European Journal of Clinical Investigation (1979) 9,239-241

EDITORIAL

Antibodies t o DNA antigens: their specificity and clinical relevance

Antibodies to nuclear antigens have been detected in a wide variety of connective tissue disorders and may offer diagnostic help by virtue of being associated with certain clinically definable diseases: RNA antibodies in scleroderma, RNAse and trypsin-sensitive ‘extractable nuclear antigen’ antibodies in mixed connective tissue disease, PM, antibodies in polymyositis, antibodies to lymphocyte cellular antigens A, B and C in Sjogren’s syndrome, soluble nuclear non-nucleic acid antigen (Sm) antibodies in SLE, and DNA antibodies in SLE. It should be stressed, however, that while the detection of such antibodies may support a clinical diagnosis, they cannot be assumed to be pathognomonic of any disease at this stage. Indeed, knowing the wide clinical overlap of many of these disorders and the tendency for disease patterns to change with time, it would be naive to expect any single antibody to be confined solely to one disorder. A glance at the history of the biochemistry of nucleic acids may be reassuring to today’s immunologist. The painful step-by-step elimination of methodological errors, the gradual development of more refined techniques, the few vital milestones signalling a quantum leap in understanding, all have their counterparts in the history of the development of our understanding in the field of antinuclear antibodies. The quantum leaps in understanding were provided by Hargreaves, who offered the springboard from which the entire field developed with his description of the LE cell phenomenon, by the description of ‘antinuclear factors’ and by the subsequent demonstration that these were a collection of antinuclear antibodies. The development of the immunofluorescent antinuclear antibody test in the 1950shas now largely displaced the LE cell test and the variations in nuclear staining patterns observed pointed to the heterogeneity of antinuclear antibodies. This prompted investigation into the purification and isolation of the antigens involved, and such studies led to the demonstration of free DNA, antibodies to DNA and complement fixing DNA-anti DNA complexes in both the sera and tissue, focusing attention on this system as a putative pathological mechanism. A wide variety of techniques have been applied to the detection of anti-DNA antibodies in sera. Precipitin reactions in gel, either as Ouchterlony reactions or by immuno-electroprecipitation, and methods depending upon complement fixation or haemagglutination have become less popular on the grounds of 001 4-2972/79/0800-0239S02.00

01979 Blackwell Scientific Publications

technical difficulty, insensitivity, or lack of reproducibility. While some of these methods suffer from the drawback of being qualitative rather than quantitative, it should be emphasized that they may provide information on the characteristics of antibody-antigen reaction not necessarily available from the more recently developed techniques. The use of radiolabelled nuclear antigens has led to the introduction of more reproducible and sensitive assays for detecting nuclear antibodies. A variety of different in uitro (external ) and in vivo (internal) chemical and biological radiolabelling techniques have been developed. Calf thymus DNA has been labelled in vitro with 3H actinomycin D or dimethyl sulphate and 1251 using thallium chloride. These procedures radiolabel not only double-stranded but also single-stranded DNA and run the risk of also radiolabelling any nuclear protein impurities which may be present. Biological radio-iodination in vivo using iododeoxyuridine (a thymidine analogue) will label only nucleic acids and has the advantage of being a gamma emitter. The inevitable lower specific activity is balanced by a reduction in the likelihood of radiation damage caused by the radiolabel. The most widely accepted radiolabelling technique at present in the incubation of growing cells with I4C thymidine. The wide variety of sources of DNA used is reflected by a recent Multicentre Study [ 11 in which no less than twelve sources of DNA were used, including bacteria (E. coli, B. subtilis, M . lysodeikiticus), protozoa (Crithidiu lucifiue), bacteriophages (PM2, T4), and mammalian sources (calf thymus, human cell lines KB and HeLa). Different sources may show minor differences in reaction kinetics, but of greater importance is the molecular weight of the DNA, and it has been shown that the percentage binding activity of sera is linearly dependent on the molecular weight of DNA up to lo7 Dalton’s [2]. Probably the greatest factor influencing binding activity is whether the DNA exists in either single-stranded (SS) or double-stranded (DS) (‘native’) form. In practice, native DNA may be converted into SS DNA either by boiling and rapid cooling or by treatment with alkali. The problem is complicated by the propensity of SS DNA to reform into DS DNA and compounded by the almost invariable presence in DS DNA of SS sections or ‘frayed ends’ [3]. The lesson that DNA must be handled by only the most delicate methods was learned many years previously by biochemists, and even extrusion of samples through small bore needles may result in the formation of SS regions 239

