Peptide mimetics of immunoglobulin A (IgA) and FcαRI block IgA-induced human neutrophil activation and migration

Linear and cyclic peptide mimetics inhibit ligand binding to FcαRI

To determine whether peptide mimetics are able to block IgA binding to FcαRI, a panel of different soluble peptides was created based on described interaction sites of FcαRI and IgA (Supporting Information Fig. 1) 13, 14, 16, 17. Peptides varied in composition of amino acid residues, size (7- to 18-mers) and level of constraint (linear or cyclic peptides, Table 1 and 2). Linear peptides mimicking either FcαRI (FcαRI1-lin) or IgA (IgA1-lin) demonstrated blocking of neutrophil binding to IgA-coated wells with approximately 50% (Fig. 1A). CLIPS technology was used to cyclize the peptide and to thereby increase peptide half-life and stability 18, 19. Cyclic peptides were less effective in blocking binding as they showed blocking capacities between 5 and 25% (Fig. 1B).

Peptides reduce IgA-induced effector functions of neutrophils

Subsequently, it was investigated if blocking IgA-FcαRI interactions with IgA or FcαRI peptide mimetics inhibited IgA-induced effector functions of neutrophils. FcαRI1-lin and FcαRI8-CLIPS were able to block phagocytosis of IgA-coated beads with approximately 50% (Fig. 2A). Furthermore, H₂O₂ production was measured with an Amplex Red assay by adding neutrophils to IgA-coated wells. Several peptides were capable of blocking H₂O₂ production (Fig. 2B).

IgA-induced neutrophil migration is reduced in the presence of peptide mimetics in vitro and ex vivo

Next, we investigated whether peptide mimetics inhibit IgA-induced neutrophil migration. Linear peptides FcαRI1-lin and IgA1-lin blocked neutrophil migration to IgA-coated beads with 60–80% (Fig. 3A). CLIPS-peptides blocked migration of neutrophils between 0 and 80% (Fig. 3B). Additionally, we tested oxidated peptide variants and smaller CLIPS-peptide variants (7-13 amino-acids), but these peptides did not block IgA-induced neutrophil migration (Supporting Information Fig. 2). Importantly, neither linear nor CLIPS peptides had an effect on IL-8 induced chemotaxis (Supporting Information Fig. 3).

To mimic blocking of neutrophil migration toward aberrant IgA-antigen complexes in the skin, an ex vivo migration assay was established. Full thickness human skin grafts were injected with IgA-coated beads (or BSA-coated beads as control) and incubated for 24 h with fluorescently labeled neutrophils in the absence or presence of peptides with the best blocking capacities as demonstrated in previous in vitro migration experiments. No influx toward BSA-coated beads was observed, whereas massive influx of neutrophils toward the injected IgA-coated beads was observed (Fig 4A). The non-blocking peptide mimetic FcαRI9-ox did not inhibit IgA-induced migration (Fig. 4B). However, when IgA-peptide IgA1-lin or FcαRI-peptide FcαRI1-lin were added, neutrophil migration to IgA-beads was completely blocked (Fig. 4C). Furthermore, cyclic peptides IgA7-CLIPS and FcαRI8-CLIPS fully abrogated neutrophil migration as well (Fig. 4D).

Penetration of peptide mimetic IgA7-CLIPS in human skin

Ultimately, we aim to develop a topical therapy for patients with chronic IgA-blistering diseases, which requires an ointment containing peptides which can penetrate into the skin. Therefore, we next analysed the potential dermal delivery of one of the cyclic peptides with the best blocking capacities demonstrated in in vitro and ex vivo migration experiments. An ointment containing radioactive labelled IgA-peptides ([¹⁴C]IgA7-CLIPS) was applied to skin explants and penetration of the peptides was determined. Without a skin permeation enhancer, minimal penetration of peptides into skin was observed. However, in presence of the enhancer DDAIP, a dose dependent increase of the amount of penetrated peptide mimetic was observed (Fig. 5). Moreover, the concentration of [¹⁴C]IgA7-CLIPS in receptor fluid, which is a measure of systemic delivery, was negligible. In conclusion, topical application of a cream containing an enhancer resulted in local delivery of peptide mimetics.

