Clinical use of anti-histone antibodies in idiopathic and drug-induced lupus

Abstract

Anti-histone antibodies (AHAs) make their appearance in a number of systemic autoimmune diseases including systemic lupus erythematosus (SLE) and drug-induced lupus erythematosus (DILE). Although being known for over 50 years, they are poorly studied and understood. There is emerging evidence for their use in predicting clinical features of SLE, diversifying their clinical use. AHAs, however, are probably less prevalent in DILE than once thought owing to a move away from older DILE drugs to modern biological agents which do not appear to elicit AHAs. This review examines the historical studies that have defined AHAs and looks at some of the recent work with these autoantibodies.

Keywords:

• Anti-histone antibodies
• autoantibodies
• drug-induced lupus
• histone
• systemic lupus erythematosus

1. Basic histone biology

Histones are structural subunits that provide a core in which chromatin can be wrapped around. The core comprises of two H2A–H2B dimers and an (H3–H4)₂ tetramer (histone octamer) with an external H1 histone, a conserved subunit that assists with maintaining this ‘beads on a string’ structure (Figure 1) [1]. The histone octamer coupled to around 150 bp of DNA is known as the nucleosome and forms the basic fundamental unit of chromatin [2]. Histones not only form scaffolds for chromatin, but they also play an important role in regulating gene expression [3]. Histones themselves are encoded by multiple genes spanning a number of chromosomes including chromosomes 6, 11 and 12 in humans [4].

Figure 1. Basic subunit of DNA. A nucleosome comprises a histone octamer core with DNA wrapped around, resembling ‘beads on a string’.

Histones may become ‘exposed’ through the formation of apoptotic blebs or neutrophil extracellular traps (NETs) [5]. Either through excess formation, reduced clearance and/or post-translational modification, histones may become immunogenic [6,7]. Furthermore, recent attention has turned to the role of epigenetic modifications of these proteins in the rendering of immunogenicity and hence, the formation of anti-histone antibodies (AHAs) [8,9]. Deliberate post-translational modification of histones may be done therapeutically in inflammatory disorders. Murine systemic lupus erythematosus (SLE) models injected with an inhibitor of histone deacetylase attenuated renal pathology compared to a vehicle control [10]. This may be through, in part, the reduction of autoreactive plasma cells and autoantibodies [11].

Histone proteins are an example of a danger-associated molecular pattern (DAMPs) and possess the intrinsic ability to elicit inflammation. Notably, mice administered histones elicited systemic inflammation and suffer from multi-organ damage and eventually death, in a dose-dependent manner [12]. In in vitro experiments, H1 and H2A histones were able to induce specific T cell proliferation in SLE patients. Moreover, they stimulated the production of interferon-γ, tumour necrosis factor and anti-double stranded DNA (dsDNA) autoantibodies [13,14].

2. Anti-histone antibodies (AHAs)

Amongst the antinuclear antibodies (ANAs), AHAs represent a clinically important subset [15]. AHAs are found in a variety of immunological and infectious disorders. They may be directed against free histones or histones bound to DNA [16]. Although AHAs may be of any isotype, IgG is considered the most clinically relevant and detected isotype. IgM AHAs are less specific and may be found in a wide range of other disorders such as rheumatoid arthritis and mixed connective tissue disease. These antibodies have been typed at the peptide level by microarray analyses to define their precise specificities [17]. Although classically attributed to SLE and drug-induced lupus erythematosus (DILE), AHAs have been studied since the 1970s and are found in a large variety of pathologies including Sjögren’s syndrome, inflammatory myositides and rheumatoid arthritis [18].[16]

The functional activity of AHAs is largely unknown; although, they may possess proteolytic activity towards histone proteins [19]. Whether this directly contributes to disease pathogenesis is unclear at this stage.

3. AHA detection in the laboratory

AHAs may refer to either total histones or histone subunits; although the latter is more often seen in the research laboratory. AHAs may be noted on indirect immunofluorescence that screens for ANAs. On the commonly-used HEp-2 substrate, AHAs commonly appear as a homogenous pattern; but is not specific for these autoantibodies [20]. Some false positives have also been noted on the Crithidia luciliae immunofluorescence test (CLIFT) (which normally detects anti-dsDNA antibodies) in some patients that produce AHA that can bind Chrithidia luciliae kinetoplast [21].

