Chemokine CCR3 ligands-binding peptides derived from a random phage-epitope library

Immunology Letters 149 (2013) 19–29

Contents lists available at SciVerse ScienceDirect

Immunology Letters journal homepage: www.elsevier.com/locate/immlet

Chemokine CCR3 ligands-binding peptides derived from a random phage-epitope library Mehdi Houimel a,∗ , Luca Mazzucchelli b a b

Laboratoire de Microbiologie et d’Epidémiologie Vétérinaire, Institut Pasteur de Tunis, Tunisia Institute of Pathology Locarno, Locarno, Switzerland

a r t i c l e

i n f o

Article history: Received 27 September 2012 Received in revised form 6 November 2012 Accepted 14 November 2012 Available online 23 November 2012 Keywords: hCCR3 hCCL11 Phage display Peptides Antagonists

a b s t r a c t Eosinophils are major effectors cells implicated in a number of chronic inflammatory diseases in humans, particularly bronchial asthma and allergic rhinitis. The human chemokine receptor C-C receptor 3 (hCCR3) provides a mechanism for the recruitment of eosinophils into tissue and thus has recently become an attractive biological target for therapeutic intervention. In order to develop peptides antagonists of hCCR3-hCCL11 (human eotaxin) interactions, a random bacteriophage hexapeptide library was used to map structural features of hCCR3 by determining the epitopes of neutralizing anti-hCCR3 mAb 7B11. This mAb t is selective for hCCR3 and exhibit potent antagonist activity in receptor binding and functional assays. After three rounds of biopanning, four mAb7B11-binding peptides were identified from a 6-mer linear peptide library. The phage bearing the peptides showed specific binding to immobilized mAb 7B11 with over 94% of phages bound being competitively inhibited by free synthetic peptides. In FACScan analysis all selected phage peptides were able to strongly inhibit the binding of mAb 7B11 to hCCR3-transfected preB-300-19 murine cells. Furthermore, synthetic peptides of the corresponding phage epitopes were effective in blocking the antibody–hCCR3 interactions and to inhibit the binding of hCCL11 to hCCR3 transfectants. Chemically synthesized peptides CKGERF, FERKGK, SSMKVK and RHVSSQ, effectively competed for 125 I-hCCL11 binding to hCCR3 with IC50 ranging from 3.5 to 9.7 ␮M. Calcium release and chemotaxis of hCCR3 transfectants or human eosinophils were inhibited by all peptides in a dose-dependent manner. Furthermore, they showed inhibitory effects on chemotaxis of human eosinophils induced by hCCL11, hCCL5, hCCL7, hCCL8, and hCCL24. Specificities of all selected peptides were assessed with hCXCR1, hCXCR2, hCXCR3, and hCCR5 receptors. Peptides CKGERF and FERKGK showed inhibitory effects on eosinophil chemotaxis in a murine model of mCCL11-induced peritoneal eosinophilia. The development of peptides inhibiting the interactions between hCCR3 and its chemokine ligands will facilitate the development of small peptides antagonists with the hope of ameliorating chronic inflammatory diseases in humans. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Human eosinophils are pro-inflammatory granulocytes that play an important role in several inflammatory diseases, including asthma [1], allergic rhinitis [2], atopic dermatitis [3], eosinophilic gastroenteritis [4], and parasitic infections [5]. Eosinophils respond to a variety of CC chemokines including CCL11 (eotaxin), CCL24 (eotaxin-2), CCL26 (eotaxin-3), CCL5 (RANTES), CCL7 (MCP-2), CCL8 (MCP-3), and CCL9 (MCP-4) [6], through binding to the CC chemokine receptor-3 (CCR3), a seven transmembrane domain G

∗ Corresponding author at: Laboratoire de Microbiologie et d’Epidémiologie Vétérinaire, Institut Pasteur de Tunis, 13 Place Pasteur BP-74, 1002 Tunis-Belvédère, Tunisia. Tel.: +216 71 8783 022; fax: +216 71 791 833. E-mail address: [email protected] (M. Houimel). 0165-2478/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.imlet.2012.11.006

coupled receptor. Eosinophils express the highest amount of CCR3 and this is the dominant chemokine receptor on their surface [7]. CCR3 is also expressed on Th2 T cells [8,9], basophils [10], and mast cells [11]. The binding of CCL11 or related chemokines to CCR3 induces actin polymerization, chemotaxis, intracellular calcium flux in eosinophils, and the release of toxic reactive oxygen species (ROS) [12,13]. Blocking activation and migration of eosinophils should provide a new treatment option for inflammatory diseases. CCR3 receptor becomes an interesting target for drug intervention and antagonists development. Several peptide-derived chemokine receptor antagonists have been created by modification of the NH2 -terminal region of the chemokine as the N-terminus is crucial for activation to the chemokine receptor [14]. These modifications were constructed by deletion or extension of amino acids or by chemical modification on the NH2 -terminal residue of CC chemokines such as RANTES(3–68)

20

M. Houimel, L. Mazzucchelli / Immunology Letters 149 (2013) 19–29

[15], Met-RANTES [16], AOP-RANTES [17], and NNY-RANTES [18]. Whereas AOP-RANTES and RANTES(3–68) showed intrinsic activity (respiratory burst, chemotaxis and calcium release) for human eosinophils [15,7], Met-RANTES specifically inhibits intracellular calcium flux, actin polymerization and release of ROS of eosinophils following stimulation with RANTES, MCP-3 and eotaxin [19]. Met-RANTES antagonizes the response of eosinophils via CCR3 [19]. In a murine model of allergic airway disease, MetRANTES inhibited the influx of eosinophils in OVA-sensitized and -challenged mice [20]. Moreover, s.c. administration of MetRANTES inhibited murine eotaxin-induced eosinophil recruitment into sites of allergic inflammation in mouse skin by 68% [21]. These data suggest that Met-RANTES is able to prevent eosinophil recruitment into the lung and skin. Another antagonist of CCR3, termed Met-Ck␥7 was derived from the CCL18 or chemokine macrophage inflammatory protein (MIP-4) [22]. This antagonist, specifically binds to CCR3, and blocks eosinophil chemotaxis to CCL11 at concentrations as low as 1 nM. Finally, potent small nonpeptide molecules antagonists of CCR3 have been identified by functional screening of large numbers of organic compounds [23,24]. It was also demonstrated that the CXCR3 ligands, monokine induced by gamma IFN (Mig, CXCL9), IFN-inducible T cell (I-TAC, CXCL10), and IFN-inducible protein 10 (IP-10, CXCL11), are effective CCR3 antagonists [25]. Initial evidence that blocking of CCR3 results in inhibiting eosinophil activation in vitro and in vivo came from studies using inhibitory mAbs against CCR3 [26,27]. Anti-CCR3 monoclonal antibody (mAb) can significantly inhibit airway eosinophilia and mucus overproduction in asthmatic [28,29]. Among inhibitory mAbs, the anti-CCR3 mAb 7B11, that is selective for CCR3 and has the properties of a true receptor antagonist. mAb 7B11 blocked binding of various radiolabeled chemokines to either CCR3 transfectants, or eosinophils. Pretreatment of eosinophils with mAb 7B11 blocked chemotaxis and calcium flux induced by all CCR3 ligands [27]. Defining epitope (s) of the protective mAb 7B11 may help in rapid development of CCR3 chemokines peptide antagonists. In this study, we used phage peptide library to map the amino acid residues involved in the binding of anti-CCR3 mAb 7B11. The screening of a linear hexapeptide library displayed on M13 filamentous bacteriophages led to the identification and characterization of four peptides (CKGERF, FERKGK, SSMKVK and RHVSSQ), recognized specifically by mAb 7B11 and probably essential for the binding of chemokines ligands to the hCCR3 receptor. Further biological investigations, showed that selected peptides mimicking the binding epitope on hCCR3 for mAb 7B11, specifically blocked the binding of hCCL11 to hCCR3, and they inhibited the calcium release and the chemotaxis of transfectants expressing hCCR3 and human eosinophils. These peptides inhibited the chemotaxis of eosinophils induced by a broad panel of hCCR3 ligands. Specificities of derived peptides were confirmed on hCXCR1, hCXCR2, hCXCR3, and hCCR5 receptors. The incubation of peptides CKGERF, and FERKGK with murine CCL11 before administration to healthy Balb/C mice, induced a reduction in the numbers of eosinophils found in the lavages from the peritoneal cavities. The selected peptides were able to inhibit the biological activities of hCCR3 ligands and could be used as candidates for the developments of new drugs for treatment of inflammatory diseases.

from Southern Biotechnology Inc. (Birmingham, AL). Horseradish peroxidase-conjugated anti-M13 antibody was purchased from Amersham Biosciences (Sweden). All chemical reagents were purchased from Sigma Chemical Co. (Sigma, St Louis, MO). Cell culture media and reagents were obtained from Gibco BRL, Life Technologies France.

