Cross-recognition between histones and La/SSB may account for anti-DNA reactivity in SLE patients

Abstract

Antibodies to La/SSB are detected in sera of patients with primary Sjogren's syndrome (pSS) and systemic lupus erythematosus (SLE). The vast majority of anti-La/SSB positive sera contain antibodies directed towards a linear B-cell epitope of La/SSB spanning the sequence 349-364aa (pep349-364). The aim of this study was to evaluate the fluctuation of antibody levels to major B-cell epitopes of La/SSB over time and investigate for their possible crossreactions. Sequential sera from 15 SLE and 15 pSS patients, followed from 3 to 10 years were obtained. All patients with SLE were positive for anti-Ro/SSA, anti-La/SSB and anti-dsDNA antibodies and patients with pSS were positive for anti-Ro/SSA and anti-La/SSB antibodies. Sera from 30 patients with SLE without anti-La/SSB antibodies and 30 healthy individuals served as disease and negative control respectivelly. All sera tested for the presence of anti-pep349-364 antibodies, using a specific ELISA. Specific anti-pep349-364 IgG was purified from sera of SLE patients and evaluated for cross reactivity against dsDNA and histones. In all SLE sera the levels of anti-pep349-364 antibodies varied in time and fluctuated in parallel with anti dsDNA antibodies. Anti-pep349-364 IgG purified from 7 SLE patients. Five out of 7 were found to react with calf thymus DNA in ELISA. All purified (7/7) anti-pep349-364 IgG preparations reacted with histone H1 and failed to produce a positive immunofluorescence pattern in Crithidia luciliae anti-dsDNA assay which lacks histones. Competative inhibition experiments demonstrated that histone H1 could inhibit completely the binding of anti-pep349-364 IgG to pep349-364 while pep349-364 inhibited by 70% the binding of anti-pep349-364 IgG to histone H1. These findings indicate that a subgroup of SLE patients possess cross-reacting anti-histone H1 antibodies and anti-pep349-364 antibodies, which can be faulty considered as anti-dsDNA reactivity in regular ELISA techniques.

Fig. 1

Antibody responses against pep349–364 for 12 representative patients. The x-axis holds the sequential patient sera sorted by the collection time (e.g. D1 the first serum collected, D2 the second serum collected, etc.), while the z-axis represents the 12 different patients (P1 to P12). Patients P1-P4, P5-P8 and P9-P12 participate with 5 (D1-D5), 6 (D1-D6) and 7 (D1-D7) sequential sera, respectively. The antibody responses, appear early in the disease course, vary in time and could not be correlated with the duration of the disease.

Fig. 2

Combined antibody reactivity against pep349–364, pep289–308, recLa/SSB and dsDNA for 9 representative patients. The titre of antibodies against pep349–364 of La/SSB, varied in parallel with the titre of anti-dsDNA antibodies, in serial sera from 15 SLE patients. Data for 9 representative series of sera are presented in the Figure.

Fig. 3

Inhibition experiments using SLE patient sera. Anti-dsDNA serum reactivity could be inhibited using (a) DNA as an inhibitor 90% and (b) pep349–364 as an inhibitor at 93%. Similarly, anti-pep349–364 serum reactivity could be inhibited using (a) DNA as an inhibitor at 68% and (b) pep349–364 as an inhibitor at 65%. The ODs without the inhibitor were in the range 1·7–2·0.

Fig. 4

Anti-dsDNA and anti-histone H1 reactivity of purified anti-pep349–364 antibodies. All purified anti-pep349–364 IgGs recognized histone H1 while five of seven IgGs recognized also the dsDNA preparation.

Fig. 5

Homologous inhibition of the anti-dsDNA reactivity of purified anti-pep349–364 antibodies. Inhibition of binding of anti-349–364 IgG to dsDNA using dsDNA as inhibitor (0–10 µg/ml) (ODs without the inhibitor: 1·4–1·6).

Fig. 6

Immunofluerence pattern on Crithidia luciliace anti-dsDNA assay. (a, b) anti-dsDNA laboratory reference sera; (c) Normal IgG; (d–f) purified anti-pep349–364 antibodies.

Fig. 7

Homologous and heterologous inhibition experiments using histone H1 as inhibitor. (a) the binding of anti-349–364 IgG to histone H1 almost completely inhibited (ODs without the inhibitor: 1·7–2·0) and (b) the binding of anti-pep349–364 IgG to pep349–364 inhibited at 70%– 90% (ODs without the inhibitor: 1·5–1·8).

Fig. 8

Heterologous inhibition experiments using pep349–364 as inhibitor. The recognition of histone H1 by anti-pep349–364 IgG was inhibited at 30%-70% by soluble pep349–364 (ODs without the inhibitor: 1·7–2·0).