Epitope mapping studies reveal sequences important for binding between IgA and FcαRI

Next to the generated peptides based on the known interaction sites of IgA and FcαRI, we used epitope mapping to identify potential novel peptides which block IgA-FcαRI interactions. Possibly, this second strategy allows us to discover novel interacting sequences, which are not described in current literature. Screening of a FcαRI-peptide library with IgA identified strongly binding peptides on 5 regions of FcαRI (Fig. 6A and B). These included all three regions known to be involved in binding IgA (CQAIREAYL, LKFWNETDP and YRIGHYRFR), and at least two regions unrelated to the known binding interface. In the reciprocal experiment, an IgA-peptide library was screened with both soluble FcαRI and 293T cells that had been transfected with FcαRI, with both experiments yielding similar results (Fig. 6C and D). Some binding was observed for IgA peptides which covered two out of three loops forming the previously described binding interface between IgA and FcαRI (LQGSQELPR and EALPAFTQ). However, within the complete dataset these were not the strongest binding regions. For region LEDLLLGSE no binding was observed, although this region is documented to form part of the binding interface.

Next, we investigated the specific residues important for the binding between FcαRI-mimetic GRYQACYRIGHYRFRCSD (FcαRI8-CLIPS) and IgA. This peptide is a strong binder and covers a region of FcαRI known to be important for the interaction with IgA. The analysis was performed by synthesizing a full positional replacement library for this sequence. By screening this mutated library for binding to IgA, the core binding region, critical residues and segments of the peptide that may be targets for future binding optimization studies could be identified. From this analysis region RIGHYRFR emerged as the core binding region, in which R87 and R89 showed the strongest effect on binding (Fig. 6E). When the identified core region was plotted on the structure of FcαRI and IgA, functional overlap was observed (Fig. 6F). In summary, epitope mapping studies revealed several novel sequences important for binding between IgA and FcαRI. Additionally, the core binding region of a promising FcαRI mimetic was discovered. Further studies are necessary to investigate if these peptides can block the IgA-FcαRI interaction and thereby reduce neutrophil infiltration during IgA-mediated blistering skin diseases.

Peptide library synthesis

The described interaction sites of FcαRI and IgA (Supporting Information Fig. 1) were used to create a panel of soluble peptides, based on the amino-acid sequence GRYQCQYRIGHYRFRYSD of FcαRI (Table 1) and on the amino-acid sequence SCMVGHEALPLAFTQKT of IgA (Table 2).

Synthesis of CLIPS-peptides and SS-peptides and peptide-microarrays

Synthesis of CLIPS-peptides, SS-peptides (oxidated variants) and peptide microarrays was performed as described previously 19, 43.

Pepscan-based ELISA

Binding of soluble FcαRI or IgA Ab to plate-bound peptides was tested with Pepscan-based ELISA's, which were adapted according to the method previously described 19, 43, 44. The samples were washed to remove unbound fragments after each incubation step. The 455-well credit-card format polypropylene cards containing the covalently linked IgA-peptides were incubated with blocking solution (4% horse serum, 5% ovalbumin (w/v) in PBS/1% Tween). Next, peptides were incubated with soluble FcαRI or 293T cells transfected with FcαRI and subsequently with mouse anti-human FcαRI IgG mAb (1 μg/ml; BD, Franklin Lakes, NJ). Then, peptides were incubated with rabbit anti-mouse IgG-HRP (1/1000, Dako, P0212) for 1 h at RT. Alternatively, after blocking, the covalently linked FcαRI-peptides were incubated with pooled human serum IgA (Cappel™, MP Biomedicals, Santa Ana, CA), and rabbit anti-human IgA-HRP (1 μg/mL, Dako, P0212) was added. The peroxidase substrate 2,2′-azino-di-3-ethylbenzthiazoline sulfonate (ABTS) and 2 μL of 3% H₂O₂ was added (1 h, RT). Colour development was measured, which was quantified with a charge coupled device (CCD)-camera and an image processing system. Values mostly ranged from 0 to 3000, a log scale similar to 1–3 of a standard 96-well plate ELISA-reader.

Epitope mapping

To identify the core binding region of peptide GRYQACYRIGHYRFRCSD (FcαRI8-CLIPS), a full positional replacement library was created. A set of variants was designed in which one of the 17 single residues (C residues are excluded) was replaced by one of the 19 other available L-amino acids, resulting in a dataset of 19 × 17 unique peptides. To identify the specific residues important for the interaction, this library was screened for binding to IgA as described above. The observed binding for each peptide variant was then analysed in comparison to the unmodified (“wt”) sequence. The amount of loss of binding observed for the variant, aggregated for each position in the sequence, is taken as a measure for the binding relevance for that residue. Within this library a relative value is then obtained for each position within the sequence which is used to determine the core binding region, critical residues and segments of the peptide that may be targets for future binding optimization studies.

