Antinuclear antibodies (ANA): Immunologic and clinical significance

S eminars

in and Rheumatism

Arthritis VOL.

VI,

NO.

NOVEMBER

2

Antinuclear

Antibodies (ANA): Immunologic Clinical Significance

By Felix Fernandez-Madrid

and Martha

1976

and

Mattioli

A

NTINUCLEAR ANTIBODIES (ANA) are a diverse collection of antibodies directed against numerous discrete macromolecules which are normal components of the cell nucleus. This field originated with the discovery of the lupus erythematosus (LE) cell factor by Hargraves et al., almost 28 years ago.’ This interesting observation opened a new area to which many investigators have directed their efforts. Consequently, a great deal of progress in the understanding of the immunologic nature of the LE cell has been achieved and this has led to the identification of an ever-growing number of autoantibodies. Shortly thereafter, several laboratories reported similar factors in sera from patients with systemic lupus erythematosus (SLE) as well as other diseases.2-7 In the late 1950’s and early 1960’s it became evident that the “antinuclear factors” belonged to the gamma globulin fraction of the serum2*3 and were indeed antibodies.8-‘2 These earliest works have been reviewed,13 and excellent accounts of various aspects of the antinuclear antibodies are available in the literature.‘4-‘7 Later work characterized some of the antigenic specificities of these antibodies and several investigators showed that ANA cross-react in general with nuclei from different sources, hence lacking tissue or species specificity. There are a few exceptions to this generalization, including certain sera possessing ANA reacting specifically with nuclei from leukocytes, 18--21thyroid, gastric mucosa, adrenal cortex,22 and erythrocytes.23r24 Systemic lupus erythematosus is the disease in which a variety of ANA have been more deeply analyzed. 25-32 The resolution of this heterogeneous group of antibodies has provided important information for the understanding of this disease. From the Deparimeni of Medicine, WayneState University, Detroit, Mich.. and the Department of Medicine, State University of New York, Buffalo, N. Y. Felix Fernandez-Madrid, M.D., Ph.D.: Chief Division of Rheumatology and Clinical Immunology Professor of Medicine, Wayne State University School ofMedicine, Detroit. Mich. Martha Mattioli, M.D.: Research Associaie. State University of New York, Buffalo, N. Y.; present address: Baylor College of Medicine, Dept. of Pathology, Houston, Texas. Supported by USPHS Research Grant 2-RO I-AM 10428, the Research Fund, Veterans Adminhtration Hospital, Buffalo, N. Y.. and the Michigan Chapter, Arthritis Foundation, Address for reprint requests: Felix Fernandez-Madrid, M.D.: Wayne State University School of Medicine, 540 Confield Ave., Detroit, Mich. 48201. s 1976 by Grune & Stratton, Inc. Seminars in Arthritis and Rheumatism,

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The pathogenetic role of some of these antigen-antibody reactions in SLE nephritis through the immune complex disease mechanism has been established.33-37 ANA can also be present in fluids other than serum and sometimes have been detected only in synovial fluid. 18*38 In patients with proteinuria, urine has proved to be a good source for the study of these antibodies. Lastly, it is worth noting that ANA are not restricted to humans. They have been described in several other species, including the mouse,3Q-41dog,42*43mink,44 and chicken.23,24 METHODS

TO DETECT ANA

The detection of ANA in human sera has been approached by a large number of different methods. Some of these, such as immunofluorescence, have shown to be of practical application and are used routinely in clinical laboratories. More sophisticated techniques are useful only in the hands of reliable investigators and are applied mostly on an experimental basis. As expected, all of the techniques used to detect ANA vary in sensitivity and reproducibility. Methods in current use can be separated into two groups: those that detect any type of ANA regardless of their specificities (screening methods); and those used to investigate individual ANA specificities, involving a separate test for each antigen. When purified antigen is not available, many investigators resort to treatment of the complex antigens with specific enzymes or to inhibition experiments with known substances. Some of the ANA have first been characterized by the Ouchterlony technique in agar gels with prototype serum.45p46 Screening Methods Indirect Immunofluorescent

ANA (IF-ANA)

Test

In 1957, Friou adapted the indirect immunofluorescent technique47 described by Coons and Kaplan4s for the study of ANA. The usefulness of this test was confirmed,6*4gand it soon became widely used in the clinical laboratory for the evaluation of the connective tissue diseases (CTD). The indirect IF-ANA test is very sensitive and capable of detecting many antibody specificities directed against nuclear antigens. In most of the variations of this technique5’ whole nuclei from different types of cells have been used as substrate; calf thymus,4 rat liver,4.51*52 human kidney, 53human thyroid,54 leukocytes,52.54-57 tumor,5s endometrial cells,5Q mouse spleen imprints,4Q and bovine and human epidermis.‘jO Recently, tissue culture cells have been usedel and standardized preparations of these have become commercially available.62y63Drawbacks of the indirect IF-ANA technique include the multiplicity of procedures in current use, the variations in fixation, the lack of standardization of conjugated antisera, the difficulties in interpretation, and the inherent problems of fluorescence microscopy.50*g4*65 Discrepancies in the results have been reported with the IF-ANA test when Indeed, it is possible to obtain positive different sources of nuclei are used. 24*51*54 and negative results using the same serum on different substrates.24 This problem is of practical importance for the clinician since sera from the same patient are often studied in different laboratories. In 1961, Beck had already demonstrated that different ANA titers were observed with the same serum using frog and rat liver as substrates.5’ Undoubtedly, this is due to the qualitatively different antigenie composition of frog and rat liver nuclei. It has also been demonstrated that

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the intrinsic characteristics of the fluorescent conjugate are also relevant in defining ANA titer. Beutner et al. showed that upon adjustment of the conjugate dilution, the range of one ANA titer varied from l/80 to 1/64O.64 In addition, it is known that some of the nuclear antigens are denatured by the mode of fixation of the tissue substrate. The reproducibility of visual readings made by different individuals in different laboratories has been questioned.6g However, blind studies of ANA performed by different laboratories on the same sera indicated that visual readings of titers using the same substrate can be as reproducible as any other serologic test.15 Dilution of the serum and/or conjugate,67 improvements on the purification of the conjugates, as well as control of the fluoresceine/protein (F/P) ratio have brought the problem of nonspecific fluorescence almost under full contro1.6s The appropriate dilution of conjugate (use-dilution) for indirect IF antibody tests has been determined by chessboard titrations of conjugated antiglobulin serum versus human serum ANA .67,68 We have found no difference in reading mounted slides that were left for 72 hr in the -30°C freezer when compared with the slides read immediately after mounting.6g The usefulness of the indirect IF-ANA technique is universally recognized and in our opinion this is the best screening method available, but standardization is extremely desirable if results from different laboratories are to be compared.15,64*70 The report of ANA by IF should include the pattern of nuclear fluorescence, the titer, and the substrate utilized. Soon after the application of the indirect IF technique for the Patterns. screening of ANA, it was observed that some sera consistently gave a specific type of nuclear fluorescence. Depending on the substrate used, the indirect IFANA test may or may not allow the identification of a variety of patterns of nuclear fluorescence. Friou has used the term “total ANA” to indicate ANA detected by a method which does not separate these different types of reactivity.50 Patterns that have been described include the homogeneous, speckled, and nucleolar,51 shaggy,55 peripheral, 71 rim and fibrillar,72 membranous,73 nodular and reticular,74 thready,75 and comet,76 and still other patterns representing a very low number of the reported positive ANA. Though the presence of certain antibody specificities have been correlated with reproducible patterns, it is clear that the variation in tissue substrates and fixation techniques may account for some of the morphological differences. It is also known that certain ANA appear to interfere DNA with the expression of others,50,72 and that specific antibodies to denatured (dDNA) are very rarely detected by this technique due to the small amount of single-stranded DNA normally present in mammalian cells.77 Because of the heterogeneity of ANA, mixed patterns may be seen, or different patterns may appear as dilutions are used.5Q*7z~74~78 L’k I ewise, transitions from peripheral to homogeneous, or from homogeneous to speckled, going from low to high dilution have been observed. Poor correlation has been found between fluorescent patterns and other serologic tests for specificity of ANA. 52,7gAt the present time, all these variations preclude the use of the immunofluorescent patterns as an absolute diagnostic tool of antibody specificity. The most frequently studied patterns are the homogeneous, peripheral, fibrillar, speckled, and nucleolar (Fig. 1). (1) Homogeneous pattern: Antibodies to insoluble deoxyribonucleoprotein

