High mobility group (HMG) non-histone chromosomal proteins HMG1 and HMG2 are significant target antigens of perinuclear anti-neutrophil cytoplasmic antibodies in autoimmune hepatitis

Abstract

Background: High mobility group (HMG) non-histone chromosomal proteins HMG1 and HMG2 have been identified as novel antigens of perinuclear anti-neutrophil cytoplasmic antibodies (p-ANCAs), and the existence of anti-HMG1 and anti-HMG2 antibodies in a population of patients with ulcerative colitis has been reported.

Aims: To investigate whether HMG1 and HMG2 are target antigens for p-ANCAs in autoimmune hepatitis (AIH).

Patients: Serum samples from 28 patients with AIH, 44 patients with primary biliary cirrhosis (PBC), 27 patients with chronic hepatitis C, and 23 patients with chronic hepatitis B were tested.

Methods: ANCAs were detected by routine indirect immunofluorescence (IIF). Anti-HMG1 and anti-HMG2 antibodies were assayed by enzyme linked immunosorbent assay.

Results: p-ANCAs were detected in 89% (25/28) of patients with AIH, 36% (16/44) of patients with PBC, 11% (3/27) of patients with chronic hepatitis C, and 13% (3/23) of patients with chronic hepatitis B. Anti-HMG1 and/or anti-HMG2 antibodies were detected in 89% (25/28) of patients with AIH, 70% (31/44) with PBC, 26% (7/27) with chronic hepatitis C, and 9% (2/23) with chronic hepatitis B. In AIH, anti-HMG1 and/or anti-HMG2 antibodies were detected in 96% (24/25) of p-ANCA positive patients. The p-ANCA staining pattern detected by IIF using sera from patients with AIH disappeared or decreased in titre after preincubation with a mixture of HMG1/HMG2. The presence and titres of those antibodies in AIH correlated significantly with those of p-ANCA, but not with those of anti-nuclear antibody or anti-smooth muscle antibody.

Conclusions: HMG1 and HMG2 are significant target antigens of p-ANCA in AIH.

Figure 1

Perinuclear anti-neutrophil cytoplasmic antibody activities on indirect immunofluorescence. Sera from patients with autoimmune hepatitis (A, B) and primary biliary cirrhosis (C, D), and an affinity purified anti-HMG1/HMG2 antibody (E, F) showing perinuclear staining on ethanol fixed neutrophils (A, C, E) and cytoplasmic or negative staining on formalin fixed neutrophils (B, D, F).

Figure 2

Titres of antibodies to HMG1 (A) and HMG2 (B) determined by ELISA. AIH, autoimmune hepatitis; PBC, primary biliary cirrhosis. The solid lines represent median values of antibody titres. p values by Mann-Whitney U test.

Figure 3

Competitive inhibition ELISA. Anti-HMG1/HMG2 antibody positive serum from a patient with autoimmune hepatitis was preincubated with a mixture of HMG1 and HMG2 or ovalbumin (OVA) at the concentrations indicated. In a subsequent HMG1/HMG2 ELISA, the reactivity decreased in a dose dependent manner when preincubated with HMG1/HMG2, but not with ovalbumin.

Figure 4

Correlation between the titres of anti-HMG2 antibody and perinuclear anti-neutrophil cytoplasmic antibodies in autoimmune hepatitis. A positive correlation was found between the two parameters (r = 0.8264, p<0.0001, Spearman rank correlation test).

Figure 5

Disappearance of perinuclear anti-neutrophil cytoplasmic antibody (p-ANCA) staining after preincubation with specific antigens. Serum from a patient with autoimmune hepatitis with a p-ANCA titre of 1280 was diluted at 1:160 and preincubated with an equal volume of 100 µg/ml of a mixture of HMG1 and HMG2 (B) or phosphate buffered saline (A). Subsequent IIF was performed by each solution, and photographs were taken at the same exposure. No fluorescence was visible in (B), where the only bright cell is an eosinophil with spontaneous fluorescence.