Serum IgG2 antibody multicomposition in systemic lupus erythematosus and lupus nephritis (Part 1): cross-sectional analysis

Abstract

Objectives: Serum anti-dsDNA and anti-nucleosome IgGs have been proposed as signatures for SLE and LN in limited numbers of patients. We sought to show higher sensitivity and specificity of the same antibodies with the IgG2 isotype and included IgG2 antibodies vs specific intracellular antigens in the analysis.

Methods: A total of 1052 SLE patients with (n = 479) and without (n = 573) LN, recruited at different times from the beginning of symptoms, were included in the study. Patients with primary APS (PAPS, n = 24), RA (RA, n = 24) and UCTD (UCTD, n = 96) were analysed for comparison. Anti-nucleosome (dsDNA, Histone2A, Histone3), anti-intracellular antigens (ENO1), anti-annexin A1 and anti-C1q IgG2 were determined by non-commercial techniques.

Results: The presence in the serum of the IgG2 panel was highly discriminatory for SLE/LN vs healthy subjects. Serum levels of anti-dsDNA and anti-C1q IgG2 were more sensitive than those of IgGs (Farr radioimmunoassay/commercial assays) in identifying SLE patients at low-medium increments. Of more importance, serum positivity for anti-ENO1 and anti-H2A IgG2 discriminated between LN and SLE (ROC T0-12 months), and high levels at T0-1 month were detected in 63% and 67%, respectively, of LN, vs 3% and 3%, respectively, of SLE patients; serum positivity for each of these was correlated with high SLEDAI values. Minor differences existed between LN/SLE and the other rheumatologic conditions.

Conclusion: Nephritogenic IgG2 antibodies represent a specific signature of SLE/LN, with a few overlaps with other rheumatologic conditions. High levels of anti-ENO1 and anti-H2A IgG2 correlated with SLE activity indexes and were discriminatory between SLE patients limited to the renal complication and other SLE patients.

Trial registration: The Zeus study was registered at https://clinicaltrials.gov, NCT02403115.

Keywords: LN; SLE; anti-C1q antibodies; anti-ENO1 antibodies; anti-Histone 2A antibodies; biomarkers.

Fig. 1

Correlation between anti-dsDNA, anti-C1q IgG2 and IgGs levels (commercial kits) (a) Correlation between circulating levels of anti-dsDNA IgG2 as determined by our assay and anti-dsDNA as determined by the Farr assay (Farr assay, Kodak Clinical Diagnostics) (levels on the x-xis are presented as log₁₀ values); (b) correlation between circulating levels of anti-C1q IgG2 and anti-C1q IgGs, as determined by a non‐commercial assay [27] (levels on the x-axis are reported as log₁₀ values); (c) heat map showing the correlation between the levels of each circulating IgG2 antibody, for all categories of patients considered together [the correlogram, based on Spearman’s coefficient, uses a pseudocolour scale (from red, +1, positive; to blue, –1, negative; and to white, 0, null), to represent the correlation between two antibodies]; (d) graphic representation of the correlation between anti-dsDNA and anti-Annexin A1 IgG2 serum levels (R = 0.82, P<0.0001).

Fig. 2

Circulating levels of anti-dsDNA and anti-Annexin A1 IgG2 (a) Circulating levels of anti-dsDNA and (d) of anti-Annexin A1 IgG2 in patients with LN (n = 479) and patients with SLE (n = 573) subdivided in six subgroups according to the time elapsed from the onset of the disease (0–1 months, 1–12 months, 12–24 months, 24–48 months, 48–96 months, >96 months); patients with RA (n = 24), PAPS (n = 24) and UCTD (n = 96) were included as representative of other rheumatologic conditions. (b, e) Percentage of patients with LN (black) and of patients with SLE (white) with high levels divided into subgroups according to the time from disease onset at recruitment; (c, f) ROC curves for difference in anti-dsDNA and anti-Annexin A1 IgG2 between patients with LN and healthy controls (1), patients with SLE and healthy controls (2) and patients with LN_T0–12vs patients with SLE_T0–12 (3). In all cases, antibodies were of the IgG2 isotype, and levels were calculated as Relative Intensity value (RU/ml), given the absence of WHO international standards [33]. PAPS = primary APS.

Fig. 3

Circulating levels of anti-Histone 2 A and anti-Histone 3 IgG2 (a) Circulating levels of anti-Histone 2A and (d) anti-Histone 3 IgG2 in patients with LN (n = 479) and patients with SLE (n = 573) subdivided into six subgroups according to the time elapsed from the onset of the disease (0–1 months, 1–12 months, 12–24 months, 24–48 months, 48–96 months, >96 months); patients with RA (n = 24), PAPS (n = 24) and UCTD (n = 96) were included as representative of other rheumatologic conditions. (b, e) Percentage of LN (black) and of SLE (white) patients with high levels divided into subgroups according to the time from disease onset at recruitment; (c, f) ROC curves for difference in anti-Histone 2A and anti-Histone H3 IgG2 between patients with LN and healthy controls (1), patients with SLE and healthy controls (2) and patients with LN_T0–12vs patients with SLE_T0–12 (3). In all cases, antibodies were of the IgG2 isotype and levels were calculated as Relative Intensity value (RU/ml), given the absence of WHO international standards [33].

Fig. 4

Circulating levels of anti-ENO1 and anti-C1q IgG2 (a) Circulating levels of anti-ENO1 IgG2 and (d) of anti-C1q IgG2 in patients with LN (n = 479) and SLE (n = 573) subdivided into six subgroups according to the time elapsed from the onset of the disease (0–1 months, 1–12 months , 12–24 months, 24–48 months, 48–96 months, >96 months); patients with RA (n = 24), PAPS (n = 24) and UCTD (n = 24) were included as representative of other rheumatologic conditions. (b, e) Percentage of LN (black) and of SLE (white) patients with high levels divided into subgroups according to the time from disease onset at recruitment, (c, f) ROC curves for difference in anti-dsDNA and anti-Annexin A1 between patients with LN and healthy controls (1), patients with SLE and healthy controls (2) and patients with LN_T0–12 versus patients with SLE_T0–12 (3). In all cases, antibodies were of the IgG2 isotype and levels were calculated as Relative Intensity value (RU/ml), given the absence of WHO international standards [33].

Fig. 5

Clinical correlations and volcano plots (a) Heat map showing the association between the levels of circulating IgG2 antibodies and the clinical and laboratory parameters in all categories of LN and SLE patients considered together [the correlogram, based on Spearman’s coefficient, uses a pseudocolour scale (from red, +1, positive; to blue, –1, negative; and to white, 0, null), to represent the correlation between two parameters]. Anti-ENO1 and anti-H2A IgG2 had the highest correlation with the SLEDAI index, and anti-C1q was correlated with low C3 and C4 levels (blue = inverse correlation). (b) Heat map showing the percentage incidence of target organ pathologies in all the categories of SLE and LN patients (0–1 months up to >96 months). Here the heat map indicates the correlation between two parameters (e.g. an antibody and an organ pathology) [again, the correlogram, based on Spearman’s coefficient, uses a pseudocolour scale (from red, +1, positive; to blue, –1, negative; and to white, 0, null)]. (c) Volcano plots based on fold change (Log₂) and on P value (–Log₁₀) of each antibody level identified SLE and LN patients who had experienced a renal flare in the previous 24 months. Anti-ENO1 and anti-H2A IgG2 were the antibodies with the most significant changes in LN patients.