Development of antigen-specific ELISA for circulating autoantibodies to extracellular matrix protein 1 in lichen sclerosus

Abstract

Lichen sclerosus is a common, acquired chronic inflammatory skin disease of unknown etiology, although circulating autoantibodies to the glycoprotein extracellular matrix protein 1 (ECM1) have been detected in most patients' sera. We have examined the nature of ECM1 epitopes in lichen sclerosus sera, developed an ELISA system for serologic diagnosis, and assessed clinicopathological correlation between ELISA titer and disease. Epitope-mapping studies revealed that lichen sclerosus sera most frequently recognized the distal second tandem repeat domain and carboxyl-terminus of ECM1. We analyzed serum autoantibody reactivity against this immunodominant epitope in 413 individuals (95 subjects with lichen sclerosus, 161 normal control subjects, and 157 subjects with other autoimmune basement membrane or sclerosing diseases). The ELISA assay was highly sensitive; 76 of 95 lichen sclerosus patients (80.0%) exhibited IgG reactivity. It was also highly specific (93.7%) in discriminating between lichen sclerosus and other disease/control sera. Higher anti-ECM1 titers also correlated with more longstanding and refractory disease and cases complicated by squamous cell carcinoma. Furthermore, passive transfer of affinity-purified patient IgG reproduced some histologic and immunopathologic features of lichen sclerosus skin. This new ELISA is valuable for the accurate detection and quantification of anti-ECM1 autoantibodies. Moreover, the values may have clinical significance in patients with lichen sclerosus.

Figure 1

Schematic representation of ECM1 protein and its five recombinant GST fusion fragments. The distinct molecular domains comprise a signal peptide, a cysteine-free NH₂-terminal domain, two tandem repeat domains, and a COOH-terminal domain. The position of the single cysteine residues and potential N-glycosylation sites are indicated. The full-length 1.8-kb ECM1 cDNA was initially divided into three different fragments, ΔNH₂ (33 to 226 amino acids), ΔPx/COOH (226 to 499 amino acids), and ΔS/COOH (499 to 559 amino acids), designed to span almost the entire ECM1. Two further fragments were then synthesized. The ΔS/ex7 fragment encodes part of the ΔPx/COOH domain (226 to 359 amino acids), and the ΔDs/COOH fragment encodes the distal part of the second tandem repeat domain and COOH-terminus (359 to 559 amino acids). All fragments were fused with GST-encoded cDNA at the NH₂-terminus of the recombinant proteins. The amino acid residue numbers are shown as described elsewhere (13). The predicted molecular weights (kDa) of each recombinant fusion fragment are shown on the right.

Figure 2

Purification of recombinant ECM1-GST fusion proteins. The purified ECM1-GST fusion recombinant proteins were separated on SDS-polyacryl amide gel and detected by Coomassie blue staining (top). The identical gel was immunoblotted using anti-GST antibody (bottom). Molecular weights are indicated on the left column.

Figure 3

Immunoblotting for lichen sclerosus sera by use of a series of recombinant ECM1-GST fusion fragments. The five recombinant ECM1-GST fragments, ΔNH₂, ΔPx/COOH, ΔS/COOH, ΔS/ex7, and ΔDs/COOH, were independently immunoblotted with serum samples from patients with lichen sclerosus (12 of which are illustrated) or control subjects. Each of the lichen sclerosus sera (numbered on the bottom) showed heterogeneous immunoreactivity against the recombinants. Note that ΔDs/COOH represents the most antigenic epitope recognized by the vast majority of lichen sclerosus sera. Representative results from each recombinant blot are shown. Arrows indicate the positive signals.

