Histone Autoantibodies

CHAPTER

Histone Autoantibodies

23 Sylviane Muller

CNRS Immunopathologie et Chimie Thérapeutique, Institut de Biologie Moléculaire et Cellulaire, Strasbourg, France

Historical notes The presence of antihistone antibodies (AHA) in the serum of autoimmune patients was detected for the first time by Kunkel in 1960. In the 1970s and later, numerous studies described the characteristics of this class of antibodies in terms of specificity, affinity, and clinical interest. In fact, the generic term of AHA includes a complex array of autoantibodies. Evidence for the existence of histones was revealed in 1884 from studies by Kossel who reported the isolation of a so-called acid-extractable peptone-like component from goose erythrocyte nuclei. It was long after that the description of specific oligomeric histone complexes and a tandemly repeated beaded morphology for chromatin led in 1974 to the description of nucleosome entity and the introduction of its name by Chambon in Strasbourg (France). The discovery of the histone H5 was also made in Strasbourg and reported in 1976 by Crane-Robinson, Champagne, and Daune. Using newly developed methods for isolating histone–histone and ­histone–deoxyribonucleic acid (DNA) complexes, characterization of human and murine AHAs was undertaken early in the 1980s. It was demonstrated that certain AHA subsets recognized individual histones while others preferentially reacted with histone complexes, associated or not with DNA (e.g., H2A-H2B or H3-H4 complexes, (H2A-H2B)-DNA subparticles, and nucleosomes), but not with ­individual histones.

Autoantigens The nomenclature for histone fractions was proposed and subsequently universally adopted in 1974. It was based on their chromatographic fractionation behavior. The four core histones are H2A, H2B, H3, and H4. Linker histone species (also called extranucleosomal, lysine-rich histones) are H1, H5, and H1°. In fact, each histone fraction is composed of a number of isoprotein species or variants, designed for example, as H3.1, H2A.X, H2A.Z, macroH2A1, or 2, H1b. Certain variants, such as H1°, H5, CENP-A (centromeric H3 variant), H1t, TH2B, H2BFWT, or H3t, occur in specific tissues only. Core histones are basic proteins that are highly water soluble. All except H2B and H3 are acetylated at their N-terminus (Table 23.1). It is now well established that in the nucleus, histones play signaling roles in essential regulatory events. Certain histone variants have been shown to be expressed in different cell types, such as Autoantibodies. http://dx.doi.org/10.1016/B978-0-444-56378-1.00023-X Copyright © 2014 Elsevier B.V. All rights reserved.

195

196

CHAPTER 23  Histone Autoantibodies

Table 23.1  Principal Characteristics of Calf Thymus Histones and Location of (Auto)Epitopes

Fractions

Mr

Number of residues

N-terminal residue

H1b3

26,500

220

Ac-Ser

H2A H2B H3

14,000 13,770 15,340

125 129 135

Ac-Ser Pro Ala

H4

11,280

102

Ac-Ser

Known B-cell autoepitopes1 (human/mouse) 74–94, 144–159, 170–185, 197–212, 204–218 1–20, 65–85, 91–129 1–25, 21–38 1–21, 40–55, 47–64, 53–70, 64–78, 111–130, 130–135 1–29, 28–42

Post-translational modifications that enhance autoantibody reactivity2

Ub-Lys 119 Ac-Lys 12 TriMe-Lys 27 Ac-Lys 8 and/or 12 and /or 16

Ac: acetylation; Me: methylation; Ub: ubiquitination. 1Results obtained with murine monoclonal antibodies or generated after immunizing animals with autoantigens are not indicated. Major histone epitopes recognized by T cells from SLE patients are located in residues 22–42 of H1’, 10–33 of H2B, 95–105 of H3, 16–39 and 71–94 of H4. 2Modifications in H2B, H3, and H4 are related to apoptosis. 3Main isoform.

developing male germ cells, where they alter the fine structure of nucleosome. Histones are also subject to numerous post-translational modifications including phosphorylation, methylation, deimination/ citrullination, acetylation, ubiquitination, SUMOylation, and poly(ADP-ribosyl)ation, which varies with species, tissue, and stage of cell cycle [1,2]. They are primarily located on their N-terminal tails, but also in their globular domains and more rarely in their C-termini. These modifications dramatically influence chromatin structure and DNA storage by altering histone–DNA interaction to facilitate transcription, DNA replication, and DNA repair, for example. Multiple modifications can take place on a single residue side chain, and a synergistic or antagonistic cross-talk exists that facilitates or hampers cis or trans modifications on the same or a different histone. These complex histone modifications constitute the so-called epigenetic histone code, which plays a central role in determining and stabilizing gene expression patterns from one generation to the next (heritable cell memory). Certain histone modifications appear or disappear specifically during apoptosis, and it has been postulated that apoptotic chromatin condensation and fragmentation might be a consequence of such modifications. ­Aberrant post-translational histone modifications have been found in murine models of lupus [3]. A number of specific enzymes involved in histone modifications have been identified. Histones are present in all eukaryotic nuclei with a few exceptions since, for example, yeast does not contain H1. Each mammalian diploid nucleus contains about 40% (weight/weight (w/w)) DNA (5 × 109 base pairs of DNA), 40% histones, and 20% of other compounds (nonhistone proteins and ribonucleic acid (RNA)). In nucleated erythrocytes from birds, fishes, and reptiles, H5 replaces a part of H1 during the process of genetic inactivation. H1° accumulates in nondividing cells or in cells that have been chemically induced to differentiate. Germ cell-specific histones are either absent from oocytes or present in much lower concentrations than in comparable stages of male germ cells.