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in DS DNA. Attempts to separate SS from DS DNA never provide 100% separation, and the methods most commonly used at present depend upon the differing elution characteristics of SS DNA compared to DS DNA from either hydroxyapatite or methylated albumin kieselguhr columns. Enzymatic methods using endonucleases and exonucleases have suggested that most of the single-stranded regions present in DS DNA exist at the ends of the helix [3]. Contaminating protein may be removed either chemically or by the use of enzymes such as pronase. At present the most theoretically attractive contenders as a source of DS DNA antigen are radiolabelled circular DNA extracted from PM2 bacteriophages or crithidia lucillae, which may be used in an immunofluorescent technique. Both these sources appear to be free from SS DNA breaks. Certainly, when crithidia immunofluorescent tests were used, the results appeared to be as meaningful as those obtained using radiolabelled DS DNA antigens [l]. A paper in the present issue of this Journal [4] focuses attention on the problem of finding the optimum antigen. The DS DNA antigen employed apparently provides useful separation of SLE from non-SLE sera despite its SS regions. The authors suggest that even greater definition may be obtained if the ratio of DS to SS DNA binding is considered. Separation of radiolabelled antigen-antibody complexes may be achieved in several ways, including highspeed centrifugation, the use of solid phase absorbants, and second antibody precipitation, but most laboratories today employ some modification of the Farr ammonium sulphate assay [5]. This depends on the stability of DNA-anti-DNA complexes in halfsaturated ammonium sulphate, free DNA remaining in solution while DNA-bound to globulin is precipitated. The Farr test is not a functional assay of antibody activity, nor can it detect which class of antibody is involved. It expresses the 1:l binding of strongly positively charged DNA to negatively charged protein (usually anti-DNA antibody) and is open to the criticism that low affinity antibody may be dissociated in the high salt concentration. However, it has the advantage of detecting primary antigen-antibody reaction. The results of the Farr test may be critically dependent on variations in molarity, temperature, hydrogen ion concentration, incubation volumes and timing, and contaminating proteins. Many of these may be minimized by attention to methodological detail and the inclusion of established standards [6]. The essential debate at present concerns the relative specificities of antibodies to DS DNA versus SS DNA. There is no doubt that antibodies to SS DNA are common in lupus sera and may fluctuate in proportion to disease severity. However, anti-SS DNA antibodies are also commonly encountered in a wide variety of other connective tissue diseases [7] while antibodies to DS DNA appear to be more specific for SLE and again may fluctuate with disease severity in individual patients. Depending on the monitoring system

employed, antibodies to DS DNA have been found in low frequency in conditions other than SLE such as chronic discoid lupus, chronic active hepatitis, and Sjogren’s syndrome [6]. When this occurs, such antibodies are usually present in lower titre than in spontaneous SLE. Certain groups feel that the variation in binding between SS and DS DNA is purely quantitative and claim that when the DNA binding activity is expressed in picomols DNA-bound per unit serum then the binding of SS DNA always exceeds that of DS DNA [l]. However the weight of available evidence suggests that certain patients do indeed carry antibodies with pure DS DNA binding specificity. It is important to point out that undue concentration on the problems of quantifying DNA antibody may divert attention from other characteristics of the antibody which may be of equal importance, such as the affinity of the antibody [8] and the class of immunoglobulin involved and its possible change with disease duration [9]. Moreover, attention has been drawn to the variation in affinity of DNA antibody eluted from the kidney (high) when compared to that of circulating antiDNA-antibody in the same patient (low), raising doubts about the validity of extrapolating serological findings to those of tissue pathology [lo]. The question of why DNA, which is a poor immunogen in all mammals, should apparently become immunogenic in SLE, is still unanswered. It is known that DNA is released into the circulations of patients after burns, major trauma and treatment of neoplasms without evoking an anti-DNA antibody response. SS DNA or native DNA exposed to UV irradiation, on the other hand, may be rendered immunogenic. An attractive hypothesis is that, in immunogenetically predisposed individuals, native DNA altered by a variety of exogenous agents such as a C type virus infection or UV irradiation becomes immunogenic. The animal evidence of a transmissible, filtrable agent in murine and canine lupus provides exciting support for this hypothesis. The need for a reliable and robust DNA antigen remains. At present a DS DNA antigen remains the most attractive candidate in terms of diagnostic specificity. Although most of the currently available DS antigens have disadvantages in terms of lack of homogeneity, they still continue to provide useful clinical information in diagnosing and monitoring the course of SLE patients.

I. D. GRIFFITHS W. CARSON DICK Uniilersity Department of Rheumutology, Royal Victoria Infirmary, Newcastle upon Tyne References I Maini R.N. & Holborrow E J . (eds.) (1977) Detection and measurement of circulating soluble antigen -antibody complexes and anti-DNA antibodies. A m Rheuni Di.v 36S, 1 - 142.

EDITORIAL 2 Aarden L.A.. Lakmaker F. & Feltkamp T.E.W. (1976) Immunology of DNA: the effect of size and structure of the antigen on the Farr Assay. J Immunol Merhod~10, 39-48. 3 Samaha R.J. & lrvin W.S. (1975) DNA strandedness, partial characterisation of the antigen regions binding antibodies in lupus sera. J Clin Inrrs/ 56, 446-457. 4 Couture F., Beaulieu A,, Raptis L. & Menard H. (1979) Operationally defined single and double-stranded DNA antigens in the Farr Assay: diagnostic value. Eur J Clin Inrr.sr 9, 243-25 I . 5 Wold R.T.. Young F.E. & Tan E.M. (1968) DNA antibody: a method to detect its primary interaction with DNA. Sricwt- 161, 806-807. 6 Holian J . , Griffiths I.D.. Glass D.N.. Maini R.N. & Scott J.T. (1975) Human anti-DNA antibody: reference standards for diagnosis and management of systemic lupus erythematosus. Ann Rhrur~iDis 34, 438-444.

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7 Koffler D., Carr R.I.. Agnello V., Fiezi T. & Kunkel H.G. (1969) Antibodies to polynucleotides: distribution in human serums. Science 166, 1648-1649. 8 Steward M.W., Katz F.E. & West N.J. (1975) The role of low affinity antibody in immune complex disease, the quantity of anti-DNA antibodies in NZBI’WF hybrid mice. Clin E,vp Imniunol21, 121-130. 9 Tala1 N. & Pillarisetty R. (1975) I g M and IgG antibodies to DNA. RNA and DNA-RNA in systemic lupus erythematosus. Clin Imniunol Immunopa/hol4, 24-3 I . 10 Winfield J.B., Faiferman I . & Koffler D. (1977) Avidity of antiDNA antibodies in serum and IgG glomerular eluates from patients with systemic lupus erythematosus. J Clin Inivst 59, 90-96.

Antibodies to DNA antigens: their specificity and clinical relevance.

European Journal of Clinical Investigation (1979) 9,239-241 EDITORIAL Antibodies t o DNA antigens: their specificity and clinical relevance Antibod...
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