One of the now-outdated AHA tests is the complement fixation assay. These assays rely on the ability of complement to bind AHA and histone antigen immune complexes, and therefore, do not cause the haemolysis of added sensitised sheep red blood cells. In contrast, the absence of AHA will lead to unbound complement – due to lack of antibody-antigen immune complexes – and eventual haemolysis of sensitised SRBCs. Haemolysis is measured spectrophotometrically [22].

A further historical test is the immunofluorescence assay on histone-reconstituted tissues [23]. Briefly, fixed mouse kidney cryostat sections are treated with hydrochloric acid to elute histones. A proportion of these is then reconstituted with calf thymus histones. Comparison of acid-extracted and reconstituted tissues after incubation with diluted patient sera allows the specific detection of histone antibodies [23]. The advantage of this assay is the specific detection of AHA; however, it is time-consuming and labour-intensive. The complement fixation test and histone reconstitution assays have good concordance [24].

Modern-day assays, such as the chemiluminescence assay, are based on an ELISA format with various methods of antigen presentation and secondary antibody detection, which has the advantage of being able to detect whole histone or histone subunits. These may be single- or multi-plex and afford automaticity and quick turnaround times [25]. The positivity may differ depending on assay use and if there is contaminating DNA which acts as an antigenic source for patient antibodies [26].

Western blotting has been used to traditionally detect antibodies against acid-extracted histone subunits. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) is used to separate histones out into their constituents based on molecular weights: H1 is around 23 kDa whilst the core histones (H2A, H2B, H3 and H4) range from 10–15 kDa [27]. Detection against these components using IgG-, IgA- or IgM-labelled secondary antibodies can be performed. The line immunoassay works in a similar manner. The major drawback to these assays is the histone antigens are denatured and therefore, reduced sensitivity in detecting antibodies against conformational epitopes [1].

4. AHAs in drug-induced lupus erythematosus (DILE)

DILE is a syndrome that resembles some or most pathological and clinical features of idiopathic SLE but is temporally related to the commencement of a particular drug. On cessation of the offending drug, the syndrome abates [28]. Numerous drugs and biological agents are implicated. Although hydralazine and procainamide are the most commonly implicated agents [28]; infrequent drugs associated include interferon-α, sulfasalazine and captopril [29]. The risk of DILE is generally increased in the older population and those that have been taking implicated medications for a longer period of time [30]. It is estimated to affect up to 30,000 people each year in the United States [30].

DILE is characterised by the emergence of autoantibodies, including AHAs which appear at a much higher frequency than idiopathic SLE [28,31]. In contrast, biologic agents such as anti-tumour necrosis factor (TNF) do not appear to induce AHAs as frequently, suggesting a different mechanism for the pathogenesis [32,33]. Anti-TNF DILE tends to produce more internal organ involvement and rashes compared to classic DILE [34]. In addition, AHAs are generally absent in drug-induced ANA without symptoms [35].

Overall, AHAs (mainly IgG isotype) are present in a substantial proportion of DILE patients (Table 1). In a cohort of patient samples submitted to a diagnostic laboratory, AHA had a sensitivity of 67% and specificity of 95% for the diagnosis of DILE [18]. This is in contrast to the reported prevalence of 96% in one study [36]; however, the latter was derived from a cohort of DILE patients rather than a general laboratory population. Furthermore, older studies in DILE excluded newer biological agents that cause DILE without AHA, so this may be somewhat biased.

Table 1. Prevalence of IgG antibodies to total histones and histone subunits in selected disorders.

Histone component	Disease	Average prevalence, % (range)	References
Total histone	Rheumatoid arthritis	5	[50]
	Various malignancies		[50]
	Inflammatory myositides	17	[20]
	Systemic sclerosis	30 (29–33)	[41,48]
	Schizophrenia	23	[51]
	Systemic lupus erythematosus (SLE)	53 (24–81)	[24,39,43,50,52,53]
	Drug-induced lupus erythematosus (DILE)	96	[36]
H1	Human immunodeficiency virus		[54]
	Inflammatory myositides	17	[20]
	Mixed connective tissue disease (MCTD)		[55]
	SLE	32 (6–49)	[36,39,42,53]
	DILE	9	[36]
H2A	SLE	34	[53]
	DILE	67	[35]
	Systemic sclerosis		[56]
	MCTD		[55]
H2B	SLE	63	[53]
	Systemic sclerosis		[56]
	MCTD		[55]
H2A-H2B complex	SLE	9	[26]
	DILE	91	[36]
H2A-H2B-DNA complex	SLE	10	[26]
	DILE	30	[57]
H3	SLE	42 (37–56)	[42,53]
	DILE	13 (H3–H4 complex)	[36]
H4	SLE	1 (1–22)	[39,53]