2. Materials and methods

2.5. Phage libraries and production of phage peptide

2.1. Antibodies and reagents

Phage hexapeptide library construction was previously described in [33]. It contains about 5 × 108 random hexapeptideencoding inserts. For phage production, TG1 bacteria containing phage were grown for 16 h at 30 ◦ C in 200 mL of 2× TY medium containing 15 ␮g tetracycline/mL and 1% glucose. The culture was

Monoclonal anti-human CCR3 receptor antibody (clone 7B11, IgG2a) was kind gifts of Millenium Pharmaceuticals, Inc. (Boston, MA). FITC-conjugated goat F(ab’)2 anti-mouse was obtained

2.2. Chemokines Human CCL11 (eotaxin-1), hCCL24 (eotaxin-2), hCCL5 (RANTES), hCCL7 (MCP-1), hCCL8 (MCP-2), hCXCL8 (IL-8) and hCXCL10 (IP-10) were chemically synthesized using tBoc solidphase chemistry purified by HPLC and analyzed by electron spray mass spectrometry as described by Clark-Lewis [30]. Murine recombinant eotaxin (mCCL11) was purchased from PreproTech (Rocky Hill, NJ). Interleukin-5 (IL-5) was purchased from R&D Systems (Minneapolis, MN). 2.3. Chemokine receptor transfectants Chemokine receptor-expressing cells were obtained by transfecting murine pre-B 300-19 cells with the corresponding encoding cDNA for human CCR3, hCXCR1, hCXCR2, hCXCR3, or hCCR5 cloned into the SmaI/XbaI site of SR␣puro vector as previously described in Loetscher [31]. Transfected cells were grown in humidified 5% CO2 atmosphere at 37 ◦ C and maintained in RPMI 1640 supplemented with 10% FCS, non-essential amino acids (0.1 mM), sodium pyruvate (1 mM), penicillin (100 U/mL), streptomycin (100 ␮g/mLl), L-glutamine (2 mM) and 1.5 ␮g/mL puromycin. Expression of receptors has been checked regularly by fluorescence active cell sorter analysis using specific antibodies and transfectants showing a decrease of expression have been replaced. 2.4. Preparation of human eosinophils Eosinophils were separated with the method of Hansel et al. [32]. Briefly, venous blood (50 to 100 mL) from healthy individuals was collected into 10 to 20 mL of acid-citrate-dextrose anticoagulant. White blood cells were obtained by sedimentation with 3% hydroxyethyl starch, layered onto Ficoll, and centrifuged at 500 × g for 30 minutes at 18 ◦ C. The mononuclear cell layer was discarded and the pellet containing granulocytes and red blood cells was washed in HBSS. Contaminating red blood cells were lysed by hypotonic lysis. The remaining granulocytes were washed, counted, and resuspended in 300 ␮L RPMI 1640 containing 2% FCS and 5 mM EDTA (RPMI/FCS/EDTA). Eosinophils were purified from neutrophils with use of immunomagnetic anti-CD16 antibodyconjugated beads (1 ␮L of beads per 2 × 106 neutrophils). After the addition of beads, cells were incubated at 4 ◦ C for 40 minutes before resuspension in 6 mL of RPMI/FCS/EDTA. The mixture was loaded onto a separation column positioned within a magnetic field and eluted with 40 mL of RPMI/FCS/EDTA. The CD16+ cells (i.e., neutrophils) were retained by the column, whereas the eluted eosinophils were collected, washed in RPMI 1640, counted, and then resuspended at 106 cells per milliliter. Eosinophil purity was >99% as assessed by microscopic examination in Kimura stain. The cells were cultured for 48 h (+37 ◦ C, 5% carbon dioxide) in RPMI 1640 medium with 10% FCS plus antibiotics.

M. Houimel, L. Mazzucchelli / Immunology Letters 149 (2013) 19–29

centrifuged at 4000 × g for 20 min at 4 ◦ C and the supernatant containing phage particles was precipitated twice with 0.2 vol. of 20% (w/v) polyethylene glycol (PEG) 6000, 2.5 M NaCl on ice for 1 h. After centrifugation at 4000 × g for 20 min at 4 ◦ C the pellet containing phages was resuspended with PBS pH 7.4. All produced phages were mixed, passed through a 0.45 ␮m pore filter and stored at 4 ◦ C. The titer of each production was estimated at 1.0 × 1012 transducing units (TU) per mL. 2.6. Selection of anti-hCCR3 mAb 7B11-binding peptide phage Selection procedures were performed with a phage peptide library displaying a random linear hexapeptide. Briefly, 1.0 × 1012 phage particles were added to immunotubes (Maxisorp, Nunc International, Denmark) coated with mAb 7B11 at 10 ␮g/mL concentration for the first round, 1 ␮g/mL and 0.1 ␮g/mL for the second and third round respectively, and incubated for 2 h at room temperature under rotation. Unbound phages were removed by washing the immunotube 20 times with PBS containing 0.05% Tween-20 (PBST), and 20 times with PBS. Bound phages were then eluted with 1 mL of 100 mM triethlyamine pH 12, immediately buffered with 1 mL of 1 M Tris–HCl, pH 7.4, propagated in exponentially growing E. coli strain TG1, and grown selectively with 15 ␮g/mL of tetracycline for 16 h at 30 ◦ C. Amplified phages were purified by precipitation with 20% PEG 6000, 2.5 M NaCl and used in the next cycle. After three rounds of selection, individual colonies were picked up randomly and subjected to analysis by phage ELISA and DNA sequencing, following amplification in E. coli TG1. 2.7. Phage binding assay In total, 94 phage-peptide clones were tested for binding to mAb 7B11 by monoclonal phage ELISA as previously described in [33]. Microtiter plates (Maxisorp, Nunc) were coated overnight at 4 ◦ C in PBS with 10 ␮g/mL of mAb 7B11 or a control mouse IgG and then blocked with 4% skim milk in PBS. The phage-peptides were added to the coated wells (1 × 1010 phages) and the plate was incubated for 2 h at room temperature under shaking and washed 3 times with 0.05% Tween-20/PBS. Bound phages were detected by incubation with HRP-conjugated anti-M13 antibody (diluted 1:5000) for 1 h at room temperature followed by washing and the addition of a peroxidase substrate 3,3 ,5,5 -tetramethylbenzidine in 100 mM sodium acetate pH6.0 containing 0.01% (v/v) H2 O2 . The reaction was stopped with 50 mL of 2 N H2 SO4 ant the absorbance was measured at 450 nm. The ELISA assay was performed in triplicate and repeated at least twice. 2.8. DNA sequencing and peptides synthesis The random peptide sequences of phage clones giving the strongest ELISA signal were deduced after PCR amplification and sequencing of the unique nucleotide region of the pIII gene as previously described [33]. The selected hexapeptides were synthesized using solid phase Fmoc chemistry with free amino- and carboxytermini and subjected to analytical HPLC and MALDI-TOF mass spectrometry. For bioassays, the peptides were dissolved in PBS pH 7.4 at 1 mg/mL and the pH was checked. 2.9. Peptide ELISA The reactivity of mAb 7B11 with the chemically synthesized peptides was tested by ELISA. Microtiter plates were coated with 0.1 mg/mL peptides in PBS pH 7.4, and then blocked with 4% skim milk in PBS. mAb 7B11 was added (5 ␮g/mL) and the plates were held for 2 h at room temperature. Bound antibody was detected

21

using HRP-conjugated anti-mouse IgG antibody with 3,3 ,5,5 tetramethylbenzidine as a peroxidase substrate, as described in “phage ELISA” section. As control we used the following linear hexapeptide MRFIAW. The ELISA assay was performed in triplicate and repeated at least twice.

2.10. Competitive inhibition assay For competition experiments, mAb 7B11was coated on microtiter plates at 1 ␮g/mL of concentration. Coated antibody was then incubated for 1 hr at room temperature with 100 ␮L of synthetic or control peptides at 1 mg/mL in PBS. Phage clones (1010 tu/well) were added for further 1 hr incubation without removing the peptide solutions. Bound phages were detected by incubation with HRP-conjugated anti-M13 antibody with 3,3 ,5,5 tetramethylbenzidine as a peroxidase substrate, as described under “phage binding assay”.