Autoantibodies

197

Whole histone mixtures and individual histones, which are unmodified or display unique modifications, are currently available from several suppliers. Commercial histones are generally prepared from calf thymus by the acid method or are of recombinant origin. They should be checked for purity and net protein content. When evaluating the purity of histone fractions, it should be kept in mind that each histone type presents different affinities for the usual protein stains (Coomassie blue, amido black) and, therefore, do not stain equally. In clinical practice, individual histones are rarely used for studying the presence of AHA. Purified individual histones can be kept in lyophilized form for years under dry conditions at 20 °C. When resuspended in solution, however, histones should not be stored longer than 1 or 2 weeks at 4 °C since they form homoaggregates, especially at high ionic strength and concentration, leading to changes in their antigenic properties. The purity of histone fractions can be evaluated by electrophoresis in different gel systems including sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and acetic acid–urea gels with or without Triton. These procedures, however, are not very sensitive and it is not uncommon to find that fractions that appear to be pure by biochemical criteria are in fact contaminated by as much as 1–2% of other histone classes. In some cases, and depending on the conditions used for immunoassays, this degree of contamination can be sufficient to influence the results. ­Synthetic peptides are used classically for epitope mapping because they can be chemically controlled and ­produced in large amounts at relatively low cost. Improvements in solid-phase peptide synthesis allow producing peptides bearing natural or non-natural modifications [4].

Autoantibodies AHA are part of the so-called antinuclear autoantibodies family. They can be of the immunoglobulin (Ig)M or IgG type and comprise a heterogeneous set of antibodies (see above). Major linear autoepi­ topes of histones have been identified. They are mainly located in the N-terminal end of H2A, H2B, H3, and H4 and in the C-terminal end of H3 and H1 [5] (Table 23.1), which also encompass most of the sites of post-translational modifications (Fig. 23.1) and are particularly exposed in chromatin. These antigenic regions are generally the same as those recognized by antibodies raised in New Zealand white (NZW) rabbits against nucleosomes and a total histone mixture complexed with RNA. Administered in the absence of RNA, however, histone mixtures generate antibodies that react very rarely with histonederived antigenic determinants, supporting the idea that chromatin or histone–DNA complexes ­represent the antigenic stimulus giving rise to AHA. AHA can be visualized as a homogeneous diffuse nuclear staining on HEp-2 cells upon indirect immunofluorescence (IIF) analysis. However, antidouble-stranded deoxyribonucleic acid (dsDNA) autoantibodies can yield the same pattern; therefore, it is necessary to perform complementary testing. Western immunoblotting (WB), dot-immunoassays, and enzyme-linked immunosorbent assay (ELISA) provide easy, efficient, and sensitive methods for detecting AHA. Those three techniques have been extensively compared regarding their sensitivity for AHA detection. ELISA tests were generally found to be slightly less sensitive than WB and dot-immunoassays (e.g., about 50% vs. 70% in a comparative study using sera from lupus patients), but it sometimes allows detecting cases that appear negative using the other methods. This is due to the fact that different types of epitopes (linear vs. conformational) are detected using the three different techniques. Although both ELISA and WB essentially reveal reactivity to partially denatured histones, discrepancies have even been found between the

198

CHAPTER 23  Histone Autoantibodies

FIGURE 23.1 Location of post-translational modifications of the four core histones. The modifications include acetylation (Ac), methylation (Me), phosphorylation (P), and ubiquitination (Ub). They mainly occur in the N-terminal regions of histones.