IgG and IgM antibodies to the H2A–H2B and H2A–H2B–DNA histone complexes have been noted in patients with procainamide DILE [37,38]. In contrast, patients with hydralazine and chlorpromazine DILE had predominantly IgM directed against DNA-free histone complexes, namely to H2A–H2B, H1, (H3–H4)₂ tetramers [37]. These data reinforce the heterogeneous immune response in DILEs, yet the specific reasons for targeting these histone complexes are unclear.

5. AHAs in systemic lupus erythematosus

In contrast to DILE, SLE ANAs are more heterogeneous and include AHAs and non-histone antibodies, perhaps reflecting the complex pathogenesis. IgG, IgA and IgM to individual core histones are primarily directed against H1, H2A, H2B and H3; very little is directed against H2A–H2B complex or H4 [39]. Antibodies to these histone subunits are generally more diverse and reflect epitope spreading than DILE AHAs [38], and may also be directed to histone complexes (e.g., H2A–H2B) and histone coupled to DNA [26].

Studies reveal the presence of antibodies against total histone in approximately half of the patients with SLE (Table 1). Compared to healthy controls and non-SLE ANA-positive patients, total histone autoantibody detection has a diagnostic sensitivity of 55–92% and specificity of 69–82% for SLE [40,41]. H4 antibodies are relatively rare in SLE but when present, they have excellent diagnostic sensitivity (95%) and specificity (90%) for SLE when compared to healthy controls [25]. This is analogous to H1 and H3 specific antibodies which also demonstrate high specificity (94–96%) for the diagnosis [42]. In general, AHA has lower sensitivity for SLE than anti-nucleosomes and lower specificity than anti-dsDNA for the diagnosis [40].

AHAs often co-exist with anti-dsDNA and anti-nucleosome antibodies, particularly in lupus nephritis (LN), and may reflect more severe renal involvement than those LN patients negative for these autoantibodies [43]. AHA quantitation appears to correlate with LN severity and may be predictive of flares [43–45]. Although total IgG AHA may fluctuate with disease activity, levels do not reliably correlate with overall disease activity [16]. Clinically, studies show that AHA is associated with oral ulceration, neuropsychiatric symptoms, lymphopaenia and fatigue [39,41,46]. There is no association with arthritis or other cutaneous features [46]. As of yet, there appears not to be clinical significance ascribed to the differential expression of antibodies to histone subunits [47].

6. Conclusions

Although AHAs were classically attributed to DILE and SLE, there is an expanding spectrum of the association of these autoantibodies to other autoimmune and infectious disorders. The value of these may be to help predict and prognosticate clinical features in disorders such as SLE and systemic sclerosis [48]. Subunit histone antibody analyses may also help distinguish SLE from certain cases of DILE but this is not routinely performed in the diagnostic laboratory and probably does not add significant diagnostic value. Moreover, the prevalence of AHAs in DILE likely is much less than reported in older literature since newer agents implicated in DILE do not elicit AHAs as frequently as older agents such as procainamide.

What can be done to further study AHAs? Firstly, the factors that make histones immunogenic are a matter of ongoing research, particularly in terms of epigenetics in recent times. In addition, there are only a handful of studies that examine isotype determination in various disorders and, apart from a research perspective, there appears to be little clinical relevance although further studies in this area are warranted. Certainly, isotypes other than IgG increase the sensitivity of picking up AHAs [39]. Furthermore, emerging mass spectrometry technology to type these autoantibodies at the amino acid and peptide levels has afforded an innovative way to study these autoantibodies at high resolution [49], increasing our understanding of their pathogenicity and the immunological epiphenomena of systemic autoimmunity. It is possible that with increasing observational studies, databases of disease associations may be established to enable clinicians and laboratorians to find value in the molecular typing of these fascinating autoantibodies.

Disclosure statement

No potential conflict of interest was reported by the author(s).