a6

Fig. 1. Patterns of nuclear fluorescence in the, IF-ANA geneous pattern. (Cl speckled pattern, (D) nucleolar pattern.

FERNANDEZ-MADRID

test. (A) Peripheral

AND MAlTlOLl

pattern,

(6) homo-

(dNP) have been found to produce the homogeneous staining of nuclear fluorescence.51~53*7**80 It was demonstrated that these antibodies have the same pattern has been specificity as the LE factor. *W However, the homogeneous described with sera in which antibodies to insoluble dNP could not be detected, and it is possible that other antibody specificities may also produce this pat-

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a7

it has been proposed that two antibody specificities producing the nodular and reticular staining may contribute to produce homogeneous staining.74 tern.53~74~s3For instance,

(2) Peripheral pattern: It seems clear that the peripheral,” shaggy,“” rim,” ring,55 and membranous73 patterns probably refer to the same type.of IF staining reported under different conditions. This pattern has been shown to be produced by sera containing anti-native DNA (nDNA) antibodies.55x72,77 Albeit infrequently, it can also be produced by some sera in which no antibody to nDNA is detectable by other techniques.52 It may also be produced by antibodies to soluble dNP.72 Studies by Rothfield and Stollar demonstrated that the peripheral pattern of nuclear fluorescence could be removed by absorption of sera with either native or denatured DNA or with dNP. However, one serum which contained only complement-fixing antibodies to dDNA and which did not react with nDNA or dNP did not produce the peripheral pattern of nuclear fluorescence. Also, the antibody to dDNA which remained after absorption with dNP did not produce the peripheral pattern of nuclear flu0rescence.77 (3) Fibrillar pattern:” This pattern is probably the same as the thready75 and the reticularT4 patterns. It can be associated with a peripheral pattern (rim and fibrillar) but it may be observed alone. This pattern can be produced by anti-nDNA antibodies as indicated by its removal by quantitative absorption by nDNA?’ In some cases this pattern may also be produced by anti-soluble dNP antibodies.‘” Thus, the observation of this type of nuclear staining with sera does not necessarily mean that anti-nDNA antibodies are present. (4) Speckled pattern: This pattern was described by Beck, who noted that the “nuclear antigen” producing this pattern of fluorescence is easily extractable by saline.“’ Later on, it was found that this pattern is the expression of several antibody specificities; some have been partially characterized and others are still unknown. Antibodies to the Sm antigen, 45to a nuclear RNA-protein (MO) antigen,R4 or reactions with the so-called extractable nuclear antigen (ENA) may produce this pattern. It is not surprising that antibodies with the first two specificities give the same pattern of nuclear fluorescence, since it has been demonstrated that in calf thymus extract, Sm and MO antigens form a molecular complex.86 Nevertheless, this type of staining is also-produced by some sera in which antibodies with those specificities are not found, suggesting that other antigens may also be implicated. Moreover, a variation in the size of the speckles, which may be the reflection of further heterogeneity of these antigens, has been reported by several authors”7+sn and also has been observed. The clinical significance of these variations is uncertain, since the biochemical characterization of the antigens has not been accomplished. At the present time, because of biochemical, immunologic, and clinical heterogeneity, the identification of the speckled pattern of immunofluorescence with only one antibody specificity, so-called “speckled antigen,” and/or a clinical syndrome, is misleading. Techniques such as those involving the use of purified antigens, enzymatic studies, or the Ouchterlony double diffusion precipitin method with prototype serum should be carried out to determine the actual specificity of the antibodies from sera giving a speckled nuclear fluorescence.45,84,85 1.5) Nucleolarpattern: The nucleolar pattern is a specific nuclear staining which

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identifies antibodies to this organelle. It is most often seen in association with other patterns as a component of the mixed type of fluorescence. Ritchie reported that antinucleolar antibodies may give different reproducible patterns of immunofluorescence in nucleoli, 8g though the significance of these findings is unknown. Because of its sensitivity to RNase and trypsin, this antigenic activity has been attributed to nucleolar RNA-protein.51 (6) Mixedpattern: Frequently, more than one pattern of nuclear fluorescence is present in the same preparation. These mixed patterns are probably due to multiple antibody specificities. With dilution of the serum one pattern usually prevails, but this is not always the case. One possible explanation for this observation could be the presence of similar concentrations of antibodies of different specificities. Titration. Important information regarding the clinical significance of a positive ANA test is obtained by titrating the sera. go Because of the lack of standard reagents, each laboratory controls its own screening dilution to determine positives and negatives. Most laboratories consider positive sera those which give titers of nuclear fluorescence between l/10 and l/30 or higher. Usually, positive sera are set up in twofold serial dilutions and the last which gives a distinct nuclear fluorescence determines the titer. Reports of intensity of fluorescence as 1+ to 4+ without mentioning the titer should be avoided because they are less accurate and depend mostly on the impression of the readers. In general, positives of less than l/80 are considered low titer and over l/500 high titer. Horseradish Peroxidase Technique The first use of enzyme-conjugated (horseradish peroxidase) antiglobulin as a substitute for fluorochrome-conjugated antiglobulin in the detection of ANA was reported by Benson and Cohen. g1 The major advantage of this technique is that special equipment is not needed since the preparations are examined with the light microscope. In addition, since the peroxidase complexes are electron dense, it has been suggested that other studies, such as electron microscopy, can be done with the same materia1.g2 Recently, several comparative studies have been performed involving the use of both techniques. g1*g3-g6From the available data, it appears that the peroxidase method might provide an acceptable alternative to immunofluorescence for routine determination of ANA. Methods

to Identify Specificity

of ANA

LE Cell Test The LE cell test was the first method developed to detect antibodies to nuclear antigens.’ Although it is being replaced in many laboratories by more sensitive and quantitative methods, it is still used in the diagnosis of patients with SLE and related diseases. An excellent review on the subject appeared recently.g7 The steps necessary for the development of the LE cell have been analyzed by several laboratories and are fairly well known. It has been proposed that 7S IgG antibodies with specificity for insoluble dNP are responsible for the LE cell phenomenon.81*82 This proposal was later confirmed by absorption studies.4Q.g8 Since its discovery by Hargraves et al., this antibody has been called the LE factor.’ There have been reports that in the presence of anti-dNP antibodies of the IgM class the