Figure 4

Preabsorption assays for affinity-purified lichen sclerosus sera with recombinant proteins. (A) Immunostaining with anti-ECM1 rabbit polyclonal antibody on normal human skin displays intracellular labeling in the lower epidermis, particularly in basal and suprabasal cell layers. Inset shows labeling of dermal blood vessels. (B) Similar staining pattern was obtained using affinity-purified IgG fractions from ΔDs/COOH-positive lichen sclerosus sera. (C) Before immunostaining, the affinity-purified IgG fractions from ΔDs/COOH-positive sera were incubated with an excess of ΔDs/COOH recombinant. This results in a marked reduction in labeling intensity (c.f. Figure 4B). (D) Before immunostaining, the affinity-purified IgG fractions from a dual ΔNH₂-positive and ΔDs/COOH-positive lichen sclerosus serum were incubated with an excess of either ΔNH₂ or ΔDs/COOH recombinants. No alteration occurred in the original labeling intensity. (E) Before immunostaining, the affinity-purified IgG fractions from a dual ΔNH₂-positive and ΔDs/COOH-positive sera were incubated with a mixture of both the ΔNH₂ and ΔDs/COOH recombinants or with (F) control recombinant protein BP180 NC16A. Note the marked signal reduction in (E) but not in (F). Scale bar: 50 ∝m.

Figure 5

Quantitative analysis of IgG reactivity against the recombinant protein encompassing the distal COOH-terminal region of ECM1 in the sera of lichen sclerosus patients. Sera from 95 patients with lichen sclerosus (LS) and from 318 comparative control subjects (161 normal volunteers, 70 with systemic lupus erythematosus [SLE], 72 with bullous pemphigoid [BP], 15 with systemic sclerosis [SSc]) were incubated with ΔDs/COOH recombinant protein immobilized onto an ELISA plate. Bound IgG was detected with alkaline phosphatase-labeled anti_human IgG antibody. Each serum sample was run in duplicate. The mean OD values from the ECM1 recombinant protein were normalized by mean OD values of the background immunoreactivity (against GST control protein) and expressed as arbitrary units (AU) on the basis of the positive and negative reference sera. Dashed line indicates a cutoff value (0.328 AU), as determined by receiver operative characteristic (ROC) analysis (inset). The area under the curve, which represents the diagnostic accuracy of the ELISA, was 0.919.

Figure 6

Comparison between ECM1-ELISA values and clinical presentation of lichen sclerosus patients. Disease severity of 35 well-defined patients with lichen sclerosus was divided into two clinical subgroups, “severe” and “mild,” and analyzed with individual ELISA scores against ECM1. The ECM1-ELISA scores were significantly higher in patients with a severe clinical phenotype compared with those with a mild phenotype (mean ELISA score ± SD; 0.665 ± 0.216 vs. 0.425 ± 0.129, respectively; Mann-Whitney U test, P = 0.031). Horizontal bars indicate the mean ELISA values in each subgroup. In the severe group, four cases had longstanding disease complicated by genital skin squamous cell carcinoma; two of whom had just genital disease (filled circles), whereas two others had both genital and extragenital involvement (filled squares).

Figure 7

Passive-transfer study using affinity-purified lichen sclerosus IgG in neonatal BALB/c mice. (A) Intradermal injection of affinity-purified lichen sclerosus IgG into BALB/c mouse caused extensive erythematous swelling with telangiectasie (left ear), whereas the control skin injected with either nonimmune human IgG exhibited no evidence of skin inflammation (right ear). This picture was taken at 28 days in a mouse that had received seven separate injections in both ears at days 0, 4, 8, 12, 16, 20, and 24. (B) Light microscopy of the mouse skin injected with affinity-purified lichen sclerosus IgG exhibited a pronounced inflammatory infiltration with edema and dilated blood vessels in the upper-middle dermis. The overlying epidermis showed mild acanthosis. (C) In contrast, the control skin injected with affinity-purified normal human IgG showed only a scanty perivascular infiltration in the dermis. (D) The mouse skin injected with affinity-purified lichen sclerosus antibodies revealed IgG deposition within the lower epidermis (arrows) and surrounding dilated dermal blood vessels (arrowheads). Asterisk indicates the injected site. Higher magnification views revealed an intracellular signal in basal keratinocytes and weaker staining in the suprabasal keratinocytes (E), as well as in the walls of dilated dermal blood vessels (F). (G) In contrast, the control injection with affinity-purified normal human IgG resulted in no immunolabeling. (H) At higher magnification, control injections only resulted in a nonspecific signal in the corneal layers. Scale bar: 50 ∝m.