2.11. FACS analysis of competitive binding between selected peptides clones and mAb 7B11 Murine pre-B 300-19 cells stably expressing hCCR3 receptor (5 × 105 cells) was incubated for 1 h at 4 ◦ C in FACS binding buffer (PBS-2% FCS) containing 1 ␮g/mL of mAb 7B11. After washing, the cells were sequentially incubated with FITC-conjugated goat F(ab’)2 anti-mouse (1:200). For competition assays, the mAb 7B11 was preincubated for 1 h at 4 ◦ C with 1.0 × 1010 tu of the selected phage-peptide clones or 0.1 mg/mL of synthetic peptides before its incubation with cells. The competition was quantitatively analyzed by FACScan® (Becton Dickinson). The competition between the binding of mAb7B11 to hCCR3 transfectants was presented as the percent of Fluorescence as calculated by 100 × [(S − B)/(T − B)], where S (sample) is the fluorescence obtained after the preincubation of mAb 7B11 with indicated peptides, B is background binding, and T is total fluorescence without competitors. Background binding was obtained by incubating cells with secondary antibody conjugated with FITC. Duplicates were used throughout the experiments and the standard deviations were always < 10% of the mean. All experiments were repeated at least three times. 2.12. Binding competition using [125 I]hCCL11 as a chemokine ligand Human CCL11 (10 ␮g or 1.2 nmol) was labeled with 100 ␮Ci of ([125 I] sodium-iodide, (Amersham Biosciences) in 10 ␮g of Iodo-Gen (Pierce Chemical Company, Rochford, MI) coated tubes for 30 min at room temperature. Uncoupled iodine was removed by gel filtration on a PD-10 column (Amersham Biosciences). The specific activity of radiolabeled hCCL11 was 11,700 cpm/ng. 125 IhCCL11 binding assays were conducted with hCCR3 transfectants pretreated with a PBS binding buffer containing 0.1% bovine serum albumin (BSA) and 0.02% NaN3 at 25 ◦ C for 2 h. A final volume of 100 ␮L, containing 5 × 105 cells, 10 nM 125 I-hCCL11 (final concentration) and 1010 phage clones/tube or 50 ␮M of synthetic peptides made up in PBS binding buffer, was added to each tube and incubated at 4 ◦ C for 2 h. Cells were washed five times with 200 ␮L of ice-cold PBS binding buffer, which was removed by aspiration and the cpm counted using a Packard gamma-counter (Packard). The percent of cell-bound 125 I-hCCL11 was determined by the cpm in the presence of phage clones or peptides divided by the cpm in the presence of PBS binding buffer only. To investigate the specific competition binding, 125 I-hCCL11 was incubated for 2 h with serial concentration (0.01, 0.1, 1, 10, 100, and 200 ␮M) of the following peptides, CKGERF, FERKGK, SSMKVK and RHVSSQ, and added to 125 I-Na

22

M. Houimel, L. Mazzucchelli / Immunology Letters 149 (2013) 19–29

hCCR3 transfectants. All experiments were performed in duplicate and repeated three times.

cell count was carried out under a microscope after fixation and staining with Giemsa dye.

2.13. Inhibition of intracellular calcium mobilization

2.16. Statistics

The calcium mobilization was assayed in hCCR3 transfectants loaded with Fura-2 according to standard protocol described elsewhere [34]. Changes in the cytosolic free intracellular calcium concentration ([Ca2+ ]i ) were recorded after stimulation at 37 ◦ C with 10 nM of hCCL11 preincubated with various concentration of the selected peptides, CKGERF, FERKGK, SSMKVK and RHVSSQ. HCCR3 transfectants were placed in a continuously stirred tube and stimulated with 10 nM hCXCL8 prealably incubated for 1 h at room temperature with synthetic peptides at concentration of 0.1 ␮M, 1 ␮M, 10 ␮M, 100 ␮M and 200 ␮M. Data were recovered every 100 ms and the relative fluorescence ratio was determined as previously described [31].

Statistical significance between the groups was determined by applying an unpaired 2-tailed Student’s t-test. p value lower than 0.05 was considered as statistically significant.

2.14. Inhibition of eosinophil chemotaxis The effect of free synthetic peptides was analyzed in cell migration assays performed in 24-well tissue culture plates with 6.5 mm diameter transwell inserts with a 5.0 ␮m pore size (Corning Costar, Cambridge, MA). Human eosinophils, hCCL11 and synthetic peptides were diluted in RPMI-1640 supplemented with 10 mM Hepes, pH 7.4, and 1% pasteurized human plasma protein. Medium containing 5 × 104 cells (100 ␮L) were added to the top of the chamber, and hCCL11 (10 nM), in the presence of various concentrations of synthetic peptides (1, 10, 50, and 100 ␮M) was added to the lower chamber of the assay wells. The plates were incubated at 37 ◦ C for 1 h in 5% CO2 . The potential inhibition of hCCR3 specific chemokines hCCL5, and hCCL7, hCCL8 and hCCL24 on eosinophils was studied in the same manner. Cell migration of chemokine receptor-transfected murine pre-B 300-19 cells [chemokine receptors/ligands (hCXCR1/hCXCL8, hCXCR2/hCXCL8, hCXCR3/hCXCL10, and hCCR5/hCCL5)] were evaluated in a 24-well by using 5-␮m pore-size transwell filters (Costar) with or without peptides. For the inhibition of migration, 5 × 105 transfected cells were plated to the upper chamber, and chemokines, hCXCL8 (10 nM), hCXCL10 (10 nM), or hCCL5 (100 nM) or were added in the lower compartment in the presence of 100 ␮M of the following peptides, CKGERF, FERKGK, SSMKVK and RHVSSQ. The plates were incubated for 2 h at 37 ◦ C in 5% CO2 after which the migrated cells were counted using a haemocytometer. The data are expressed as the percent of cells that migrated to the lower compartments divided by the total number of cells seeded to the top compartments. Independent functional experiments were repeated three times.

2.15. Mouse model of eotaxin-induced cellular influx Balb/C mice were primed with 100 ng of IL-5 (an important determinant of the activation and survival of eosinophils [35,36]), injected intravenously in the lateral tail vein 30 min prior to the intraperitoneal injection of murine CCL11 preincubated with 100 ␮M of the following peptides, L7B11/1 (CKGERF), L7B11/2 (FERKGK), equimolar mixture of L7B11/1+ L7B11/2, or control peptide MRFIAW. The mice were euthanatized with CO2 and the peritoneal cells were harvested 24 h after challenge with 5 mL of phosphate-buffered saline containing 1.0% Fetal Calf Serum (Gibco BRL, France) and 10 U/mL heparin (Sigma Chemical, St. Louis, MO). The total number of peritoneal cells from a given mouse was counted with a hemocytometer. For this purpose, 50 ␮L of the peritoneal cell suspension (5 × 105 cells/mL) was smeared on a microscope slide after centrifugation in a Cytospin. A differential