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phenomenon is not observed and the LE test is negative.Q7 It was suggested that the failure of IgM antibodies to induce LE cell formation may be due to the low complement-fixing capacity of this immunoglobulin class with nucleoprotein antigen.Qg This finding is not surprising, since although IgM usually fixes complement much more efficiently than IgG, there are many antigens that do not fix complement with a specific antibody of the IgM class. The sequence of events which culminates with the development of the LE cell can be summarized as follows:y7 First, an antigen-antibody reaction takes place. The antigen is provided by leukocytes slightly damaged by a variety of procedures, such as mixing with glass beads or passing the cells through a wire mesh. These leukocytes have their nuclei exposed and they react with the LE factor present in the patient’s serum. In the presence of appropriate amounts of complement,‘n”~‘O’ this antibody attaches to specific nuclear antigenic determinants. As a result of this reaction, nuclear swelling, loss of chromatin pattern, and basophilia occur in otherwise unremarkable white blood ceils. Once these changes have occurred, the altered nuclei are seen under the microscope as free, round, homogeneous, purplish bodies, although they might also be seen with cytoplasm attached.g7 The second part of the LE cell phenomenon involves phagocytosis, implying that at least a certain number of phagocytic cells as well as active complement components should be present in the sample. The phagocytic cell is almost always a polymorphonuclear (PMN) leukocyte. This PMN engulfs the altered nuclear material formed during the first part of the phenomenon. When the cytoplasm is still attached, it has been shown that only the nuclear material is phagocytized.‘O” The leukocytes containing the homogeneous purplish mass as an inclusion body are the LE cells. It is believed that the entire process occurs within minutes after the blood is drawnlo and that the LE factor does not react with intact cells.‘03 Two direct LE cell methods assay the serum LE factor in its capacity to produce the LE cell phenomenon with the patient’s own white blood cells. These are the glass bead and the sieve clot techniques. 104,10sMany modifications of both have been described. It has been reported that staining the preparations with acridine orange yields a larger number of LE cells than the universally used Wright’s stain.‘O’j It is essential for the accuracy of the method that the amount of heparin, age of specimen, degree of trauma, and period of incubation be properly controlled. The reading of the test requires a skilled technician who should know how to differentiate LE cells, pseudo-LE cells, and phagocytic cells with inclusion bodies.g7 Both the glass bead and sieve clot techniques are of value in demonstrating LE cells. It has been reported that some samples are consistently positive with one and not with the other technique, though the reasons for these findings are not known.y7 When the quantity of blood is insufficient to be tested by the conventional methods, an LE cell micro method or capillary technique has been described.‘07 Large comparative studies are not available to evaluate this procedure, although it might well be useful on some occasions. In cases when only serum or plasma is available, the indirect LE cell method is used. lo8 This technique studies the reaction of the LE factor on freshly prepared leukocytes from a normal donor. Again, all the factors influencing the test must be closely controlled. Although several reports indicate that antibodies with specificity for insoluble dNP (LE factor) occur in the serum from almost every patient with SLE at some time during the evo-

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lution of the disease,13s83 the LE cell test is only positive in 60%70% of the cases in the sera from acutely ill patients. It should be mentioned that while antibodies with LE factor specificity (as detected by the LE cell test) should theoretically be uniformly detected by the IF-ANA technique, there is not always a correlation between the two tests. Some reports describe occasional patients who have positive LE cell tests and are negative by the IF-ANA technique.83~10Q The reason for this discrepancy is not presently understood.

Conventional Immunological Techniques Utilizing Chemically Defined Antigens Assays for Anti-DNA antibodies. A variety of methods have been used to detect anti-DNA antibodies and any of them are also useful for the study of other antigenic specificities. Complement fixation,25-27~110~111precipitation,27~112~113 agglutination of bentonite particles,30*1’4 and passive hemagglutination techniques are frequently used in many 1aboratories.37.115 The indirect IF spot test is also widely used and is easy to perform. l16s117However, large numbers of positive DNA spot tests with negative IF-ANA have been reported.52 Several laboratories are using crithidia luciliae as a substrate for the IF-ANA test to study specifically anti-nDNA antibodies, since the kinetoplast of these trypanosomes seem to contain only double-stranded DNA. 118It seems a reliable and simple approach, and the data available correlate well with the presence of anti-nDNA antibodies detected by other techniques. 118 The most sensitive procedures for measuring anti-nDNA antibodies require the use of labeled nDNA. Commercially available labeled nDNA is of variable quality and its cost is very high. 3H- and 14C-labeled nDNA usually can be prepared from bacterial origin or from cells in tissue culture11g or, alternatively, calf thymus nDNA can be labeled with 1251.120--122Various binding assays with radioactive sulfate precipitation technique, DNA are being used. 123-12g The ammonium initially introduced by Farr,130 using albumin as the antigen, was later applied to the study of the DNA-anti-DNA system.123,125.127 It was found that 7.5% of unselected patients with SLE exhibited abnormal binding activity,125 compared with the 25%64% reported when other methods were used to detect anti-DNA antibodies.‘24 This method is very sensitive and is presumed to detect all antibodies to a given antigen. It has been shown to be helpful in the diagnosis and assessment of the course of SLE. More sophisticated techniques are being used in some laboratories but are still not adapted to the routine determination of ANA. Jones and Berg were the first to demonstrate the usefulness of nitrocellulose membrane filters in detecting DNAprotein complexes. They found that while both RNA polymerase and nDNA were filterable through such a membrane, a complex of the two was retained.131 This led to the application of these filters to study other DNA-protein complexes such as DNA-anti-DNA. Though useful as a research tool, the applicability of this method is still somewhat questionable for the routine determination of anti-DNA antibodies. Kredich et al. reported data from 33 patients with SLE using the nitrocellulose membrane filter technique for assaying anti-DNA antibodies. They showed that when the molecular weight of the DNA preparation is reduced by mechanical shearing in a high-speed homogenizer, the quantity of DNA retained in the nitrocellulose membrane by a given amount of serum decreased mark-

