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Published in final edited form as: J Allergy Clin Immunol. 2009 Sep 12;124(5):1113–5.e1-11. doi: 10.1016/j.jaci.2009.07.057

Peeling off the layers: Skin taping and a novel proteomics approach to study atopic dermatitis

Carolyn J Broccardo a, Spencer B Mahaffey a, Matthew Strand b,d, Nichole A Reisdorph a,e, Donald Y M Leung c,f
PMCID: PMC8648281  NIHMSID: NIHMS1754094  PMID: 19748658

To the Editor:

Atopic dermatitis (AD) is a chronic inflammatory skin disorder that affects nearly 17% of children and can persist into adulthood. Advances in our understanding of mechanisms underlying AD require direct sampling of AD skin. Although AD is primarily a skin disease involving infants and young children, there are no skin-based studies examining AD in this age group because of the invasiveness of skin biopsies. The goal of this pilot study was to develop a mass spectrometry (MS)–based, noninvasive skin taping technique to study AD. Large-scale proteomic profiling can provide new hypotheses regarding AD pathogenesis and potentially identify new disease biomarkers.

Five patients with severe AD1 (4 female, 1 male; median age, 45 years) and 5 normal nonatopic individuals with no history of atopic skin disease or allergies (3 female, 2 male; median age, 27 years) were recruited (Institutional Review Board protocol #HS 1962 NJ209). Four of 5 of the patients with AD had acute lesions (less than 3 days old), and all 5 had chronic (more than 3 days old) and nonlesional samples collected. All patients with AD had negative bacterial skin cultures and were required to discontinue the use of topical medications for 7 days and oral antibiotics for 10 days before sample collection.

A total of 20 standard D-Squame Skin Sampling Discs, 0.22-mm diameter (CuDerm, Dallas, Tex), were applied sequentially to each uncleansed chronic, acute, and nonlesional site of 5 patients with AD. Each disc was placed adhesive side up in its own borosilicate scintillation vial (Wheaton; Fisher Scientific, Pittsburgh, Pa) and frozen at −80°C. The same procedure was followed for the collection of tape discs from 3 nonatopic individuals at the same body locations sampled on the atopic patients (bicep, antecubital fossae, and back) and 2 nonatopic individuals from the antecubital fossae, for a total of 11 samples.

Proteins were extracted by using 0.01% 3-(3-[1,1-bisalkyloxyethyl]pyridin-1-yl)propane-1-sulfonate (Protein Discovery, Knoxville, Tenn), a MS-compatible detergent. Extraction buffer was pooled from tape disc layers (1–5, 6–10, 11–15, and 16–20) to ensure sufficient material for MS analysis. Proteins were digested according to standard procedures.2 MS analysis was performed in triplicate by using an HPLC-Chip ion trap instrument (Agilent Technologies, Santa Clara, Calif) coupled to an HPLC system (1200 series HPLC; Agilent). Protein identification was conducted by using the Spectrum Mill database search program (Agilent) to match mass spectral data to sequences in the Swiss Prot protein database. Database results were compiled for the replicate MS runs, pooled layers, patients, and lesions. The data were processed to calculate fold change between groups by using a Java (Sun Microsystems, Santa Clara, Calif) application developed in house. Proteins were further validated by using Scaffold (Proteome Software, Portland, Ore). Protein levels were compared between lesion type groups (acute, chronic, nonlesional atopic, and nonatopic) by using permutation F tests (α = 0.05). Pairwise comparisons were performed by using 2-sample permutation tests. See this article’s complete Methods in the Online Repository at www.jacionline.org.

A similar number of total proteins was identified with high confidence using the in-house Java application and Scaffold (104 vs 101 proteins, respectively; see this article’s Table E1 in the Online Repository at www.jacionline.org). Fifty-three unique proteins were identified in patients with AD (corresponding to 370 unique peptides) and 4 in nonatopic subjects (corresponding to 34 unique peptides), and 44 proteins were common between the 2 groups. The biological processes represented by the identified proteins are depicted in this article’s Fig E1 in the Online Repository at www.jacionline.org (Scaffold). Notable categories include immune response, response to stimulus, multiorganism response, and biological adhesion.