3. Results 3.1. Affinity isolation of anti-hCCR3 mAb 7B11 binding phage-peptides To map the neutralizing epitope of human CCR3 receptor that was recognized specifically by antagonistic mAb 7B11, a linear pIII-6aa phage display library was screened with this purified mAb. Phage clones binding to anti-hCCR3 mAb 7B11 were selected by incubation of the peptide library with decreasing amounts of coated antibody for three rounds of biopanning. This selection scheme was advantageous for obtaining phage clones expressing hexapeptides with the highest affinity. To evaluate the efficacy of enrichment with phage clones expressing mAb 7B11-specific binding peptides at different rounds of selection, phages were titrated for transducing units in the inputs and outputs to determine the degree of selection. The total phage number bound to mAb 7B11a was increased from 3.0 × 104 in the first round to 1.0 × 109 in the third round (Table 1). Progressive enrichment of polyclonal phage-peptide binding to mAb 7B11 was assessed by ELISA (data not shown). Independent phage clones (94 bacterial clones) from the third round of selection were grown individually and their phage retested for binding to mAb 7B11 (IgG 2a) and to murine irrelevant IgG2a. Sixty-five pIII-6aa clones bound, specifically to mAb 7B11, but no reactivity with isotype control was observed (data not shown). The DNA inserts of the 20 phage clones giving the strongest ELISA signals were sequenced, and their amino acid sequences were deduced. Four highly positively charged peptide sequences with related motifs (G/F)ER(F/K), K(G/V)K and SS(M/Q) responsible for the specific binding to mAb 7B11 were identified (Table 1). All selected phage-peptides exhibited measurable interaction on direct ELISA, showing that the avidity effect due to the two or five peptide copies expressed in the phage-peptide may be the cause of the phage binding. The phage-CKGERF (frequency: 7/20) and phage-FERKGK (frequency: 6/20) shared the (G/F)ER(F/K) motif and were most probably preferentially selected due to a favourable interaction with the antibody mAb 7B11 and also for expression in E. coli, which resulted in an over-representation of theses phage clones in the amplified phage stock. These results indicate that the use of a stringent phage display strategy results in the identification of high specific binding peptides to mAb 7B11. The selected peptides did not share any primary sequence similarity with the N-terminal domain of hCCR3 (1–35): MTTSLDTVETFGTTSYYDDVGLLSEKADTRALMAQ. 3.2. Reactivity of mAb 7B11 with phage clones and synthetic peptides by ELISA Selected peptide phage clones were tested in a direct ELISA using the selecting mAb 7B11. As expected, the phage clones showing the same insert gave similarly high reactivity with the mAb 7B11 (Table 2). The four hexapeptides corresponding to the sequence of epitopes isolated with mAb 7B11, were chemically synthesized and tested also in ELISA. They all showed reactivity to the mAb 7B11, whereas the control peptide (MRFIAW) gave no substantial reactivity (Fig. 1).

M. Houimel, L. Mazzucchelli / Immunology Letters 149 (2013) 19–29

23

Table 1 Enrichment of selected phage-peptides by three rounds of biopanning. The linear and constrained hexapeptides phage display libraries were selected for binding to antihCCR3 neutralizing monoclonal antibody mAb 7B11 by three rounds of biopanning. The values are given in transducing units (tu), representing the amount of phage-peptides eluted from the mAb-coated tubes. Target

Round of selection

Input phage (tu)

Eluted phage (tu)

Ratio

mAb 7B11 anti-hCCR3/Linear (pIII-X6)

I II III

1.0 × 1012 1.0 × 1012 1.0 × 1012

3.0 × 104 1.0 × 107 1.0 × 109

3.0 × 10−8 1.0 × 10−5 1.0 × 10−4

2 1.8

Absorbance 450 nm

1.6 1.4 1.2 1 0.8 0.6 0.4 0.2

Control peptide

RHVSSQ

SSMKVK

FERKGK

CKGERF

0

Fig. 1. The mAb 7B11 binds synthetic peptides. The synthetic peptides (0.1 mg/mL) selected against mAb 7B11 were immobilized on microtiter plates and tested by ELISA with 5 ␮g/mL mAb7B11. Bound antibody was detected with HRP-conjugated anti-murine IgG antibody and then with substrate, absorbance was measured at 450 nm after 20 min. Histograms show absorbance means (n = 3) and error bars represent ± 1 SD of the mean. The control peptide (MRFIAW) tested at 0.1 mg/mL was not reactive.

3.3. Specificity of peptides binding to mAb 7B11

3.4. Inhibition of mAb binding to cell surface expressed hCCR3

Specificity of the interaction of mAb 7B11 with the isolated peptide phage clones was further assessed by inhibition experiments. Fig. 2 shows that the reactivity of mAb 7B11 with the corresponding phage clones was inhibited up to 84% at 0.1 mg/mL of peptide L7B11/1, and all chemically synthesized peptides were also able to strongly inhibit the binding of mAb 7B11 its corresponding epitope. The specific binding of the phage peptide clones was confirmed by inhibition of their binding to mAbs by an excess of the corresponding chemically synthesized free peptide (Fig. 2). Because of the very strong reactivity of the mAb 7B11 with the corresponding epitopes (ELISA Absorbance >1.9 at 20 min) it was not possible to test the peptides at concentrations > 0.1 mg/mL, and complete inhibition with the peptides could not be demonstrated. However, there was no evidence that the inhibition had reached a plateau at 0.1 mg/mL, so that 100% inhibition would likely have been achieved if higher peptide concentrations had been attainable. At the same concentration (0.1 mg/mL), the control peptide (MRFIAW) had no inhibition reactivity with mAb 7B11.

Specificity of the interactions of the selected peptide phage clones with mAb 7B11 was also tested by their capacity to inhibit binding of antibody to hCCR3. The competitive binding-inhibition studies by FACS analysis confirmed the data obtained by ELISA test (Fig. 2), and showed that the selected phage-peptide clones and synthetic free peptides competitively inhibit the binding of the mAb 7B11 to hCCR3 transfectants (Fig. 3). Inhibition levels obtained with phage-peptide clones were slightly important than those obtained with free peptides which reflect the influence of multivalent binding interaction between phage, which display five copies of the peptide on the protein pIII and mAb 7B11.

Table 2 Epitope sequences of phage-peptides isolated by direct screening of a linear hexapeptide library on anti-hCCR3 mAb 7B11. The sequences of peptide phage clones isolated by biopanning, frequency and reactivity by ELISA are indicated. Name of selected phage-peptide

Sequence

Frequency

Reactivitya

L7B11/1 L7B11/2 L7B11/3 L7B11/4

CKGERF FERKGK SSMKVK RHVSSQ

7 6 4 3

1.848 1.800 1.732 1.628

± ± ± ±

0.10 0.05 0.01 0.02

a Reactivity indicates absorbance measured at 450 nm after substraction of background. All phage-peptides with similar sequences were highly reactive.

3.5. Epitope-library-derived peptides inhibit binding of human CCL11 to its receptor We assessed the capacity of the peptides to inhibit the binding of hCCL11 to hCCR3 and analyzed their inhibitory effects on the binding of 125 I-radiolabeled hCCL11 to the pre-B 300-19 cells expressing hCCR3. Excess of unlabeled hCCL11 (1 ␮g) markedly suppressed the specific binding of 125 I-hCCL11. Competition assay analysis showed that both phage-peptide clones and peptides inhibited the binding of hCCL11 to hCCR3 transfectants (up to 86–90% (Fig. 4a). The specificity of inhibition was confirmed by different concentrations (0.01, 0.1, 1, 10, 100, 200 ␮M) of the peptides, CKGERF (IC50 = 1.0 ± 0.1 ␮M), FERKGK (IC50 = 8.0 ± 0.5 ␮M), SSMKVK (IC50 = 10 ± 2 ␮M) and RHVSSQ (IC50 = 89 ± 1 ␮M). The potency of peptides to displace 125 I-hCCL11 binding to hCCR3 showed variable IC50 ranging from 1.0 to 89 ␮M (Fig. 4b). Whatever the mechanism of inhibition, these results suggested that excess of peptides (200 ␮M) might compete with hCCR3-N-terminal domain for binding of the hCCL11 to the receptor on the surface of transfectants.

24

M. Houimel, L. Mazzucchelli / Immunology Letters 149 (2013) 19–29 2

Absorbance 450 nm

1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2

peptide-RHVSSQ

phage-RHVSSQ

peptide-SSMKVK

phage-SSMKVK

peptide-FERKGK

phage-FERKGK

peptide-CKGERF

phage-CKGERF

0

Phage-peptides +/- free peptide Fig. 2. Synthetic peptides inhibit binding of mAb 7b11 to phage clones. The mAb 7b11 immobilized on microtiter plates w incubated with phage clones with or without 0.1 mg/mL of different synthetic peptides, CKGERF, FERKGK, SSMKVK and RHVSSQ. The binding of selected phage clones (1010 tu/well) was evaluated by detection of bound phages by anti-M13 antibody horseradish peroxidase conjugate. Absorbance was measured at 450 nm after 20 min. Data shown are absorbance means of triplicate samples ± SD.