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edly.13” These data indicate that the sensitivity of the assay of DNA antibodies by this method is affected by the molecular weight, and that different batches of the DNA used in the assay may give different results. Thus, it seems that the mass of DNA bound by a constant amount of antibody is a function of the molecular weight of the DNA used. In this regard, the use of a closed circular reactive DNA of uniform size could be considered as a means of averting the variation in molecular weight found with ordinary DNA preparations. It has also been suggested that the nitrocellulose membrane method cannot be used to measure antibodies to single-stranded DNA, 132since the latter, unlike native DNA, is retained in such membranes even in the absence of binding proteins.‘33 These features should be considered when evaluating results obtained by this technique during the follow-up of patients or when comparing results obtained from different laboratories. Another rapid-binding assay capable of detecting antibodies to nDNA and dDNA, uses the ability of glass fiber filters to bind antibody-DNA complexes without additional precipitation by ammonium sulfate or antiglobulin antiserum.‘34 Still another approach to analyzing the problem of DNA-anti-DNA was reported by M. Tan and Epstein. These authors developed a solid-phase immunoassay using polynucleotides absorbed to plastic cups to measure antibodies to DNA and RNA.135 This method, which is a modification of the solid-phase radioimmunoassay technique for the quantitative determination of immunoglobulins,‘“” seems to distinguish antibodies to dDNA from antibodies to nDNA. The advantage of this method is that each of the components of the solid-phase system is fixed in sequence to the plastic surface. In this way, protein interactions which occur in liquid-phase systems, problems related to nonspecific binding, and precipitation and anticomplementary effects of the serum are avoided. A sensitive binding radioimmunoassay enabling detection of antibodies to both soluble dNP and nDNA was described by Robitaille and E. Tan.13’ A preparation of soluble dNP was isolated from calf thymus nuclei and labeled with lz51. Sera with antibody to nDNA reacted with the DNA moiety of soluble dNP and bound ‘““I-soluble dNP, but his binding was completely inhibited by addition of unlabeled nDNA. Conversely, the reaction was not inhibited by unlabeled nDNA when the antibodies were directed against soluble dNP. Thus, antibodies to soluble dNP and nDNA could be differentiated by the use of a single isotopically labeled antigen.13’ Radioimmunoassays by the double-antibody method are being used for a variety of laboratory tests, including the detection of anti-DNA antibodies. This is a sensitive and reliable method, since only specific complexes are precipitated with specific anti-human gamma globulin serum. Precipitation techniques have also been applied to analyze the DNA-anti-DNA system. The Ouchterlony immunodiffusion technique is known to be relatively insensitive to detecting this type of antigen-antibody reaction. Instead, counterimmunoelectrophoresis (CIE) has been found to have sensitivity comparable to other useful techniques. It has been used extensively to demonstrate hepatitis associated antigens,13* and now an increasing number of investigators are reporting on their findings studying ANA specificities. 139-143Davis reported antibodies to DNA in 66% of the SLE sera tested by CIE.13g Similar results were reported by Klajman et al. These authors found a positive reaction in 16 of 29 SLE sera

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tested.140 Dorsch and Barnett compared results obtained by CIE and those by the Farr technique as a means of detecting antibodies to DNA. From 91 positive sera, 29 were positive by both techniques. They found 18 positive by binding alone, 15 positive by CIE alone, and 29 negative in both tests.‘43 Though some problems in interpreting results due to the frequency with which nonspecific bands are observed, it seems that CIE can be useful in the analysis of the DNA-anti-DNA system. Assays for ANA With Other SpeciJicities. Several methods have been applied to detect anti-dNP antibodies, including complement fixation (CF)101,*44 and the antiglobulin consumption test.3 CF is a quantitative method which has been widely used for the study of ANA. Anticomplementary activity of some sera limits its application as a routine test. Moreover, ANA belonging to the Ig classes or subclasses not fixing complement are not detected by this method, though this may be a desirable feature. Nevertheless, it is a reliable tool to confirm specificities of antibodies detected by other techniques, and as with other quantitative methods, it is useful in following levels of antibodies which may correlate with disease activity. Recently Henderson et al. published their results on the applicability of CIE for the detection of antinucleoprotein antibodies. They studied 35 SLE sera, of which 28 gave very strong positive reactions and 7 were weakly positive. In 17 of 65 control sera, only weak reactions were observed. 14’ Anti-dNP antibodies can also be assayed by the commercially available latex nucleoprotein test and by the spot test of Friou.116 Both are simple to perform and neither require expensive equipment. Some studies demonstrate that the latex nucleoprotein (Hyland LE test) is Consequently, a negative only positive in 30% of individuals with LE cells. 145*146 test has no diagnostic significance. On the contrary, the spot method for anti-dNP is rarely negative in patients with positive LE cells.“’ At the present time a new test kit is available (Lederle LE test), but more data are needed to evaluate its usefulness as a routine procedure. CF and the spot test seem to be equally sensitive in detecting antinucleoprotein antibodies. The antiglobulin consumption test has yielded extremely valuable information but it is a laborious technique, difficult to standardize in a clinical laboratory. Routine Detection

of ANA

No attempt will be made to recommend a technical routine to determine ANA. The methods to be accepted by each laboratory should be appropriate for that particular medical facility and should provide accurate results useful for patient care. As an example, from the large number of laboratory methods available for the detection of ANA, the following are used in our laboratory for routine diagnosis of the CTD. (I) ANA by IF: The strategy involved is to use the ANA by IF as a screening procedure; if this test is positive, other specificities are then investigated, including the LE cell test. (2) Anti-DNA antibodies by complement jixation using purified nDNA and dDNA: This test detects the kind of anti-nDNA antibodies that may have a pathogenetic role and are associated with high incidence of nephritis. (3) CZE: This is a promising technique which may detect precipitating antinDNA antibodies that may have a role in the pathogenesis of lesions.

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(4) Double antibody methodfor anti-nDNA: This method is sensitive; its specificity depends on the purity of the antigens used and has a very low background. (5) Hemagglutination: coating red blood cells with ENA, followed by RNase treatment. (6) Double diffusion Ouchterlony plates: These are used to detect anti-MO and anti-Sm with prototype antiserum, using calf thymus nuclear extracts as a source of antigen. IMMUNOLOGIC lmmunoglobulin

Class

ASPECTS

of ANA

The Ig classes and subclasses involved in ANA have been analyzed by several laboratories. Goodman et al. first investigated this problem and found the presence of both 7s and 19s IgM ANA.3 Later, Barnett et al. found ANA of the IgA class,14’ and there are reports that ANA are also present in IgD’48-L50 and IgE classes of Ig151 and are heterogeneous on the basis of light chain types.‘52 ANA of more than one Ig class are usually simultaneously present in the sera of Gonzalez and Rothfield the majority of SLE patients. 71,7g,‘47.153For instance, found that IgG ANA were present in 96%, IgM in Sl%, and IgA in 51% of SLE sera.71 Also, ANA have been found in the 7S IgM fraction.‘“4 The IgG subclasses having ANA activity have been investigated with the expectation of finding a relationship between Ig subclasses, antibody specificity, and incidence or severity of nephritis in SLE patients. Kacaki et al. found ANA in patients with SLE, rheumatoid arthritis (RA), and drug-induced LE in all four classes of IgG in a distribution similar to that found in normal sera.‘55 Tojo et al. found that in patients with active lupus nephritis there was a trend toward a predominance of IgG subclasses that fix complement more efficiently, though ANA were found in all IgG subclasses and the distribution was proportional to the concentration of the individual IgG subclasses in the serum.‘56 However, Schur et al. found that antibodies giving a peripheral pattern in the IF-ANA test were primarily of the IgG, and the IgG, subclasses, antibodies giving a speckled pattern were primarily of the IgG, subclass, and antibodies giving a homogeneous pattern were primarily of the IgG, subclass, and less so of the IgG, subclass. This work suggested that ANA of different specificities may be restricted to certain IgG subclasses.‘57 It is interesting that anti-nDNA antibodies that have been associated with SLE nephritis and hypocomplementemia tend to belong to the IgG, and IgG, subclasses, which are known to fix complement more efficiently. Several reports seem to indicate that in patients with RA and positive ANA, IgM is the predominant Ig class. 7gArana and Seligmann showed that some of the anti-nDNA antibodies found in SLE can also belong to the IgM class.‘SR Cracchiolo and Barnett found that 65% of the ANA found in the synovial fluid from patients with RA belonged to the IgM class and 35% to the IgG class. In two cases, ANA were found only in the synovial fluid, both times of the IgM class.“” It has also been shown that granulocyte specific (GS) ANA may also show subclass restrictions.‘“g A very interesting observation was described by Nagaya et al., who reported that 7 of 10 patients with severe pulmonary interstitial fibrosis of unexplained etiology had only IgM ANA in their sera. I60 At the present time, no definite role can be assigned to any of the Ig classes, al-