results obtained with these two methods: for example, anti-H3 antibodies were mostly detected in WB whereas anti-H1 antibodies preferentially reacted with H1 when coated onto ELISA plates. Specificity of ELISA and dot-immunoassays is obviously highly dependent on the purity of the proteins used and it should be kept in mind that commercial histones frequently do not achieve a high percentages of purity. Furthermore, sera should be pre-incubated with DNase in order to digest circulating DNA and avoid unwanted formation of DNA–histone complexes. Some histone-binding serum proteins such as C-reactive protein, nucleolin, actin, or myosin, for example, can also affect the detection of circulating AHA. AHA are present with significant frequencies in several systemic and organ-specific autoimmune diseases (e.g., SLE, rheumatoid arthritis (RA), juvenile chronic arthritis (JCA), primary biliary ­cirrhosis (PBC), autoimmune hepatitis, dermatomyositis/polymyositis, and scleroderma). They are also detected in drug-induced lupus (DIL; reversible lupus-like syndrome induced by drugs such as procainamide, hydralazine, or more recently by antitumor necrosis factor (TNF)-α agents), in neurologic diseases (e.g., subacute sensory neuropathy or Alzheimer disease), and certain infections. AHAs have been detected in HIV-infected individuals, in patients with infectious mononucleosis, and in dogs with viscerocutaneous leishmaniasis. The current view, however, is that AHA are poorly pathogenic. A number of studies trying to assess the involvement of AHA in the development of lupus have been performed in humans and animal lupus models such as (NZBxNZW)F1 and MRL/Mp-lpr/lpr mice. It was observed that serum AHA levels often decrease significantly just before the appearance of ­glomerular injury and proteinuria, suggesting that AHA may bind to unsaturated epitopes of the autoantigen in target organs. Antigenic deposits containing histones have effectively been detected (even more frequently than deposits detected with anti-DNA or antinucleosome antibodies) in both glomerular and epidermal basement membranes of a large number of lupus patients [6]. Histones are probably deposited as part of nucleosomal structures, which bind via histone positively charged N-termini to heparan sulfate (HS) present in basement membranes. This “planted autoantigen” accessible to circulating autoantibodies might serve as the starting point of in situ immune complex formation leading to a cascade of inflammatory events. Another possibility is that already formed circulating immune ­complexes bind to HS or other constituents of the basement membranes. It could be argued that

Clinical utility

199

nucleosome–AHA complexes may display a low affinity for HS since AHA will neutralize positive histone charges, and, therefore, that AHA may not be able to promote pathogenic effects in target organs. More recently, antibody-secreting cells present in different organs of (NZBxNZW)F1 mice have been characterized and it was shown in diseased mice that compared to normal and pre-diseased mice, the frequency of cells secreting antibodies reacting with the fragment 1-25 of histone H2B was much higher in their spleen, bone marrow, and locally, in nephritic kidneys [7]. AHA can be eluted from glomeruli of lupus-prone mice harboring glomerulonephritis. However, in contrast to anti-dsDNA and nucleosome autoantibodies, the small amount of AHA collected does not directly correlate with the severity of nephritis. Several studies have described apoptosis-specific post-translational modifications, which appear to be determining for their recognition by AHA in lupus. They are, for example, acetylation sites in H4 and H2B, methylation in H3, and ubiquitination in H2A (Table 23.1). Assays based on such modified histones or histone synthetic peptides might greatly help improve the detection of relevant AHA subclasses and shed light on pathophysiologic effects that have been ignored until now [8]. Interestingly, it was reported in 2008 by Neeli and Radic that histone H3 is citrullinated in neutrophil extracellular traps (NETs). This first description was confirmed and further extended by others showing that histones are subjected to extensive post-translational modifications in NETs [9]. Knowing that the process of socalled NETosis seems to be decisive in the development of human autoimmune responses, research is strongly reactivated to understand the possible involvement or requirement of these unique histone modifications in the process. Very little is known about the genetics of AHA production. An association has been described between production of anti-H3 antibodies and the HLA-A2-DR4 haplotype in a group of American patients with type 1 autoimmune hepatitis. AHA have also been associated with HLA-A2 in children with pauciarticular onset JCA, but this association might simply reflect the known association between A2 and this form of the disease. No extended genetic studies have been initiated in AHA-positive and -negative asymptomatic relatives of patients with SLE and RA or in AHA-positive twins. V regions of the light and heavy chains of a number of monoclonal AHA derived from lupus-prone mice and SLE patients have been sequenced [10]. Altogether these results showed that i) there is no restricted usage of VH, DH, JH, Vκ, and Jκ gene families, suggesting that certain gene segments are not critical for histone recognition; ii) the V regions of monoclonal antibodies to histones, nucleosomes, and DNA bear striking similarities, suggesting that common pathways lead to the expansion of B-cell clones reactive with several chromatin components; iii) most of the V sequences of AHA show extensive somatic mutations, in agreement with the fact that histones (in nucleosome structures) presumably play a key selecting role in the generation of high-affinity autoantibodies; and iv) the complementarity determining regions of monoclonal AHA contain a high number of negatively charged amino acid ­residues that may play an important role in recognition and binding to cationic histones; this contrasts with the cationic charge often observed in anti-DNA antibodies.

