Antihistone and antispliceosome antibodies

Histones are key protein components of chromatin

Histones are nuclear proteins bound to genomic DNA and organized into a macromolecular complex referred to as native chromatin. In addition to DNA and histones, which constitute 80% of its mass, chromatin contains nonhistone proteins, many of which are also autoantibody targets in rheumatic diseases—proteins such as the centromere proteins and high-mobility group proteins. Histones and DNA constitute the repeat subunit of chromatin called the nucleosome, which consists of two molecules of each of the “core” histones—H2A, H2B, H3, and H4—forming an octamer, along with a histone H1 intricately bound to approximately 200 base pairs of DNA. Antibodies to DNA are reviewed in Chapter 26 .

Anti-histone antibodies

The identification of lupus erythematosus (LE) cell phenomenon by Hargraves et al. led to the recognition of autoimmune reactivity to intracellular components as a major feature of SLE. The LE cell phenomenon—which was based on the observation that cellular components (particularly nuclei) released during cell death can be phagocytosed by neutrophils in the milieu of certain plasma factors—is a classic immunoassay that was included in the early versions of classification criterion for SLE. It was concluded from these early studies that autoantibodies to deoxyribonucleoprotein recognize histone-DNA complexes. Subsequent studies showed that antibodies to histones were a key requirement for the LE cell phenomenon and they hold a distinguished position in the recognition of the autoimmune nature of SLE that leads to the discovery of many other autoantibodies to cellular antigens referred to as antinuclear antibodies (ANA).

Assays for anti-histone antibodies

In SLE most autoantibodies to the histone-DNA complexes can initially be detected as a homogeneous or diffuse indirect immunofluorescence (IIF) staining pattern of the nuclei and the condensed mitotic chromatin ( Fig. 28.1 A), referred to as the AC-1 (AC, anti-cell) HEp-2 cell pattern designated by the International Consensus on ANA Patterns committee (ICAP, www.ANApatterns.org ). The AC-1 pattern is also seen with other autoantibodies, notably those directed against dsDNA. This IIF pattern must be discriminated from the dense fine speckled pattern (AC-2), which also stains interphase nuclei and metaphase chromatin and is the hallmark of autoantibodies to DFS70 antigens; monospecific anti-DFS70 autoantibody is relatively common in apparently healthy individuals and is comparatively uncommon in ANA-associated rheumatic diseases. Sera with antibodies to certain histone classes (e.g., H1, H3, H4) or hidden determinants (cryptotopes) on native or on denatured histones may show a weak or even a negative ANA; thus more specific assays employing purified analytes are preferable. Anti-histone antibodies are detected by a variety of solid phase immunoassays, such as enzyme-linked immunosorbent assays (ELISA), line immunoassay (LIA), and addressable laser bead immunoassays (ALBIA).

Solid phase assays for anti-histone antibodies

ELISAs are often used to detect specific anti-histone and anti-chromatin antibodies and this platform can be adapted to measure reactivity to individual histones, macromolecular histone complexes, and chromatin. Most immunoassays for the detection of anti-histone antibodies in the past two decades have relied on histones purified from various cells or tissues, such as calf thymus. Subnucleosomal structures have also been adapted to ELISA formats, which allow measurement of autoantibodies requiring these higher-ordered structures. For example, an artificial complex of the H2A-H2B dimer and DNA can be formed in vitro at physiological conditions. This (H2A-H2B)-DNA complex is an important antigenic target in patients with SLE and some patients with drug-induced lupus (DIL). Many laboratories have adopted high throughput, solid phase multianalyte array technologies such as LIA and ALBIA that include histones as an analyte.

Problems and discrepancies in measuring anti-histone antibodies

There are many possible explanations for discrepancies in the literature on the prevalence and fine specificity of anti-histone antibodies. The quality of histones used as antigens can be highly variable, and histones from commercial sources were often degraded or contaminated with non-histone proteins. In addition, the propensity for histones to bind nascent DNA in serum and other biological fluids can result in artifacts, such as false-positive reactions with anti-DNA antibodies. Such factitious binding to histone/DNA complexes that are formed in vitro is a phenomenon that is generally indistinguishable from bona fide anti- histone antibody reaction. DNA existing in serum in the form of mono- and oligonucleosomes may also have pathologic significance in that circulating nucleosome binding to the negatively charged residues on heparin sulfate of the glomerular basement membrane may mediate the binding of DNA and anti-DNA antibodies to the glomerulus.

Antibodies to purified histones detected by most immunoassays are common in SARD and appear to have limited value as disease-specific diagnostic biomarkers. Other assays which use histone-DNA complexes, including LE cells, chromatin, soluble (H1-stripped) chromatin, (poly)nucleosomes, and (H2A-H2B)-DNA complexes would more likely be detecting reactivity to native autoepitopes. In some immunoassays there may be overlap of available epitopes in purified histones and native (DNA-bound) histones; however, for the most part, autoantibodies directed to histones and to nucleosome-related antigens should be considered distinct and one cannot be substituted for the other. In the context of autoimmunity, the terms chromatin, nucleosome and polynucleosome tend to be used interchangeably.

Prevalence and disease association of anti-histone and anti-nucleosome antibodies

Reports of anti-histone antibodies in various diseases are summarized in Table 1 . Although anti-histone antibodies have been observed in various rheumatic diseases, most studies have focused on SLE or DIL. Reported prevalence ranged from 17% to 95% in SLE and from 67% to 100% in DIL. Anti-histone antibodies have also been reported in rheumatoid arthritis and juvenile idiopathic arthritis. In some cases, a remarkably high prevalence was observed in other diseases, especially in primary biliary cirrhosis, autoimmune hepatitis, and ANA-positive neoplastic diseases.

In addition to the antibodies reactive with isolated histones, up to ∼75% of patients with lupus-like disorders have autoantibodies to chromatin (nucleosomes). In this context, it is important to appreciate that anti-nucleosome and anti-dsDNA responses appear to be particularly sensitive to corticosteroid therapy. Other studies from various geographic regions reported 38%–86% sensitivity of anti-nucleosome antibodies in SLE. Most patients with lupus induced by procainamide, penicillamine, isoniazid, acebutolol, methyldopa, timolol, and sulfasalazine also have anti-nucleosome antibodies, predominately reactive with the (H2A-H2B)-DNA complex. However, it is important to recognize that some of the drugs initially associated with DIL (i.e., procainamide, hydralazine) are no longer widely used and newer biological therapeutic agents associated with DIL have a much lower prevalence of anti-histone antibodies and a higher frequency of anti-DNA. Several groups reported anti-nucleosome antibodies in almost half the patients with autoimmune hepatitis. Anti-nucleosome antibodies have also been reported in 25%–50% of patients with systemic sclerosis (SSc), but the specificity of these antibodies may be different from bona fide anti-chromatin antibodies as discussed as follows. A high prevalence of anti-nucleosome antibodies have generally not been reported in other SARD and are remarkably rare in healthy cohorts, resulting in an overall sensitivity for SLE of 63% and when compared to other SARD, a specificity for SLE of 95%.