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though the trend of producing ANA diseases deserves further investigation. clincal phenomena. Specificity

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of certain Ig class and subclass in some Eventually, it may correlate with specific

of ANA

Valuable information has resulted from experiments carried out on the DNA-anti-DNA and dNP-anti-dNP systems. The accessibility of purified antigens has facilitated these studies greatly and some preparations are readily obtainable commercially. Fine specificities of other ANA have been less well analyzed, primarily because of difficulties in isolating the antigens. A n tibodies to dNP Insoluble Form. As mentioned above, antibodies to particulate DNA-histone, insoluble in isotonic saline and sensitive to trypsin and DNase, have been conclusively demonstrated to possess the LE factor specificity. The antigen has been reconstituted from isolated DNA and histone in vitrozg Stollar has shown that anti-insoluble dNP antibodies react with different antigenic determinants of the insoluble DNA-histone molecule. This variation in fine specificity of the same group of antibodies (LE factor) is even observed in individual sera, and it seems that some SLE sera might react only with DNA-histone formed by DNA and the specific histone fraction F2a2. 161Antibodies to insoluble dNP cannot usually be absorbed completely with DNA or with histone, indicating that both macromolecules are somehow involved in the antigenic determinant.“j2 Soluble Form. Tan has described antibodies to a soluble antigen extracted from insoluble dNP, also sensitive to DNase and trypsin in the sera from patients with SLE. Antibody activity could be partially absorbed by DNA and histone. These absorption experiments demonstrated that this group of antibodies may also produce a peripheral pattern by immunofluorescence.46 The antigen is present in normal tissues and it has been detected in 58% of rheumatoid and 61% of nonrheumatoid control synovial fluids. The corresponding antibodies have been found in 26% of the fluids from patients with RA and in only 4% of the contro1s.‘63 Antibodies

to DNA

Early studies reported the presence of anti-DNA antibodies in the sera of patients with active SLE.25-27.164 These observations were subsequently confirmed using a variety of techniques with bacterial and mammalian DNA as antigens. The specificities of anti-DNA antibodies have been studied by inhibition with DNA from different sources, with purine and pyrimidine bases, oligonucleotides from enzyme digests, nucleosides, and DNA denatured by different procedures.‘65 A phosphodiesterase specific for dDNA and unreactive with nDNA has been useful in differentiating antibodies to the two forms of DNA.‘66,167 Sera containing antiDNA antibodies can be characterized according to their reactivity: (1) sera that react only with one form of DNA, either native or heat denatured; and (2) sera that react with both equally, or slightly better with one or the other form. It is clear that anti-DNA antibodies are heterogeneous and their specificities are directed to distinct antigenic sites on the DNA molecule.‘26~143,158,165,166This large antigenic variability probably explains in part the discrepancies in the results

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reported in earlier studies. At the present time, extensive surveys have reported similar conclusions on the specificities of these autoantibodies in different diseases.37~126~168--170Nevertheless, in studying specificities of anti-DNA antibodies, regardless of the method used, the origin and method of preparation of DNA seems to be of prime importance. Reactions with commercial DNA preparations are not absolutely equivalent to reactions to nDNA, since it is known that the former may contain variable amounts of denatured DNA. Even highly purified preparations of mammalian DNA have been found by methylated albumin kieselguhr chromatography, combined with enzymatic digestion by nucleases, to be heterogeneous mixtures of nearly homogeneously double stranded DNA and double stranded DNA with significant single stranded regions.“l The immunogenicity, hapten functions, and serologic reactions of nucleic acids, including native and denatured DNA, have been extensively reviewed by Stollar.‘67 The only available source of antibodies to nDNA are patients with SLE*” and the spontaneous autoimmune diseases developed in the NZB/NZW F, mice17” and Aleutian minks;173 attempts to experimentally produce them by immunization have thus far been unsuccessful. Antibodies to dDNA in various forms can be obtained in laboratory animals, enlarging the possibilities for further analysis of this antigenic specificity.‘74 Some of the antibodies induced by DNA, modified by different procedures, react only with the modified immunogen and show no reactivity with unmodified or other forms of DNA. Antibodies

to Histone

ANA with specificity for pure histone have been detected in the sera of patients with SLE by CF but they are rare.4g Because these antibodies are infrequent and their relationship to disease is unclear, they have not attracted the interest of many laboratories. In 1969 Stollar studied the specificities of three SLE sera that reacted with free histone by CF. The results demonstrated that the sera varied in their relative reactivities with the different histone fractions (Fl A, F2A, F2A2).lG’ Even in a small number of sera, it is possible to find a heterogeneity similar to that observed with other antibody specificities such as DNA and DNA-protein. Antibodies

to Soluble

Nuclear

Antigens

ANA in lupus sera as well as in sera from patients with other diseases have been found to react with antigens in saline extracts of different tissues.45,84.85.17d Cell fractionation techniques have been useful in analyzing these reactions.“” Though it is difficult to isolate pure subcellular fractions, relatively purified preparations have been obtained, which have permitted the recognition of antibodies directed to both nuclear and cytoplasmic components. Partial characterization of these antigens has led to the immunologic identification of different nuclear macromolecules. Although many antigen-antibody reactions have been observed to occur with different sera, three have been studied the most. All three are related to the speckled pattern of nuclear fluorescence. Because the chemical composition of the soluble antigens has not been completely defined, these antigens are known as extractable nuclear antigen (ENA), MO, and Sm. The last two antigens bear the first two letters of the last name of the patient in which the serum antibody was first identified.