3.6. Functional effects of anti-hCCR3-binding peptides on calcium mobilization inhibition We next investigated whether results from binding experiments were correlated with functional responses by using cytosolic free calcium change as a functional readout of cell activation. To determine the ability of the mAb 7B11 binding peptides to antagonize the calcium mobilization induced by hCCL11 (10 nM), the synthetic peptides (1–1000 ␮M) were added to Fura-2-loaded hCCR3 transfectants 60 s before stimulation with 10 nM hCCL11. As shown in Fig. 5, the synthetic peptides inhibited the transient rise in calcium induced by hCCL11 in a dose dependent manner. The IC50 was calculated for the following peptides, CKGERF (IC50 = 4.4 ± 0.2 ␮M), FERKGK (IC50 = 9.5 ± 1 ␮M), SSMKVK

(IC50 = 11.7 ± 1 ␮M), and RHVSSQ (IC50 = 25 ± 1 ␮M). The hCCL11 reactivity was unaffected by 1 mM of control peptide (MRFIAW). Stimulation of the transfectants with the peptides alone (1 mM) or control peptide failed to induce [Ca2+ ]i mobilization (data not shown). 3.7. Inhibition of eosinphils migration by the selected peptides The effects of peptides on hCCR3-mediated chemotaxis were examined on human eosinophils. Different concentrations of each peptide were co-incubated with hCCL11 in the lower compartment of a transwell migration assay. The data presented in Fig. 6 show that migration of human eosinophils attracted to hCCL11 were inhibited by all tested peptides with IC50 ranging from 10 to

100

% Fluorescence

80

60

40

control peptide

control phage

peptide-RHVSSQ

phage-RHVSSQ

peptide-SSMKVK

phage-SSMKVK

peptide-FERKGK

phage-FERKGK

peptide-CKGERF

phage-CKGERF

0

mAb 7B11 (antihCCR3)

20

Fig. 3. Phage clones and synthetic peptides inhibit binding of mAb 7B11 to hCCR3. The peptide-phage clones (1010 TU) selected on mAb 7B11 and their corresponding synthetic peptides (0.1 mg/mL) were able inhibit the interaction of mAb 7B11 with hCCR3 receptor. The mAb 7B11 was preincubated for 1 h at 4 ◦ C with the selected phage clones or the corresponding synthetic peptides in 100 ␮L of FACS buffer and added to human CCR3 transfectants for additional 1 h at 4 ◦ C. Labeled cells were analyzed by flow cytometry and expressed as % of relative fluorescence. The control phage and the control peptide (MRFIAW) did not show any inhibition. Data shown are % florescence means of duplicate samples ± SD.

M. Houimel, L. Mazzucchelli / Immunology Letters 149 (2013) 19–29

a

25

100

% Binding of 125I-hCCL11

90 80 70 60 50 40 30 20 10 control peptide

control phage

peptide-RHVSSQ

phage-RHVSSQ

peptideSSMKVK

phage-SSMKVK

peptideFERKGK

phage-FERKGK

peptide-CKGERF

phage-CKGERF

mAb 7B11 (antihCCR3)

hCCL11 (1 µg)

125I-hCCL11

0

125I-hCCL11 bound (%)

b 100 90 80 70 60 50 40 30 20 10 0 0,01

SSMKVK FERKGK CKGERF RHVSSQ

0,1

1

10

100

1000

Peptide concentration ( µM) Fig. 4. (a) Inhibition of hCCL11 binding to by phage clones and synthetic peptides. The peptide-phage clones (1010 TU) selected on mAb 7B11 and their corresponding synthetic peptides (0.1 mg/mL) were able to inhibit the interaction of 125 I-hCCL11 with hCCR3 receptor. The 125 I-hCCL11 was preincubated with the selected phage clones or the corresponding synthetic peptides and added to hCCR3 transfectants. Cell-associated radioactivity was determined after washing. Cold hCCL11, and mAb B11 served as positive controls. Control phage and control peptide (MRFIAW) did not show any inhibition. (b) Binding of 125 I-hCCL11 to hCCR3 transfectants was inhibited in a dosedependent manner by indicated concentrations of selected peptides. The IC50 was determined for each peptide: CKGERF (IC50 = 1.0 ± 0.1 ␮M), FERKGK (IC50 = 8.0 ± 0.5 ␮M), SSMKVK (IC50 = 10 ± 2 ␮M) and RHVSSQ (IC50 = 89 ± 1 ␮M). All data present percentage means of raw cpm of duplicate samples ± SD.

103 ␮M, CKGERF (IC50 = 10 ± 1 ␮M), FERKGK (IC50 = 11.9 ± 0.1 ␮M), SSMKVK (IC50 = 89.7 ± 0.3 ␮M), and RHVSSQ (IC50 = 103 ± 2 ␮M). Consistent with our previous findings on calcium release inhibition (Fig. 5), preincubation of hCCL11 with synthetic peptides significantly decreased chemotactic activity compared to incubation with control peptide (MRFIAW). Both of these results are in agreement with the 125 I-hCCL11competition (Fig. 4). 3.8. Selectivity of peptides inhibitory activity The specificity of all derived peptides was also investigated by studying their inhibitory effect on chemotaxis of murine pre-B cells stably transfected with hCXCR1, hCXCR2, hCXCR3, or hCCR5 to their corresponding chemokine hCXCL8 (IL-8), hCXCL10 (IP-10), or hCCL5 (RANTES) respectively. As can be seen in Fig. 7a, peptides inhibit the migration of only hCCR5transfectants, but they have no effect on migration of transfectants bearing hCXCR1, hCXCR2 or hCXCR3 to their appropriate chemokines. As several chemokines bind to hCCR3, we also studied the ability of these peptides to inhibit eosinophils migration induced by natural ligands other than hCCL11. As shown in Fig. 7b, migration induced by hCCL5, hCCL7, hCCL8, or hCCL24 was markedly inhibited by prior incubation of these chemokines with 100 ␮M of peptides. Taken together, these

data demonstrate the feasibility of antagonizing the hCCR3 ligands by selected peptides. 3.9. Inhibition of eosinophils migrationand accumulation in mice To evaluate the potency of L7B11/1 and L7B11/2 peptides in vivo, we developed a mCCL11-induced eosiophilia influx model in mice. When injected intrapritoneally, mCCL11 causes massive eosinophils infiltration into the peritoneal cavity, and the leukocyte accumulation reaches a plateau 24 h later (data not shown). Initially, we pre-incubated 100 ␮M of peptides, CKGERF, and FERKGK or control peptide MRFIAW, with mCCL11 (10 ␮g/kg) before intraperitoneal injection in Balb/C mice. Twenty four hours later, the peritoneal cavity was washed with PBS and the extent and types of leukocyte infiltration was counted. As shown in Fig. 8, when preincubated at a dose of 100 ␮M, the peptides caused a reduction in the numbers of eosinophils (p < 0.01) found in the lavage from the peritoneal cavities of the mice injected with mCCL11. In this model, there are limited macrophage or PMN contributions to the total cells in the lavage fluid, and no changes in the numbers of these cells were observed (data not shown). A profound reduction of eosinophils accumulation was observed in both CKGERFand FERKGK-treated mice, whereas the control peptide MRFIAW

26

M. Houimel, L. Mazzucchelli / Immunology Letters 149 (2013) 19–29

% Inhihibition of calcium release

100

Control peptide SSMKVK

90

FERKGK

80

CKGERF 70

RHVSSQ

60 50 40 30 20 10 0

1

10

100

Peptide to mAb 7B11 anti-hCCR3 (µM) Fig. 5. Inhibition by synthetic peptides of hCCL11 induced intracellular calcium release. hCCR3-transfected cells were tested for their ability to release intracellular calcium in response of various concentrations of synthetic peptides. Various concentrations of synthetic peptides (1 to 200 ␮M) were added to Fura-2 loaded hCCR3 transfectants 60 s before stimulation with 10 nM of hCCL11. The calculated IC50 was varying form 4 ␮M to 25 ␮M, CKGERF (IC50 = 4.4 ± 0.2 ␮M), FERKGK (IC50 = 9.5 ± 1 ␮M), SSMKVK (IC50 = 11.7 ± 1 ␮M), and RHVSSQ (IC50 = 25 ± 1 ␮M). A control peptide (MRFIAW) was used as a negative control. Responses are expressed in percent of the maximal calcium release induced by ionophore Fura-2 and represent means ± SEM of three independent experiments.

failed to show any inhibitory effect. The number of eosinophils was slightly reduced in mice having received concomitant injection of CKGERF and FERKGK peptides. The dose of 100 ␮M of peptides led to a high degree of inhibition, whereas 10 ␮M had no effect (data not shown). 4. Discussion Phage display is a cheap and rapid method to map epitope of the hCCR3 that is involved in specific interaction with the protective antibody mAb 7B11. The identification of epitopes is essential for hCCR3-chemokines antagonists and/or–agonists development. Since linear continuous epitopes are often six amino acids in length, the screening of phage display hexapeptide library on anti-hCCR3