Proteins that met the inclusion criteria (see the Online Repository, Protein Selection) for novel, increased, and decreased protein levels between patient groups/lesion types were subjected to statistical analyses (dermcidin precursor, serpin B3 [squamous cell carcinoma antigen 1], triosephosphate isomerase, epidermal fatty acid binding protein [e-fabp], αenolase, caspase 14 precursor, keratin type 1 cytoskeletal 10, and keratin type 2 cytoskeletal 1; see this article’s Table E2 in the Online Repository at www.jacionline.org). All proteins yielded permutation F tests with P = .05 for overall group differences, except dermcidin (P ≤ .15; see this article’s Table E3 in the Online Repository at www.jacionline.org; Fig 1).

FIG 1.

FIG 1.

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Quantification of proteins of interest. Figures represent average spectral count values for patients between groups. Significant differences between 2 groups using the Benjamini-Hochberg false discovery rate procedure with false discovery rate=0.05 within each protein indicated by bracket endpoints over those columns. See Methods for statistical analysis details. A, e-fabp. B, α-Enolase. C, Triosephosphate isomerase. D, Serpin B3. E, Keratin 1 type II cytoskeletal 1. F, Dermcidin. Avg, Average; nonles., nonlesional.

Consistent with a previous report by Yamane et al,3 2 proteins were uniquely identified in patients with AD: e-fabp and α-enolase (Table E2). Triosephosphate isomerase and serpin B3 were increased in patients with AD compared with nonatopic controls (Table E2). Four proteins were decreased in the acute lesions of patients with AD compared with nonatopic subjects: keratin type I cytoskeletal 10, keratin type II cytoskeletal 1, caspase 14 precursor, and dermcidin precursor (Table E2). The elevated levels of serpin B3, a previously identified biomarker for AD,4 and decreased levels of the sweat-derived antimicrobial peptide dermcidin5 are consistent with the literature and serve to validate this proteomics technique further.

As reported in a previous study,3 α-enolase was found exclusively in the skin of patients with AD. It is plausible that human exposure to α-enolase on the surface of pathogens leads to development of autoantigens in the human body. In support of this theory, α-enolase has been shown to be autoantigenic in patients with psoriasis,6 and such an autoimmune reaction may perpetuate the chronic inflammation of AD.

In accordance with a previous study,3 e-fabp was highly expressed in acute and chronic lesions of patients with AD, was minimally expressed in the nonlesional skin of patients with AD, and was absent in nonatopic individuals. Fatty acid binding proteins are abundant intracellular proteins that bind and transport otherwise insoluble long-chain fatty acids and appear to be essential for normal keratinocyte differentiation.7 It has been proposed that fatty acid binding proteins may serve as master regulators of inflammatory signaling pathways.8 In support of this, e-fabp has been shown to bind and stabilize leukotriene A4 and may modulate the production or metabolism of bioactive eicosanoids, which have been found in the urine of patients with AD.9,10 Limited studies have been published on the effects of leukotriene antagonists/synthesis inhibitors on the treatment of AD.

In conclusion, this proof-of-principle pilot study has revealed new proteins possibly involved in AD pathogenesis. These may serve as biomarkers for the development and persistence of AD. Studies are planned to assess a larger population of age-matched and sex-matched patient groups, especially children.

Supplementary Material

1

Acknowledgments

Supported by National Institutes of Health/National Institute of Allergy and Infectious Diseases contracts #N01-AI-40029 and R01 AR41256, National Center for Research Resources (NCRR) UL1 RR025780, and NCRR S10RR023703. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Disclosure of potential conflict of interest: S. B. Mahaffey is the owner of Laboratory Information Management Solutions. N. A. Reisdorph has received research support from National Jewish Health and the National Institutes of Health. The rest of the authors have declared that they have no conflict of interest.

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Supplementary Materials

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NIHMS1754094-supplement-1.pdf (252.5KB, pdf)

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