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Anti-ENA antibodies. The phosphate-extractable nuclear antigen was described by Holman.‘75 Recent work on this material utilizing the extract to coat red blood cells for a hemagglutination reaction has shown that it consists of at least two distinct antigenic components. One component was identified as Sm antigen and the other as an RNA-protein. 177Antibody activity to the RNasesensitive portion of ENA is probably equivalent to anti-Mo,*4 since with specific antibody, MO antigen is detected in these preparations by CF.“” Antibodies with this specificity detected by hemagglutination are found in a group of patients with mixed connective tissue disease (MCTD) and SLE. The incidence of antibodies to these antigens in other diseases is extremely low. lzg Partial analysis of ENA composition has shown that this material is formed by approximately 26% RNA, 70% protein, 1.5 % lipids, and less than 1.5 % DNA.177 Anti-Sm antibodies. Tan and Kunkel described a group of SLE patients who possess serum antibodies to a soluble antigen called Sm.45 This antigen is identified by double diffusion in agar and has been characterized as a non-nucleic acid-periodate sensitive substance. Later experiments revealed that with some sera part of the antigenicity is sensitive to trypsin. It is present in extracts of normal human and animal tissues. Antibodies to this antigen seem restricted to patients with SLE and have not been found in other CTD or normal subjects when identified by gel precipitation with prototype serum.45,46*84,17gLately, it has been reported that anti-Sm antibodies can be found in other CTD, including some cases of drug-induced lupus erythematosus by the hemagglutination technique using ENA-coated red blood cells.‘80~‘s’ Since ENA seems to be a mixture of several characterized and some still unknown antigens, material resistent to RNase cannot be equated with the Sm antigen, and the specificity of these reactions cannot be known with certainty at the present time. Thus, results that are not confirmed by other techniques which assure specificity should be interpreted cautiously. Anti-MO antibodies. Further characterization of antibody specificities to soluble antigens in the sera of patients with SLE led to the identification by Mattioli and Reichlin of a precipitating ribonucleoprotein antigen called Mo.*~ This macromolecule is present in normal human and animal tissues. The antigenic activity of MO antigen is destroyed by RNase and trypsin, hence its chemical characterization as RNA-protein. MO antigen is physically closely related to the Sm antigen, and as previously mentioned, the Sm and MO antigens (nuclear RNAwas demonstrated by protein) form a molecular complex. 86 Their association absorption experiments and it is now clear that the Sm-Mo complex is a major antigenic component of ENA. These features might explain in part the high frequency of cases of SLE in which this pair of antibodies is found together in sera.16 Antibodies to MO antigen are specific for this macromolecule and do not crossreact with other characterized RNA-protein antigens.“’ Northway and Tan described sera with antibody to RNA-protein which is also present in ENA, but its relation to the reported MO-RNA-protein has not yet been studied. Other antibodies with RNA-protein specificity have been described but little identification of the antigen is available.37 Antibodies reacting with nucleoli were first Antinucleolar antibodies. described in human sera by Beck.“’ Probably due to technical difficulties in isolation of the nucleoli, little work has been reported about the fine specificities of these antibodies. Watanabe et al. reported two sera from patients with SLE that

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reacted with an RNA-protein antigen in the nucleoli of mononuclear cells and la3 Recently, Miyawaki and Ritchie seemed to be related to ribosomal RNA. isolated from rat liver a phenol extractable nucleolar 7S RNA that reacted with 16 sera from patients with CTD by precipitation in gels.ls4 Reactive sera had been previously characterized as having antinucleolar antibodies by IF. In contrast to the antigen previously described present in mononuclear cells, it seems that this antigen is not related to ribosomes. Pinnas et al. have reported the isolation of a low-molecular-weight 4-6s RNA from partially purified nucleoli and the presence of precipitating antibodies to nucleolar antigens. The precipitating activity was destroyed by spleen phosphodiesterase.‘s” Granulocyte

SpeciJic

ANA

Originally described by Faber et al. in the serum of a patient with Felty’s syndrome,‘* GS-ANA are able to combine in vitro with the nuclei of mature PMN in the indirect IF-ANA test. These antibodies neither react with precursors of granulocytes or monocytes nor with mature lymphocytes or other cells of human origin. The nuclear antigens involved in the reaction are unknown. Several morphologically different fluorescent staining patterns have been recognized, probably representing reactions between different nuclear antigens and the corresponding antibodies.‘R” The heterogeneity of GS-ANA has been demonstrated, antibodies occurring in the IgG, IgM, IgA, and IgD classes of Ig, the four subclasses of IgG, and K and X light chains. 15g,186-188 Nevertheless, restrictions have been observed related to this specificity. For example, certain sera studied by Wiik and Munthe showed GSANA specificity in only one IgG subclass, and in some of these, only one type of light chain could be detected.‘5g GS-ANA have now been found in the sera of all patients with Felty’s syndrome, and recently they have been reported as components of the cryoglobulins observed in this syndrome.‘sg These ANA have been found in the sera of about 75% of patients with RA, one third of patients with SLE 1g~zo~‘90 occasionally in drug-induced lupus and scleroderma,8* and in low titer in 4i of healthy subjects.21 GS-ANA, in general, have not been found related to leukopenia in Felty’s syndrome. However, a correlation has been reported between complement-fixing GS-ANA and the presence of neutropenia in patients with Felty’s syndrome and RA with neutropenia. lg’ Though a universal relationship between titers of antibody and leukopenia was not found, this interesting study suggested a direct autoaggressive activity of complement-fixing GS-ANA on the neutrophilic granulocytes. It is important to recognize these organ-specific antibodies since many laboratories that are using WBC as substrate for screening ANA by IF would detect these antibodies in their reports of positive ANA. CLINICAL Diagnostic

SIGNIFICANCE

OF ANA

Potential

SLE

For the past several classification of SLE. tory criterion included been found to be an

years attempts have been made to establish criteria for the A positive LE cell test was considered as the single laborain those preliminary works. 3g2-‘g4Recently, total ANA has acceptable alternative criterion to LE cells, and there has

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been support for the inclusion of a titer of l/64 or higher in SLE criterialg5 since the sensitivity of the criteria for classification was found to be higher using ANA than LE cell tests. A recent study has reported a high degree of correlation of proposed SLE criteria in clinically diagnosed SLE patients with levels of antiDNA antibodies.lg6 Due to the lack of precision in the definition of the disease, these attempts are still preliminary, although it is increasingly apparent that laboratory parameters should be included in classification and/or diagnostic criteria. Therefore there is an urgent need to standardize these tests. In reference to the indirect IF-ANA test, we suggest that efforts should concentrate on the selection of a single substrate, the use of a uniform F/P ratio, and preparations of antiwhole human gamma globulin antiserum. Data obtained by different CTD study centers have included ANA detected by IF and by DNA binding.lg7 The results of ANA tests should always be evaluated in the light of the clinical picture, though there are some circumstances in which the laboratory can provide important diagnostic information in completely asymptomatic patients. An increased incidence of ANA in relatives, including spouses of probands affected with SLE (pointing to genetic and environmental influences in the development of this serologic finding), has been reported by several investigators.‘g8-200 A positive ANA has to be evaluated in conjunction with the pattern of nuclear fluorescence, the titer, and the complement-fixing ability of ANA. The diagnostic significance of positive ANA tests in untreated patients with undiluted or very low serum dilution is uncertain, becausethe majority of positive ANA tests reported in normal individuals and in various other conditions have been of relatively low titer.7g~*3~20’-204 Results on prevalence of ANA in the “normal” population have indicated that a small proportion of the population has ANA at low titer and that there is an increased prevalence in females over males at all ages.204-207 In contrast, high titer ANA are usually very significant clinically. According to several reports, ANA titers higher than l/500 are found significantly more often in cases of SLE and drug-induced lupus; in some patients with chronic active hepatitis, scleroderma, Sjiigren’s syndrome, and MCTD; and in a few cases of juvenile Thus, a high titer ANA should first suggest rheumatoid arthritis (JRA). 16~83*85*208 these diagnoses. It has been reported that administration of high doses of corticosteroids may produce a significant reduction in the titers of ANA in all three Ig classes, and this reduction can also be seen during remissions.7g~10g~20g A negative result of an IFANA test done in a reputable laboratory on a nontreated patient makes the possibility of SLE very unlikely. 7g However, there might be reasons for the inability to detect ANA, even if they are present in the serum of patients with SLE (hidden ANA).210 Occasionally, patients fulfilling the preliminary criteria for the classification and/or diagnosis of SLE may persistently have negative IF-ANA tests. In these patients serologic tests usually allow the detection of other autoantibodies also found in the sera of patients with SLE. I6 In patients with pleurisy of unknown pericarditis, and chronic glomerulonephritis, the finding of cause, “idiopathic” anti-dNP in significant titers significantly supports the diagnosis of SLE.13 At the present state of our knowledge a high titered ANA should be considered one of many criteria in the diagnosis of SLE and should not be given undue weight. The value of the pattern of nuclear fluorescence is controversial. The patterns described above, in our opinion, are suggestive of certain conditions but not diag-