6000 Number of migrated eosinophils

control peptide SSMKVK

5000

FERKGK CKGERF

4000

RHVSSQ

3000 2000 1000 0 1

10

10 0

100 0

Peptide concentration (µM) Fig. 6. Inhibition of eosinophils chemotaxis by synthetic peptides. Human eosinophils (5 × 104 cells) were assayed for their ability to migrate towards hCCL11 (10 nM) in the presence of various concentrations of synthetic peptides (1–200 ␮M) selected against mAb 7B11. A control peptide (MRFIAW) was used as a negative control. The data represent the mean numbers of migrated eosinophils in duplicates for each peptide concentration ± SD.

mAb7B11 is favourable to affinity select peptides that exactly match the primary structure of the epitope on hCCR3. In this paper, we describe the identification of two series of peptides that interact with the hCCL11 (eotaxin-1) and related chemokines, hCCL5 (RANTES), hCCL8 (MCP-2), or hCCL24 (eotaxin-2), and prevent their binding to hCCR3 receptor. The first series of selected peptides contains the motif (G/F)ER(F/K) that compete with hCCL11 binding to the hCCR3 receptor, CKGERF (IC50 = 1.0 ± 0.1 ␮M) and FERKGK (IC50 = 8.0 ± 0.5 ␮M). The second, less potent, series of peptides contains the motif SS(M/Q) motif and inhibit the binding of hCCL11 to hCCR3 with low affinity, SSMKVK (IC50 = 10 ± 2 ␮M) and RHVSSQ (IC50 = 89 ± 1 ␮M). Although neither series of antagonist peptides share any primary sequence similarity to N-terminal domain of hCCR3 (1–35): MTTSLDTVETFGTTSYYDDVGLLSEKADTRALMAQ, it is tempting to speculate the peptides adopt a structure that presents the appropriate functional epitopes in the correct spatial orientation to mimic the key contact points on hCCR3 important for the hCCL11 binding. In a previous biochemical and biophysical studies Ye et al. [37] demonstrated that the extracellular domain of hCCR3 plays an important role in hCCL11 binding, and showed that the isolated peptide corresponding to the N-terminal extracellular domain of hCCR3 called CCR3 [1–34] binds to hCCL11 with 80 ± 40 ␮M affinity. Although linear or cyclic peptides corresponding to the three extracellular loops of CCR3 (designated E1, E2, and E3) do not bind to hCCL11 [37]. Binding and structure analyses between CCL11 and the Nterminal peptide fragment CCR3[1–34], revealed several amino acids which seem to be important for the binding to CCR3 [37]: in the N-loop region (Leu (L)-13, Ala (A)-14, Asn (N)-15, Arg-(R)16, Lys (K)-17, and Ile (I)-18), in the 310 -helical turn (Leu (L)-20, Gln (Q)-21, and Arg (R)-22) and the ␤3-strand (Ala (A)-46, Lys (K)-47, Asp (D)-48, Ile (I)-49, and Ala (A)-51). In the same study [37], the authors et al. showed that the N-loop, 310 -helical turn, and the ␤2–␤3 hairpin together form an extended groove on one face of the CCL11 structure. The base of the groove is defined by hydrophobic residues (L-13, A-14, I-18, L-45, and I-49), whereas the edges of the groove are highly hydrophilic (defined by N-12, N-15, R-16, K17, Q-21, R-22, K-47, and D-48). The authors suggested that the surface electrostatic potential of eotaxin is strongly positive in the vicinity of the groove [37]. Therefore, it appears likely that the negatively charged N-terminal receptor sequence of CCR3 (Y-16, Y-17, D-18, D-19) lies in the groove of CCL11, forming hydrophobic contacts with the base of the groove, and electrostatic interactions with residues on the edges of the groove. Although the N-terminus of chemokine receptors seems to be important for chemokine recognition, it is noteworthy that a second region of the chemokine receptor is required for high affinity binding. This second region is most likely to be formed by one or more of the extracellular loops of the receptor but may also involve one or more of the transmembrane segments if the chemokine enters into the transmembrane bundle of the receptor [37]. Chimeras of hCCR3 with the chemokine receptor hCCR1 indicate that the N-terminal segment and the E3 loop of CCR3 both participate in hCCL11 recognition [38]. These two elements are predicted to be linked by a disulfide bond [39]. Furthermore, over the same range of concentrations as they complexes the hCCL11 (eotaxin) for the hCCR3 binding, all characterized peptides are able to block calcium release and eosinophils chemotaxis in a dose-response manner. The peptides were able to block several key biological steps of the inflammatory process such as calcium release and chemotaxis. The peptides abrogate dose-dependently the release of intracellular calcium in human eosinophils induced by hCCL11 with an IC50 values ranging between 4.4 ␮M and 25 ␮M. The IC50 was calculated for each the following peptides, CKGERF (IC50 = 4.4 ± 0.2 ␮M), FERKGK (IC50 = 9.5 ± 1 ␮M), SSMKVK (IC50 = 11.7 ± 1 ␮M), and

M. Houimel, L. Mazzucchelli / Immunology Letters 149 (2013) 19–29

a

27

Number of migrated cells

40000 35000 30000 25000 20000 15000 10000

hCCR5 + SSMKVK

hCCR5 + FERKGK

hCCR5

hCCR5 + CKGERF

hCXCR3 + RHVSSQ

hCXCR3 + SSMKVK

hCXCR3 + FERKGK

hCXCR3

hCXCR3 + CKGERF

hCXCR2 + RHVSSQ

hCCR3 + FERKGK

hCXCR2 + SSMKVK

hCXCR2

hCXCR2 + CKGERF

hCXCR1 + RHVSSQ

hCXCR1 + SSMKVK

hCXCR1 + FERKGK

hCXCR1

hCXCR1 + CKGERF

hCCR3 + RHVSSQ

hCCR3 + SSMKVK

hCCR3 + FERKGK

6000

5000

4000

3000

2000

hCCL24 + CKGERF

hCCL24 + FERKGK

hCCL8 + CKGERF

hCCL8 + FERKGK

hCCL7 + FERKGK

hCCL7 + CKGERF

hCCL5 + FERKGK

hCCL5 + CKGERF

control peptide

0

hCCL11 + FERKGK

1000

hCCL11 + CKGERF

Number of migrated eosinophils

b

hCCR3

0

hCCR3 + CKGERF

5000

Fig. 7. Chemotaxis inhibition by selected peptides. (a) Transwell migration assays were performed using hCCR3-, hCXCR1-, hCXCR2-, hCXCR3-, and hCCR5-transfected murine preB-300-19 cells. hCCL11 (10 nM), hCCL7 (10 nM), hCXCL8 (10 nM), hCXCL10 (10 nM), hCCL24 (10 nM), and hCCL5 (100 nM) were incubated for 15 min with an excess (100 ␮M) of CKGERF or FERKGK prior cells stimulation and migration. (b) Effect of the peptides on eosinophils migration induced by hCCR3 ligands. hCCR3 specific chemokines, hCCL11 (10 nM), hCCL7 (10 nM), hCCL8 (10 nM), hCCL24 (10 nM), and hCCL5 (100 nM) were incubated with 100 ␮M of indicated peptides. Human eosinophils were counted after migration. The data represent the mean numbers of migrated cells in duplicates for each chemokine ± SD.

Eosinophls Counts

70000 60000 50000 40000

*

*

*

mCCL11 + CKGERF

mCCL11 + FERKGK

mCCL11 + CKGERF + FERKGK

30000 20000 10000 0 IL-5 primed mice not treated with mCCL11

mCCL11

mCCL11 + contol peptide

Fig. 8. Inhibition mice eotaxin-induced peritoneal eosinophilia by L7B11/1 (CKGERF) and L7B11/2 (FERKGK) peptides. Mice were primed intravenously with IL-5 (100 ng) in the lateral tail vein 30 min prior to the injection of mCCL11. Mice were administered systemically via intraperitoneal injection with murine CCL11 preincubated with L7B11/1, L7B11/2, mixture of L7B11/1 + L7B11/2 or with control peptide MRFIAW at concentration of 100 ␮M each. Twenty-four hours later, the mice were euthanatized with CO2 and the peritoneal cells were harvested with PBS containing 1% FCS, and 10 U/mL heparin. The infiltrated eosinophils were counted microscopically as described in Materials and Methods. Data are expressed as mean ± SEM of eosinophil counts in five high power fields. Each bar represents the mean of eosinophil count of mice ± SD. * indicates statistical significant differences (p < 0.01) compared with mice injected with control peptide (MRFIAW).

