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. 2017 Oct;47(10):1835-1845.
doi: 10.1002/eji.201646782. Epub 2017 Sep 6.

Peptide Mimetics of Immunoglobulin A (IgA) and FcαRI Block IgA-induced Human Neutrophil Activation and Migration

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Free PMC article

Peptide Mimetics of Immunoglobulin A (IgA) and FcαRI Block IgA-induced Human Neutrophil Activation and Migration

Marieke H Heineke et al. Eur J Immunol. .
Free PMC article

Abstract

The cross-linking of the IgA Fc receptor (FcαRI) by IgA induces release of the chemoattractant LTB4, thereby recruiting neutrophils in a positive feedback loop. IgA autoantibodies of patients with autoimmune blistering skin diseases therefore induce massive recruitment of neutrophils, resulting in severe tissue damage. To interfere with neutrophil mobilization and reduce disease morbidity, we developed a panel of specific peptides mimicking either IgA or FcαRI sequences. CLIPS technology was used to stabilize three-dimensional structures and to increase peptides' half-life. IgA and FcαRI peptides reduced phagocytosis of IgA-coated beads, as well as IgA-induced ROS production and neutrophil migration in in vitro and ex vivo (human skin) experiments. Since topical application would be the preferential route of administration, Cetomacrogol cream containing an IgA CLIPS peptide was developed. In the presence of a skin permeation enhancer, peptides in this cream were shown to penetrate the skin, while not diffusing systemically. Finally, epitope mapping was used to discover sequences important for binding between IgA and FcαRI. In conclusion, a cream containing IgA or FcαRI peptide mimetics, which block IgA-induced neutrophil activation and migration in the skin may have therapeutic potential for patients with IgA-mediated blistering skin diseases.

Keywords: Autoimmune blistering skin disease; CD89; Epitope mapping; Fc alpha receptor; Immunoglobulin A; Neutrophil; Peptide mimetic.