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nostic. The clinical significance of these antibodies is emerging from the study of the individual specificities of ANA. Valuable information can be obtained if titer, pattern, and Ig class are considered in conjunction with the clinical picture. The IgG peripheral pattern, when found in high titer, is often present in active SLE. often correlates with the presThis pattern, when produced by IgG antibodies,” ence of complement-fixing anti-nDNA antibodies and with low levels of total 71*20g though occasionally it may be found in significant titers in serum complement, patients with RA, drug-induced lupus, or scleroderma in the absence of antinDNA antibodies. In these cases, serologic methods fail to detect anti-nDNA antibodies. Thus, since the peripheral pattern can be found occasionally in patients in whom anti-nDNA antibodies cannot be detected, the finding of this pattern should prompt the determination of anti-nDNA antibodies by an alternate method. The peripheral pattern due to ANA of the IgM class has not been found to correlate with the presence of complement-fixing anti-nDNA antibodies.” The correlation between the presence of anti-nDNA antibodies and active SLE was first noted by Seligmann using the CF technique.211 This observation was confirmed by several investigators using other techniques.25-“7~31~77~1’0*164~2’2*213 The finding of anti-nDNA antibodies is of prime importance in the diagnosis of SLE because when found in high titers the specificity of these antibodies is almost 100% for SLE.124.214 A low incidence of these antibodies has been detected in a few other diseases using very sensitive techniques. lzg It should be kept in mind that with passive agglutination and radioimmunoassays there is a background of reactivity with normal serum at very low dilutions, so that a given dilution or a given amount of binding have to be defined as a threshold for abnormal reactivity. In these sensitive assays, it is important to distinguish true antibody activity from exaggerated binding by nonantibody proteins. Other studies on several thousand sera from healthy subjects and patients with a variety of diseases have not demonstrated reactivity with nDNA.‘“’ Since ANA with specificity for dDNA are also found in patients with SLE as well as other CTD,215 the method should be specific enough to differentiate between nDNA and dDNA. For these purposes the preparation of nDNA should be carefully purified and free of contaminating dDNA. Kredich et al. suggested that nDNA antibody levels quantitatively assayed by the nitrocellulose membrane technique correlate with disease activity in the individual patient,‘32 and Schur and Sandson reported that in some SLE patients an increase in serum levels of anti-nDNA antibodies precedes a flare of clinical activity (renal involvement in particular) by several days to several weeks.“O Thus, serial determinations may be more valuable than an isolated value as an index of activity, and since this antibody changes more rapidly than others after initiation of therapy,s3 it is a useful parameter in the management of patients with SLE, The depression of complement associated with high titers of ANA in severe cases of SLE nephritis” suggests that ANA react with nuclear antigens in vivo and fix complement during the exacerbations of the disease. Considerable evidence supports the central role of the activation of the complement system in immune complex-mediated tissue injury.216 Several studies have suggested that the determination of the complement-fixing ability of circulating ANA may be an important prognostic tool in SLE nephritis.“0,156,217 U sing a very sensitive technique, Kredich et al. have found occasional patients with active disease who had significant levels of anti-nDNA antibody for up to 6 mo before developing a positive LE

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cell test and ANA.13* Anti-nDNA antibodies occur in about two-thirds of patients with SLE, usually in association with the more acute phase of the disease;55,71p218 since they are absent from the serum of about one-third of documented cases of SLE, a negative anti-nDNA test does not exclude this diagnosis. One of the most characteristic ANA found in SLE has specificity for insoluble dNP. This is the antibody responsible for the LE cell phenomenon and seems to occur almost invariably in the sera of SLE patients.‘3,g7*158 Its absence in patients not being treated with corticosteroids is strong evidence against the diagnosis of SLE. l3 However, since it also occurs frequently in the sera of patients with other CTD, its demonstration does not have absolute diagnostic value. There are reports suggesting that quantitative changes in the level of anti-dNP may correlate with clinical activity.83’111,137 H owever, this finding has not been universal, and in general there has been poor correlation between clinical activity and antibody titers. 13,‘lg Indeed, it is known that high titers of this antibody may be present in asymptomatic individuals. 111,220Because titers of anti-dNP tend to be higher in SLE than in RA and other conditions, a high titer of this antibody has greater diagnostic significance than a low titer. It is also well known that even multiple negative LE cell preparations do not rule out the diagnosis of SLE. The significance of other antinuclear antibodies in the constellation of immunologic phenomena in SLE is also emerging. Anti-Sm antibodies detected by immunodiffusion with prototype serum have been suggested as potentially valuable in the diagnosis of SLE since so far they have not been detected in other CTD using this method.46 The report on the value of anti-MO (RNA-protein) in segregating a group of SLE patients who have a significantly lower incidence of nephritis and a mild disease course178 is now being confirmed by other laboratories.1*1~221 ANA in Other Diseases The widespread use of the indirect IF technique for the detection of ANA has unraveled their presence in a variety of different conditions. Two considerations are important in analyzing the data available in the literature. ANA can occasionally be detected in completely asymptomatic individuals, and their incidence in the “normal” population is definitely age dependent.73~201~202~z2zIt is well known that ANA are markers for the CTD in general. Positive ANA have been found in patients with RA, scleroderma, dermatomyositis, myasthenia gravis, polyarteritis nodosa, SjSgren’s syndrome, and MCTD, and in patients with Raynaud’s For instance, about one_ phenomenon with variably frequency. 79,83.85,87,89,223-2*6 third of the patients with definite RA present positive IF-ANA tests,7grg0 the homogeneous staining being the most frequently found.13,s3,g0 As can be expected from these findings, anti-dNP has been found in about 15% of these patients, and some of them also have positive LE cell tests.13 Studies on the significance of positive ANA tests in patients with RA have suggested that such patients have more severe disease,*03 and as a group they are likely to develop immune deposits in the skin blood vessels.227 In spite of the presence of ANA, these patients usually behave clinically as having RA and not SLE.13 Several investigators have reported ANA in significant titers in 4%-60% of Petty et al. found ANA significantly more their patients with JRA. 7g~228--231 frequently in girls, in patients with early onset of disease or who presently are young, and in patients with polyarticular disease or monoarticular disease and