Generation of soluble FcαRI recombinant protein

To generate a recombinant HIS-tagged soluble FcαRI protein, full length FcαRI inserted in pMG-FcαRI-IRES-hFcR γ-chain 45 was used as template. The extracellular part of FcαRI was amplified and cloned into pcDNA/v5-his topo vector (Invitrogen, Life Technologies Europe BV, Bleiswijk, The Netherlands), making use of a Kozak sequence (CACCATG) at the end of the 5′ primer (gtc agc acg gcc acc atg gac cc) and 3′ primer (tgt cga gct agc tta gat caa gtt ctg cgt c). The vector construct was verified by sequence analysis. For protein expression, 293T cells were stable transfected with pcDNA3.1/v5-sCD89 using FuGENE 6 reagent (Promega, Madison, USA), according to the manufacturer's recommendations and cultured under selection of hygromycin B. Protein expression of soluble FcαRI was verified by SDS-PAGE and Western blotting using an anti-FcαRI antibody (kindly provided by Prof. dr. C. van Kooten, LUMC, The Netherlands) 46. Furthermore, functionality of soluble FcαRI proteins was confirmed by FACS staining using soluble FcαRI protein as blocking agent for standard FcαRI staining on BAF3 transfected cells as described in Bracke et al. 47 with commercial anti-FcαRI antibody (mouse anti-human FcαRI-PE; BD Pharmingen, Breda, The Netherlands).

Isolation of human neutrophils

Neutrophils were isolated from heparinized peripheral human blood from healthy donors after informed consent was given, using standard Lymphoprep (Axis-shield, Dundee, Scotland) density gradient centrifugation. Erythrocytes were removed by hypotonic lysis, after which neutrophils were resuspended in RPMI 1640 (Gibco BRL, Paisley, UK) supplemented with 10% heat-inactivated fetal calf serum, L-glutamine, and antibiotics. Neutrophils were labeled for 30 min at 37°C with 1 μmol/L calcein-acetoxymethylester (Molecular Probes, Eugene, OR) for binding assays or with PKH-67 (Sigma-Aldrich, St. Louis, MO) for migration experiments according to the manufacturers’ instructions. Migration assays were performed in medium without fetal calf serum. Studies were performed according to the guidelines of the Medical Ethical Committee of VU University Medical Center, The Netherlands, in agreement with the Declaration of Helsinki.

Preparation of immunoglobulin-coated beads

N-hydroxysuccinimide (NHS)-activated sepharose beads (GE Healthcare, Uppsala, Sweden) were coated with pooled human serum IgA (300 μg/mL, Sigma-Aldrich) according to the manufacturer's instructions.

Ligand binding assay

Plates (Nunc-ImmunoMaxiSorp™, Roskilde, Denmark) were coated with IgA, or BSA (as control) (10 μg/mL, 3 h, 37°C), washed and pre-incubated with FcαRI-peptides (1 μg/mL, 20 min, 4°C). Wells were subsequently incubated with calcein labeled neutrophils (2.5 × 10⁵ cells/well) for 20 min (37°C). Alternatively, calcein labeled neutrophils were pre-incubated with IgA-peptides (1 μg/mL, 20 min, 4°C) and subsequently added to IgA or BSA coated wells. Plates were thoroughly washed to remove non-bound cells. Attached cells were lysed and fluorescence of supernatant was measured with a fluorimeter (485 nm excitation/520 nm emission filters; Fluostar Galaxy, BMG Labtechnologies, Offenburg, Germany), as measure of binding. All experiments were performed in triplicate.

Phagocytosis assay

Phagocytosis assays with fluorescent latex beads were performed as described previously 48. IgA-coated beads were added in ET ratio of 100 to neutrophils for 30 min at 37°C and fluorescence was measured with flow cytometry (FACS Calibur, BD Biosciences). Beads or neutrophils were pre-incubated with peptides (1 μg/mL) for 20 min on ice. Phagocytic index was calculated as the percentage of cells that had phagocytosed, multiplied by the geometric mean of fluorescent cells.

