategy is compelling. Our approach, although similar to these recent studies, is a simplified and more natural design. Our lead peptide is a mere 10 aa long, making it a quick, cheap, and easy molecule to synthesize therapeutically. In addition, the data gathered confirms the chemokine–GAG interaction as a biologically relevant target.

We thank Dr. Andrew Herman and the flow cytometry department of Bristol University, including for the loan of Atto 425. We thank the Peptide Protein Research, Ltd. team of peptide chemists for assistance in synthesis and analytical techniques and the team of A.J.K. at the University of Graz, Austria, for help with IFT experiments. We thank Dr. Becky Foster for helpful scientific discussions.

M.F. and R.B. are employees at Peptide Protein Research Ltd. A.K. is chief executive officer of Antagonis. The other authors have no financial conflicts of interest.