28

M. Houimel, L. Mazzucchelli / Immunology Letters 149 (2013) 19–29

RHVSSQ (IC50 = 25 ± 1 ␮M). These results support the view that the inhibition of calcium release by peptides was clearly linked to the amino acid residues involved in the binding of hCCL11. Using human eosinophils, our peptides inhibited hCCL11mediated chemotaxis with IC50 values ranging from 10 to 103 ␮M, CKGERF (IC50 = 10 ± 1 ␮M), FERKGK (IC50 = 11.9 ± 0.1 ␮M), SSMKVK (IC50 = 89.7 ± 0.3 ␮M), and RHVSSQ (IC50 = 103 ± 2 ␮M). The selected peptides demonstrate high antagonistic potency, since they were able to inhibit human eosinophils chemotaxis induced by optimal concentrations of hCCL7, hCCL8, hCCL5 and hCCL24. These results correlated with the effectiveness of these peptides to compete with hCCL11 and structurally related hCCR3 ligands suggesting the presence of similar binding domains on hCCR3 to these chemokines. The most probable mechanism of action is the binding of the selected peptides to a broad spectrum of hCCR3 chemokine ligands which are shared with other chemokine receptors such as hCCL5. Indeed, the evidence that blocking peptides are able to inhibit hCCL5-promoted migration of hCCR5 transfectants. Interestingly, elements of the N-loop appear to be structurally conserved between CCL5 and CCL11 and may provide the necessary interactions for CCR3 selectivity [40,41]. The selected peptides showed ␮M levels of inhibition on binding of hCCL11 to hCCR3, on calcium release and on eosinophils chemotaxis. These results are predictable because short peptides usually cannot hold conformation needed for high-affinity biomolecular interaction. A general affinity maturation strategy would involve an initial selection of relatively low affinity binding motif derived from the selected peptide sequences, which will then be used as template to design secondary library. Inhibition of eosinophil chemotaxis was further verified in an in vivo inflammation in mice model. In fact, the administration of peptides CKGERF and FERKGK led to significant suppression of mCCL11-induced eosinophils infiltration to the peritoneal cavity. The incomplete inhibition, even at higher dose of 100 ␮M of peptide CKGERF, peptide FERKGK or of an equimolar mixture of CKGERF + FERKGK, may reflect that eosinophils infiltration may not depend solely on mCCL11. The reduced activity of the hCCL11 blocking peptides on mCCL11 could be explained by the low sequence homology (61%) between murine and human CCL11. The fact that the used hCCL11 blocking peptides did not completely abrogate the chemotactic activity of mouse eosinophils supports the possibility that such additional chemotactic factors act independently of mCCR3. In agreement with these observations, it was shown that classical chemoattractants such as C5a and FMLP had an additive effect on CCR-3-mediated transendothelial chemotaxis of eosinophils [42]. The murine eosinophils are known to migrate in response to human and murine eotaxin-1 (mCCL11) and -2 (mCCL24), but not human eotaxin-3 (hCCL26) [43]. As a chemoattractant both in vivo and in vitro, mCCL11 exhibits potent activity on eosinophils, but not on mononuclear cells or neutrophils. The cytokine IL-5 was injected to Balb/C mice to prime murine eosinophils to respond optimally to the mCCL11. As Shown in Fig. 08, the number of eosinophils increased by 177% after the injection of 10 nM of mCCL11 in comparison with the number of eosinophils primed with IL-5. This result indicates that murine eosinophils receive signals for chemotaxis directly from the mCCL11, and shows a clear synergy between IL-5 and mCCL11. The preincubation of mCCL11 with an excess of blocking peptides CKGERF, and FERKGK has the ability to inhibit the chemotaxis of murine eosinophils. Intraperitoneal administration of mCCL11 + CKGERF led to an inhibitory activity of 74.5% at 24 h. In addition the peptide FERKGK was also able to inhibit the chemotaxis of murine eosinophils (67% versus control peptide MRFIAW 0%). A significant inhibition (82.2%) of migrated murine eosinophils was observed when mCCL11 was preincubated with a mixture of both peptides.

The in vitro and in vivo finding provide evidence that inhibition of hCCR3 by the selected peptides is sufficient to prevent eosinophils chemotaxis mediated by hCCL11 and confirms that CCR3 receptor plays a major role in eosinophils migration. The main feature of hCCR3 antagonists is to displace the natural ligands from its receptor; therefore cells are not activated at the site of inflammation. Therefore, several issues must be addressed before drawing conclusions on the potential therapeutic applications of peptides. For instance, a functional effect was obtained only at relatively high concentrations, most likely because short peptides might fail to mimic a complex structure. Using hexamer approach is quite limited for clinical applications. The in vivo experiment confirmed the limitations (low affinity, low specificity, and low serum persistence) of such peptides. These limitations could be improved by the use of multivalent peptide (PEGylated peptide) to increase the half-live and the serum persistence of the injected peptides. In this study, we presented a phage display approach to develop peptides as antagonists against the interaction between hCCR3 and its ligands. We demonstrated that the selected peptides are able to bind and to inhibit the interaction between hCCL11 and hCCR3. CCR3 chemokine ligands binding peptides may represent respectable alternatives due to a better tolerance, and may open new approaches for the development of therapeutic drugs by using the peptide sequences as scaffolds for the rational design of new chemical antagonist molecules.

Acknowledgements We thank Drs Pius Loetsher and Marco Baggiolini for helpful discussions. This work was supported by grants from the Swiss National Science Foundation.

References [1] Kroegel C, Liu MC, Hubbard WC, Lichtenstein LM, Bochner BS. Blood and bronchoalveolar eosinophils in allergic subjects after segmental antigen challenge: surface phenotype, density heterogeneity, and prostanoid production. J Allergy Clin Immunol 1994;93:725–34. [2] Durham SR. Mechanisms of mucosal inflammation in the nose and lungs. Clin Exp Allergy 1998;28:11–6. [3] Leung DY. Pathogenesis of atopic dermatitis. J Allergy Clin Immunol 1999;104:99–108. [4] Bischoff SC, Mayer JH, Manns MP. Allergy and the gut. Int Arch Allergy Immunol 2000;121:270–83. [5] Meeusen EN, Balic A. Do eosinophils have a role in the killing of helminth parasites? Parasitol Today 2000;16:95–101. [6] Ying S, Meng Q, Zeibecoglou K, Robinson DS, Macfarlane A, Humbert M, et al. Eosinophil chemotactic chemokines (eotaxin, eotaxin-2, RANTES, monocyte chemoattractant protein-3 (MCP-3), and MCP-4), and C-C chemokine receptor 3 expression in bronchial biopsies from atopic and nonatopic (Intrinsic) asthmatics. J Immunol 1999;163:6321–9. [7] Elsner J, Mack M, Bruhl H, Dulkys Y, Kimmig D, Simmons G, et al. Differential activation of CC chemokine receptors by AOP-RANTES. J Biol Chem 2000;275:7787–94. [8] Sallusto F, Mackay CR, Lanzavecchia A. Selective expression of the eotaxin receptor CCR3 by human T helper 2 cells. Science 1997;277:2005–7. [9] Gerber BO, Zanni MP, Uguccioni M, Loetscher M, Mackay CR, Pichler WJ, et al. Functional expression of the eotaxin receptor CCR3 in T lymphocytes colocalizing with eosinophils. Curr Biol 1997;7:836–43. [10] Uguccioni M, Mackay CR, Ochensberger B, Loetscher P, Rhis S, LaRosa GJ, et al. High expression of the chemokine receptor CCR3 in human blood basophils. Role in activation by eotaxin, MCP-4, and other chemokines. J Clin Invest 1997;100:1137–43. [11] Romagnani P, De Paulis A, Beltrame C, Annunziato F, Dente V, Maggi E, et al. Tryptase-chymase double-positive human mast cells express the eotaxin receptor CCR3 and are attracted by CCR3-binding chemokines. Am J Pathol 1999;155:1195–204. [12] Dulkys Y, Schramm G, Kimmig D, Knoss S, Weyergraf A, Kapp A, et al. Detection of mRNA for eotaxin-2 and eotaxin-3 in human dermal fibroblasts and their distinct activation profile on human eosinophils. J Invest Dermatol 2001;116:498–505. [13] Zimmermann N, Conkright JJ, Rothenberg ME. CC chemokine receptor-3 undergoes prolonged ligand-induced internalization. J Biol Chem 1999;274:12611–8.