Amplex Red assay

Production of H₂O₂ was determined with Amplex™ Red hydrogen peroxide assays (Invitrogen, A-12221). ELISA plates (Nunc) were coated with IgA (1 ug/mL). Neutrophils were incubated in 100 μL Hepes⁺ buffer (132 nM NaCl, 20 mM hepes, 6 mM KCl, 1 mM MgSO₄·7H₂O, 1.2 mM K₂HPO₄·3H₂O, 1 mM CaCl₂, 0.5% BSA, 1 mg/mL glucose). Neutrophils or plate were pre-incubated with peptides (5 μg/mL) for 20 min on ice. Neutrophils were added to plates and Amplex Red reaction mix (200 μM Amplex red reagent and 4 U/mL horse radish peroxidase) was added. Fluorescence of the produced resorufin was measured every 1 min for 2 h at 37°C in a fluorimeter with an excitation of 550 nm and an emission of 590 nm.

Neutrophil migration assays

In vitro migration assays were performed as previously described 4. All experiments were performed in triplo. Migration towards IL-8 (30 ng/mL) was measured after pre-incubating neutrophils with peptides (1 μg/mL) or anti-IL-8 blocking antibody (10 μg/mL, Pharmingen).

For ex vivo human skin migration assays, full thickness mammary skin grafts (epidermis and dermis) were placed in an ex vivo tissue incubation chamber with the dermis face up 6, 49. IgA-coated beads were pre-incubated with FcαRI-peptides (1 μg/mL, 20 min, 4°C) and injected intracutaneously via the dermis. BSA-coated beads were used for control. Next, PKH-67 labeled neutrophils (4 × 10⁶ cells/well) were added on the dermis. Alternatively, IgA-coated beads were injected intracutaneously and PKH-67 labeled neutrophils (4 × 10⁶ cells/well) that were pre-incubated with IgA-peptides (1 μg/mL, 20 min, 4°C) were added onto the dermis. Cells were supplemented with IFN-γ to prevent early apoptosis (300 units/mL; Boehringer Ingelheim, Ingelheim am Rhein, Germany). Skin was incubated overnight at 37°C, after which biopsies of the injected skin were taken and snap frozen. Cryosections of 6 μm were analysed with a Nikon eclipse e800 (Nikon instruments Europe BV, Amsterdam, The Netherlands).

Ex vivo dermal absorption of [¹⁴C]IgA7-CLIPS through human skin

The experiments were based on the protocol ‘OECD Environmental Health and Safety Publications, Series on Testing and Assessment no. 28. Guidance document for the conduct of skin absorption studies, Paris, March 2004’ and performed by Netherlands Organisation for Applied Scientific Research (TNO). In brief, human skin membranes were placed in Franz diffusion cells (PermaGear Inc., Riegelsville, PA). The skin surface temperature was kept at 32°C, at ambient humidity. The receptor fluid consisted of PBS containing 0.01% sodium azide. Radioactive labeled peptide mimetic [¹⁴C]IgA7-CLIPS was made from Fmoc-[1-¹⁴C]glycine (Quotient Bioresearch (Radiochemicals) Ltd, Cardiff, UK). The material was purified by high performance liquid chromatography (HPLC) and the radiochemical purity was 99.3%. [¹⁴C]IgA7-CLIPS was applied topically to the skin membranes in a Cetomacrogol-cream (prepared at VU University Medical Center, Amsterdam, The Netherlands) in different concentrations and with or without skin permeation enhancer dodecyl-2-(N,N-dimethylamino)propionate (DDAIP) 50. The concentration and homogeneity of [¹⁴C]IgA7-CLIPS in the formulations was checked by taking additional weighed aliquots before and directly after dosing. After 24 h of application, the mass balance of the test substance was determined. Skin membranes were separated in epidermis and dermis using tweezers. Skin fractions were digested in a 1.5 M KOH solution with 20% ethanol for 24 h. Radioactivity in all samples was determined by liquid scintillation counting (LSC) on a Tri-Carb 3100TR liquid scintillation counter using QuantaSmart™ software (PerkinElmer, Waltham, MA). All counts were converted to DPM (disintegration per minute) using tSIE/AEC (transformed Spectral Index of external standards coupled to Automatic Efficiency Correction).

Statistical analysis

All analyses were performed using GraphPad Prism version 4.03 for Windows (GraphPad Software, San Diego, CA). Data are shown as mean ± standard deviation. Statistical differences were determined using two-tailed unpaired Student's t-test (comparing 2 groups) or ANOVA (comparing >2 groups). Significance was accepted when p< 0.05.