Rheumatology Section, Division of Medicine, Imperial College School of Medicine, Hammersmith Hospital, London W12 0NN and
1 Division of Immunology, Hammersmith Hospital NHS Trust and Kennedy Institute of Rheumatology, 1 Aspenlea Road, London W6 8LH, UK
Autoantibodies to nuclear antigens, including DNA, were discovered over 40 yr ago and have become the hallmark of systemic rheumatic disease. Antinuclear antibodies (ANA) are present in the sera of 9599% of patients with the prototypic autoimmune disease, systemic lupus erythematosus (SLE), but may also be found frequently in many other connective tissue disorders. ANAs are useful aids in the diagnosis of many rheumatic diseases, but have also shed light on the role of nuclear antigens in autoimmune disease. In this short article, we will highlight the key experiments that led to the discovery of these antibodies.
The LE cell phenomenon was first observed in 1948 by Hargraves et al. [1] in blood from patients with SLE. It was noted that, when leucocytes were incubated with serum from such patients, nuclear alterations developed together with phagocytosis of nuclear remnants by mature polymorphonuclear leucocytes. This crucial observation went on to receive considerable attention in the ensuing years.
Hargraves and others demonstrated that the LE cell phenomenon was related to a serum factor reacting with nuclear material [2, 3], subsequently termed antinuclear factor (ANF). Serum from patients with SLE was added to bone marrow preparations from normal subjects which, when compared with control preparations using normal sera, induced the formation of clumps of polymorphonuclear leucocytes around amorphous masses of nuclear material. This phenomenon was specific for SLE when compared with discoid lupus. Haserick et al. [4] then went on to demonstrate that the responsible factor in the serum from SLE patients was present in the gamma-globulin fraction, and in vitro could cause swelling and disintegration of the nuclei of mature polymorphs with phagocytosis of nuclear remnants. Histochemical techniques were then used to confirm the presence of altered nuclear material in the tissues of patients with SLE [5].
The more precise relationship between the LE cell phenomenon and this factor present in the gamma-globulin fraction of the serum was the next area to be explored. Miescher and Fauconnet [6] demonstrated that absorption of lupus serum with nuclei prevented its ability to induce the LE cell phenomenon, suggesting that a globulin in the sera was reacting with or destroying nuclei. Friou et al. [7] then used a fluorescent antibody technique to confirm that lupus serum contains a globulin factor with affinity for nuclei. Affinity for calf thymus nuclei and mammalian tissues, such as normal human kidney and mouse spleen, was examined using sera from 100 subjects, 28 of whom had SLE. Nine of these patients had a positive LE cell preparation. A fluorescently labelled anti-human globulin was added to nuclear preparations after incubation with test sera and increased binding was shown when SLE sera were used. The most marked nuclear staining occurred with sera from patients with active disease.
Holborrow et al. [8] confirmed this observation using a number of human tissues obtained after post-mortem, including adult kidney cortex, neonatal thyroid and skin. Sections were incubated with LE-positive or normal sera then with anti-human globulin labelled with fluorescein isothiocyanate. Specific nuclear staining was demonstrated using lupus sera. Buffy coat preparations were also used to demonstrate the fluorescence of leucocyte nuclei. It was concluded that the LE cell factor was an antibody with specificity for tissue cell and leucocyte nuclei.
The recognition of antibody reactive with DNA occurred at about the same time. An Italian group investigated whether DNA preparations would react with human pathological sera [9]. They studied sera from patients with SLE and discoid lupus, from normal controls and from patients with various diseases associated with hypergammaglobulinaemia, using DNA obtained from both normal and leukaemic leucocytes. A complement fixation test was used to demonstrate a factor in the serum of one SLE patient that specifically reacted with purified DNA. It did not discriminate between DNA from different species.
Robbins et al. [10] performed similar, independent experiments using a complement fixation test with calf thymus and salmon sperm DNA or calf thymus nuclei as antigen and fresh guinea-pig serum as the complement source. Positive complement fixation occurred with nuclei and 22 of 30 lupus sera, all of which also induced the LE cell phenomenon. Sera from normal controls or patients with rheumatoid arthritis failed to react. Most lupus sera that showed complement fixation with nuclei also reacted with DNA. Furthermore, absorption of lupus serum with nuclei removed its ability to induce the LE cell phenomenon, but complement fixation with DNA was unaffected. However, after absorption with DNA, complement fixation was substantially reduced while the LE cell phenomenon and complement fixation with nuclei remained unaffected. Thus, it appeared that at least two distinct serum factors were present in sera from patients with SLE.
Further evidence for distinct anti-DNA antibodies came subsequently using immunoprecipitation [11] and agglutination [12]. Later biochemical observations demonstrated the presence of antibodies reactive with native (double-stranded) and denatured (single-stranded) DNA. A number of groups described antibodies that reacted with native DNA but did not cross-react with single-stranded DNA or vice versa [13, 14]. In the late 1950s and early 1960s, extensive research was carried out into the pathogenic mechanisms involved in immune complex-mediated vasculitis and with the aid of immunofluorescence techniques the fundamental role of anti-DNA antibodies in this process began to be elucidated. A number of clinical observations ensued, including the increase in anti-DNA antibody titres with disease flares [15] and the association between these antibodies and renal disease [16].
The early techniques of indirect immunofluorescence used in the discovery of antinuclear factor have evolved and remain widely used in the detection of ANAs. Whilst early methods made use of cryopreserved tissues and organs as substrates, tissue culture cell lines have now replaced them. Figure 1 shows some of the common staining patterns obtained with different sera. Multiple staining patterns have been described, and it has been noted that these can often occur with the same sera as a result of ANAs reacting with different nuclear or nucleolar antigens. Other key developments include the discovery of anti-Sm antibodies specific for SLE [17] and the description by Pincus et al. of the DNA-binding assay [18, 19]. Numerous antibodies to non-histone nuclear proteins and RNAprotein complexes were described in the 1970s and 1980s, some of which were also found to be relatively disease-specific. A description of the discovery of various antibodies to extractable nuclear antigens is beyond the scope of this article, but the interested reader is directed to a review by Tan [20].
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Notes
Correspondence to: A. L. Hepburn.
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