Clinical utility Contradictory and/or nonconsistent observations have been published concerning the precise clinical significance of AHA. The latter not only occur in a number of autoimmune and nonautoimmune clinical conditions but also show a striking patient-to-patient variability with regard to their fine reactivity

200

CHAPTER 23  Histone Autoantibodies

with histone subfractions. On average, AHA are produced by 50% of lupus patients and are classically associated with DIL (more than 90% of patients affected by DIL possess AHA). However, although a negative screen makes a DIL unlikely, production of AHA does not allow distinction between SLE and DIL. Likewise, no definite association between the levels of circulating AHA and the overall disease activity has been demonstrated. One study, based on the use of glomerular proteome arrays, showed that, in the case of SLE, AHA are part of an IgG antibody cluster that is not associated with disease activity, in contrast to the cluster encompassing anti-DNA/chromatin IgG [11]. One study, however, has described a strong correlation between the presence of antibodies recognizing H1 (and more precisely a major H1 epitope encompassing residues 204–218) and lupus disease activity. Another study suggested that antibodies directed toward core histones (mainly H2B) might be associated with severe clinical features in scleroderma. Frequencies of patients producing AHA varies widely from one disease to the other, and also within one type of disease, depending on the criteria used to define cohorts of patients and on the detection method used. Thus AHA are present in the serum of patients with SLE (30–70%), DIL (90–95%), RA (5–50%), JCA (from 10–20% in a study performed in India to >50% in other studies), scleroderma (5–45%), poly/dermatomyositis (20%), PBC (60–80%), and autoimmune hepatitis (35%). In contrast to antibodies reactive with epitopes requiring the native histone/DNA structure (e.g., antinucleosome antibodies), autoantibodies to denatured histones do not appear to be diagnostically useful in SLE, DIL, or any other (autoimmune) disease. Moreover, although major histone epitopes recognized by circulating AHA have been defined at the level of short peptide sequences, this k­ nowledge has not allowed assisting neither diagnosis nor prognosis.

Take-home messages • A  HA are frequently produced by patients suffering not only from SLE and DIL but also from other autoimmune, neurologic, and infectious diseases. • In contrast to ANA, AHA are poorly pathogenic. • Presence of AHA in patients’ sera does not have any diagnosis or prognosis value. • Apoptosis-specific post-translational modifications of histones are central targets for AHA in SLE. Such modified histones might represent relevant probes in AHA assays.  

References [1]  Cosgrove MS, Boeke JD, Wolberger C. Regulated nucleosome mobility and the histone code. Nature Struct Mol Biol 2004;11:1037–43. [2]  Margueron R, Trojer P, Reinberg D. The key to development: interpreting the histone code? Curr Opinion Gen Dev 2005;15:163–76. [3]  Garcia BA, Busbi SA, Shabanowitz J, Hunt DF, Mishra N. Resetting the epigenetic histone code in the ­MRL-lpr/lpr mouse model of lupus by histone acetylase inhibition. J Proteome Res 2005;4:2032–42. [4]  Briand J-P, Muller S. Synthetic peptides for the analysis of B-cell epitopes in autoantigens. In: Pollard KM, editor. Autoantibodies and Autoimmunity: Molecular Mechanisms in Health and Disease. Weinheim, ­Germany: Wiley-VCH; 2006. p. 189–224.

References

201

[5]  Fournel S, Muller S. Synthetic peptides in the diagnosis of systemic autoimmune diseases. Curr Protein Pept Sci 2003;4:261–76. [6]  Grootscholten C, van Bruggen MCJ, van der Pijl JW, de Jong EMGJ, Ligtenberg G, Derksen RHWM, et al. Deposition of nucleosomal antigens (histones and DNA) in the epidermal basement membrane in human lupus nephritis. Arthritis Rheum 2003;48:1355–62. [7]  Lacotte S, Dumortier H, Décossas M, Briand JP, Muller S. Identification of new pathogenic players in lupus: autoantibody-secreting cells are present in nephritic kidneys of NZB/W mice. J Immunol 2010;184:3937–45. [8]  Dieker J, Muller S. Epigenetic histone code and autoimmunity. Clin Rev Allergy Immunol 2010;39:78–84. [9]  Liu CL, Tangsombatvisit S, Rosenberg JM, Mandelbaum G, Gillespie EC, Gozani OP, et al. Specific ­post-translational histone modifications of neutrophil extracellular traps as immunogens and potential targets of lupus autoantibodies. Arthritis Res Ther 2012;14; R25. [10] Monestier M, Decker P, Briand JP, Gabriel JL, Muller S. Molecular and structural properties of three ­autoimmune IgG monoclonal antibodies to histone H2B. J Biol Chem 2000;275:13558–63. [11] Zhen QL, Xie C, Wu T, Mackay M, Aranow C, Putterman C, et al. Identification of autoantibody clusters that best predict lupus disease activity using glomerular proteome arrays. J Clin Invest 2005;115:3428–39.