M. Houimel, L. Mazzucchelli / Immunology Letters 149 (2013) 19–29 [14] Loetscher P, Pellegrino A, Gong JH, Mattioli I, Loetscher M, Bardi G, et al. The ligands of CXC chemokine receptor 3, I-TAC, Mig, and IP10, are natural antagonists for CCR3. J Biol Chem 2001;276:2986–91. [15] Struyf S, De Meester I, Scharpe S, Lenaerts JP, Menten P, Wang JM, et al. Natural truncation of RANTES abolishes signaling through the CC chemokine receptors CCR1 and CCR3, impairs its chemotactic potency and generates a CC chemokine inhibitor. Eur J Immunol 1998;28:1262–71. [16] Proudfoot AE, Power CA, Hoogewerf AJ, Montjovent MO, Borlat F, Offord RE, et al. Extension of recombinant human RANTES by the retention of the initiating methionine produces a potent antagonist. J Biol Chem 1996;271:2599–603. [17] Simmons G, Clapham PR, Picard L, Offord RE, Rosenkilde MM, Schwartz TW, et al. Potent inhibition of HIV-1 infectivity in macrophages and lymphocytes by a novel CCR5 antagonist. Science 1997;276:276–9. [18] Mosier DE, Picchio GR, Gulizia RJ, Sabbe R, Poignard P, Picard L, et al. Highly potent RANTES analogues either prevent CCR5-using human immunodeficiency virus type 1 infection in vivo or rapidly select for CXCR4-using variants. J Virol 1999;73:3544–50. [19] Elsner J, Petering H, Hochstetter R, Kimmig D, Wells TN, Kapp A, et al. The CC chemokine antagonist Met-RANTES inhibits eosinophil effector functions through the chemokine receptors CCR1 and CCR3. Eur J Immunol 1997;27:2892–8. [20] Gonzalo JA, Lloyd CM, Wen D, Albar JP, Wells TN, Proudfoot A, et al. The coordinated action of CC chemokines in the lung orchestrates allergic inflammation and airway hyperresponsiveness. J Exp Med 1998;188:157–67. [21] Teixeira MM, Wells TN, Lukacs NW, Proudfoot AE, Kunkel SL, Williams TJ, et al. Chemokine-induced eosinophil recruitment. Evidence of a role for endogenous eotaxin in an in vivo allergy model in mouse skin. J Clin Invest 1997;100:1657–66. [22] Nibbs RJ, Salcedo TW, Campbell JD, Yao XT, Li Y, Nardelli B, et al. C–C chemokine receptor 3 antagonism by the beta-chemokine macrophage inflammatory protein 4, a property strongly enhanced by an amino-terminal alanine-methionine swap. J Immunol 2000;164:1488–97. [23] Sabroe I, Peck MJ, Van Keulen BJ, Jorritsma A, Simmons G, Clapham PR, et al. A small molecule antagonist of chemokine receptors CCR1 and CCR3. Potent inhibition of eosinophil function and CCR3-mediated HIlV-1 entry. J Biol Chem 2000;275:25985–92. [24] White JR, Lee JM, Dede K, Imburgia CS, Jurewicz AJ, Chan G, et al. Identification of potent, selective non-peptide CC chemokine receptor-3 antagonist that inhibits eotaxin-, eotaxin-2-, and monocyte chemotactic protein-4-induced eosinophil migration. J Biol Chem 2000;275:36626–31. [25] Loetscher P, Pellegrino A, Gong JH, Mattioli I, Loetscher M, Bardi G, et al. The Ligands of CXC Chemokine Receptor 3, I-TAC, Mig, and IP10, Are Natural Antagonists for CCR3. J Biol Chem 2000;276:2986–91. [26] Sabroe I, Conroy DM, Gerard NP, Li Y, Collins PD, Post TW, et al. Cloning and characterization of the guinea pig eosinophil eotaxin receptor, C-C chemokine receptor-3: blockade using a monoclonal antibody in vivo. J Immunol 1998;161:6139–47. [27] Heath H, Qin S, Rao P, Wu L, LaRosa G, Kassam N, et al. Chemokine receptor usage by human eosinophils. The importance of CCR3 demonstrated using an antagonistic monoclonal antibody. J Clin Invest 1997;99:178–84. [28] Das AM, Vaddi KG, Solomon KA, Krauthauser C, Jiang X, McIntyre KW, et al. Selective inhibition of eosinophil influx into the lung by small molecule CC

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

[40]

[41]

[42]

[43]

29

chemokine receptor 3 antagonists in mouse models of allergic inflammation. J Pharmacol Exp Ther 2006;318(July (1)):411–7. Shen HH, Xu F, Zhang GS, Wang SB, Xu WH. CCR3 monoclonal antibody inhibits airway eosinophilic inflammation and mucus overproduction in a mouse model of asthma. Acta Pharmacol Sin 2006;27(December (12)):1594–9. Clark-Lewis I, Moser B, Walz A, Baggiolini M, Scott GJ, Aebersold R. Chemical synthesis, purification, and characterization of two inflammatory proteins, neutrophil activating peptide 1 (Interleukin-8) and neutrophil activating peptide. Biochemistry 1991;30:3128–35. Loetscher P, Seitz M, Clark-Lewis I, Baggiolini M, Moser B. Both interleukin8 receptors independently mediate chemotaxis. Jurkat cells transfected with IL-8R1 or IL-8R2 migrate in response to IL-8, GRO alpha and NAP-2. FEBS Lett 1994;341:187–92. Hansel TT, Pound JD, Pilling D, Kitas GD, Salmon M, Gentle TA, et al. Purification of human blood eosinophils by negative selection using immunomagnetic beads. J Immunol Methods 1989;122:97–103. Houimel M, Mach JP, Corthesy-Theulaz I, Corthesy B, Fisch I. New inhibitors of Helicobacter pylori urease holoenzyme selected from phage-displayed peptide libraries. Eur J Biochem 1999;262:774–80. von Tscharner V, Prod’hom B, Baggiolini M, Reuter H. Ion channels in human neutrophils activated by a rise in free cytosolic calcium concentration. Nature 1986;324:369–72. Stern M, Meagher L, Savill J, Haslett C. Apoptosis in human eosinophils. Programmed cell death in the eosinophil leads to phagocytosis by macrophages and is modulated by IL-5. J Immunol 1992 Jun 1;148(11):3543–9. Sugaya H, Abe T, Yoshimura K. Eosinophils in the cerebrospinal fluid of mice infected with Angiostrongylus cantonensis are resistant to apoptosis. Int J Parasitol 2001;31(December (14)):1649–58. Ye J, Kohli LL, Stone MJ. Characterization of binding between the chemokine eotaxin and peptides derived from the chemokine receptor CCR3. J Biol Chem 2000;275:27250–7. Pease JE, Wang J, Ponath PD, Murphy PM. The N-terminal extracellular segments of the chemokine receptors CCR1 and CCR3 are determinants for MIP-1alpha and eotaxin binding, respectively, but a second domain is essential for efficient receptor activation. J Biol Chem 1998;273(32): 19972–6. Blanpain C, Lee B, Vakili J, Doranz BJ, Govaerts C, Migeotte I, et al. Extracellular cysteines of CCR5 are required for chemokine binding, but dispensable for HIV1 coreceptor activity. J Biol Chem 1999;274(27):18902–8. Crump MP, Rajarathnam K, Kim KS, Clark-Lewis I, Sykes BD. Solution structure of eotaxin, a chemokine that selectively recruits eosinophils in allergic inflammation. J Biol Chem 1998;273:22471–9. Skelton NJ, Aspiras F, Ogez J, Schall TJ. Proton NMR assignments and solution conformation of RANTES, a chemokine of the C-C type. Biochemistry 1995;34:5329–42. Kitayama JM, Carr MW, Roth SJ, Buccola J, Springer TA. Contrasting responses to multiple chemotactic stimuli in transendothelial migration: heterologous desensitization in neutrophils and augmentation of migration in eosinophils. J Immunol 1997;158:2340–9. Borchers MT, Ansay T, DeSalle R, Daugherty BL, Shen H, Metzger M, et al. In vitro assessment of chemokine receptor-ligand interactions mediating mouse eosinophil migration. J Leukoc Biol 2002;71(June (6)):1033–41.