Figures

Figure 1
Figure 1
Peptide mimetics inhibit IgA binding to FcαRI. Percentage of ligand binding between IgA and FcαRI (on neutrophils), either in the presence or absence of peptide mimetics. Fluorescently labelled human neutrophils, isolated from blood, were added to IgA‐coated plates, and the number of neutrophils attaching to the plate was measured with a fluorimeter. IgA binding to FcαRI was normalized to 100% (indicated with dotted line). Neutrophils or plates were pre‐incubated with either linear peptides (A) or cyclic CLIPS‐peptides (B) mimicking FcαRI‐sequences (white bars) or IgA‐sequences (black bars). Data are representative of three independent experiments, performed in triplicates. Mean is shown, error bars refer to SD (n = 3). Statistical analysis: ANOVA*p< 0.05, **p < 0.01.
Figure 2
Figure 2
Peptide mimetics inhibit IgA‐induced ROS production and phagocytosis of IgA‐beads. (A) Neutrophils, isolated from blood, were incubated with fluorescent IgA‐coated beads, in the presence or absence of peptide mimetics. Phagocytic index was calculated as the percentage of neutrophils that had phagocytosed beads, multiplied by the geometric mean of fluorescent cells. IgA binding to FcαRI was normalized to 100% (indicated with dotted line). Data are pooled from three independent experiments, performed in triplicates. Mean ± SD is shown (n = 5). Statistical analysis: ANOVA *p< 0.05. (B) Production of H2O2 as after adding neutrophils to IgA‐coated plates, as determined with an Amplex Red hydrogen peroxide assay. Neutrophils or plates were pre‐incubated with indicated peptides. Data are representative of three independent experiments, performed in triplicates. Mean of one representative experiment is shown (n = 3).
Figure 3
Figure 3
Peptide mimetics block IgA‐induced migration in vitro. Percentage of migration of fluorescently labelled neutrophils to IgA‐coated beads, either in the presence or absence of peptide mimetics. The number of migrated neutrophils was determined with a fluorimeter. Neutrophil migration to IgA was normalized to 100% (dotted line). Neutrophils or beads were pre‐incubated with (A) linear or (B) cyclic peptides mimicking FcαRI‐sequences (white bars) or IgA‐sequences (black bars). Data are representative of three independent experiments, performed in triplicates. Mean ± SD is shown. Statistical analysis: ANOVA *p< 0.05, **p < 0.01.
Figure 4
Figure 4
Peptide mimetics block neutrophil migration and penetrate the dermis in an ex vivo human skin model. Migration of green‐fluorescent neutrophils to BSA‐ (left panel) or IgA‐ (right panel) coated beads (indicated with circles) in the dermis of ex vivo skin explants (A). Migration of green‐fluorescent neutrophils to IgA‐coated beads (indicated with circles) after pre‐incubation with non‐blocking peptide FcαRI9‐ox (B), linear peptides (C) or cyclic CLIPS‐peptides (D). Images are representative of three independent experiments, performed in duplicate. Scale bar = 250 μm. Images are 10× magnified.
Figure 5
Figure 5
Penetration of peptide mimetic IgA7‐CLIPS in human skin. Penetration of radioactive labeled peptide mimetic ([14C]IgA7‐CLIPS) in ointment in ex vivo skin (white bars) and receptor fluid (striped bars). Recovery of peptide after 24‐h exposure is presented as ng/cm2 skin. Cream was applied with (+) or without (‐) nominal 5% permeation enhancer dodecyl‐2‐N, N‐dimethylaminopropionate (DDAIP). Data are shown as mean ±SD from a single experiment, performed in duplicates.
Figure 6
Figure 6
Epitope mapping studies reveal binding sequences of FcαRI and IgA. (A) Amino‐acid sequence of FcαRI. Vertical lines indicate different domains. Amino‐acids that are involved in binding with IgA are highlighted in gray. NCBI accession number for FcαRI is P24071 and based on Maliszewski et al.20 (B) IgA binding to FcαRI peptide library. Peptide positions indicate the sequence position of the N‐terminal residue of each 15‐mer peptide. Signal is the observed quantitative binding of the screened sample to the peptide. Gray regions are sequence areas predicted to be involved in the IgA‐FcαRI interaction. (C) Amino‐acid sequence of the constant regions of IgA. Gray highlighted residues have been documented to be involved in binding to FcαRI. NCBI accession number for IgA is P01876 and based on Woof et al.21. The IgA sequence is numbered according to the commonly adopted scheme used for human myeloma IgA1 protein Bur.22 (D) Screening of soluble FcαRI against the IgA peptide library, visualized as in B. (E) In‐depth analysis of peptide GRYQACYRIGHYRFRCSD (FcαRI8‐CLIPS). Residues involved in core binding are indicated, as mutation of these residues resulted in loss of function (binding). Data are shown as mean ±SD from a single experiment, performed in duplicate. (F) Schematic model of interaction of the amino‐acids of the FcαRI‐peptide GRYQACYRIGHYRFRCSD (FcαRI8‐CLIPS) with IgA‐Fc. Residues YRIGHYRFR are highlighted as “stick” visualization.
Figure 7
Figure 7
Working model: peptide mimetics prevent activation of neutrophils. IgA autoantibodies directed against skin epitopes activate FcαRI on neutrophils (A). This leads to release of LTB4 (B), initiating chemotaxis of neutrophils to the skin (C). These neutrophils get activated and also release LTB4, thereby initiating a positive feedback loop (arrows). Activated neutrophils release ROS and other toxic molecules (D), ultimately leading to tissue damage and blister formation (E). Linear and cyclic peptide mimetics of IgA (F) or FcαRI (G) in a topical ointment block the interaction between IgA‐FcαRI and thereby prevent neutrophil activation and migration. Of note, as example we depicted a sub‐epidermal blistering disease such as linear IgA bullous disease. However, IgA‐induced neutrophil activation and migration can also occur in epidermal blistering skin diseases such as IgA pemphigus, in which case neutrophil influx and blisters are located in the intraepidermal area (not shown).

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