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iridocyclitisz3’ Schaller et al. reported an interesting association between positive ANA and the occurrence of chronic iridocyclitis in patients with JRA: positive ANA titers of l/50 or greater were found in 51 of 58 patients (88%) with chronic iridocyclitis and JRA, while only 40 of 133 patients (30%) with JRA but without iridocyclitis presented this serologic abnormality.232 They proposed that ANA may be useful for identifying patients with JRA at risk for chronic iridocyclitis. The association of chronic iridocyclitis and positive tests for ANA in a juvenile patient with joint disease makes very unlikely the diagnosis of ankylosing can be prospondylitis, which often masquerades as JRA. 232Valuable information vided by the GS-ANA which is present in the sera of all patients with Felty’s syndrome. In the differential diagnosis of RA with polyarthritis of other etiologies, the finding of GS-ANA strongly suggests RA. Anti-dNP has also been found in patients with dermatomyositis and scleroderma, and some with necrotizing vasculitis of the small vessel type.‘” It has been reported that nucleolar fluorescence is more common in but it is not restricted to this disorder. scleroderma than in other CTD, 13*14,R7*Rg Miyawabi and Ritchie reported on the clinical features of 16 patients with high and four had Raynaud’s titers of antinucleolar antibodies.‘“’ Six had scleroderma phenomenon with changes less typical of scleroderma. Titers over l/ 1000 of pure antinucleolar antibodies can be considered highly suggestive of scleroderma, though occasionally they can be observed in the sera of patients with Sjiigren’s syndrome and Raynaud’s phenomenon without scleroderma.‘5’~‘X” The speckled pattern can be found in a variety of clinical syndromes and consequently has little diagnostic significance.8”,2”3 It has been found most frequently in scleroderma73~X3~“7*23”and in MCTD,8” but it is also seen less frequently in SLE, and other CTD.45,83,17R,23.5Patients drug-induced LE, Raynaud’s phenomenon, with a clinical picture described as MCTD usually have a speckled pattern of nuclear fluorescence and antibodies to the RNase-sensitive moiety of ENA.Xj The speckled pattern has also been described in elderly individuals and in a small percentage of “normal” adults. x7*235An antinuclear antibody very characteristic of polymyositis has been recently described by precipitin reactions. This ANA, which was found in the sera from 7 of 11 patients, may be a marker for dermatomyositis or polymyositis.23” The finding of ANA in conditions other than SLE always poses a complex clinical problem. In general, the clinical picture is of prime importance for the diagnosis. The laboratory findings have to be evaluated with great caution, since in addition to the clinical and immunologic overlapping in the CTD, ANA can be found in other diseases and can be induced by drugs commonly used in therapy. Conditions in which ANA have been reported include a variety of lung conditions such as interstitial pulmonary fibrosis,‘60,237 asbestosis,““x pneumoconiosis,““” and silicosis.“” These findings and the reports of rheumatoid factors (RF) in patients with “idiopathic” interstitial pulmonary fibrosis have raised the speculation that this condition may have an autoimmune pathogenesis.‘60.241,242 ANA have been repeatedly described ‘3,83,234,213 in various liver diseases since Saint et al. reported the LE phenomenon in patients with chronic active hepatitis244 and MacKay coined the name “lupoid hepatitis” to stress the serologic similarities between these patients with chronic liver disease and those with SLE.245 Though this name has been criticized,246 it is unquestionable that ANA are useful markers in this

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group of patients ‘OSin whom high titers of anti-nDNA antibodies,s3s’10 positive LE tests, anti-dNP,13 and all patterns of nuclear fluorescence have been reported. Some sera from patients with cryptogenic cirrhosis, mostly elderly women, also have ANA, but titers in this disease have been found to be lower.208 The significance of ANA in this condition is uncertain since there is an increased incidence of ANA of relatively low titer in the elderly population.83~20’~202~204~222~247 An interesting correlation with positive ANA has also been found in some female patients with chronic interstitial cystitis, though the significance of this finding is unknown. 248Many reports have linked ANA and a variety of infections. For instance, epidemiologic studies using ANA as a tool have linked ANA and malaria. A survey in West Africa has shown a high prevalence of speckled ANA in sera from apparently healthy Africans. This study proposed an association between speckled ANA and malarial antibody levels in Nigerians.24g In a survey of ANA in East African populations with similar ethnic, sociologic, and nutritional status, Voller et al. reported that individuals living in malarious areas frequently have ANA of the speckled type and malaria antibodies.250 Leprosy251 provides another example of the presence of ANA in a chronic disease.251 There are some reports suggesting that viral infections may, in some individuals, elicit the appearance of some of the serologic manifestations of lupus, including ANA.252-257 An increased prevalence of ANA has also been reported in psychiatric patients. 258 Unfortunately, the patient sample was too small to allow statistical analysis which takes age and sex into consideration.25g The significance of ANA found in a random psychiatric population is difficult to interpret because the results might include some patients with serologic abnormalities due to spontaneous SLE with psychiatric manifestations, or to the medications, such as phenothiazines, frequently used to treat psychiatric patients.“j’ A high incidence of ANA has been reported to be associated with neoplasia.261-264 In one study, positive ANA were reported in 65 of 342 patients (19%) with malignant disease or a past history of a malignant tumor, while only 2 of 201 blood donor controls (0.99%) demonstrated a positive test.264 It was suggested that a positive ANA test to a significant titer in a patient without features of a CTD and with no history of exposure to drugs which may be associated with induction of ANA should raise the question of a malignancy.264 An alarmingly high incidence of positive IF-ANA tests (52.2%) has been reported in pregnant women,265 though these findings have not been confirmed.“j6 The true incidence of ANA in most of these conditions is uncertain at present. A general criticism that applies to many of the reports on the incidence of ANA in nonrheumatic diseases is that age,83~201~202~222 sex,83,204*247and /or drugs which could have been important in the induction of serologic abnormalities were not considered. Clinical and Experimental

Induction of ANA

The use of therapeutic agents has been found to be associated with the occurrence of ANA. Symptoms resembling those produced by SLE and positive LE cell tests were first reported in patients with hypertension who received high doses of hydralazine.267*268 In most instances large doses were required, but it has occurred in patients receiving as little as 75 mg daily. 26g It was also found that asymptomatic individuals receiving this drug may have positive LE preparations and

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ANA.267J70 Though the homogeneous pattern is most frequently found by the IFANA test, any other pattern can be observed in drug-induced LE. Drug-induced LE has also been reported as a consequence of treatment with isoniazid (INH),‘” and subsequently ANA were shown to develop in patients with tuberculosis who were receiving this drug.272,273 In one study, 10 of 55 patients (19%) receiving INH developed ANA, while only 2% of patients with tuberculosis not receiving antituberculosis therapy had this finding.272 In a prospective study Rothfield et al. found that 22 of 98 patients (22%) had ANA levels changing from less than l/10 prior to therapy, to greater than l/10 when tested l-12 mo after starting therapy with INH. The difference in incidence of ANA before and after therapy was statistically significant. 274In both studies, most of the patients were taking more than one drug, although the only constant drug used was INH.273,274 At the present time, procainamide is the most commonly implicated drug in the induction of LE, conseveral laboratories firming the original observation of Ladd. 275-280Subsequently, have reported ANA in patients being treated for cardiac arrhythmias with procainamide.2”‘-2s3 While about 20% of patients treated with INH develop ANA,272 approximately 50%-75% of those receiving procainamide develop this serologic abnormality. 281*282It has been reported that every patient that has received this drug for 1 yr or longer demonstrates elevated ANA titer.284 Although procainamide, hydralazine, and INH are most frequently associated with the development of ANA, other drugs have been reported as well. Anticonvulsant medications of dissimilar chemical nature such as diphenylhydantoin, mephentoin, trimethadione, and ethosuximide have been implicated in the induction of ANA and drug-induced LE.180,285-2g2However, drug-induced SLE seems to be infrequently related to the use of these chemicals and some of the reported cases, especially those associated with anti-DNA antibodiesZs8 may be related to the fact that some patients with SLE have convulsions as the first manifestation of this disease involving the central nervous system. g7 Phenothiazine derivatives have also been implicated in the production of the LE phenomenon.260 Berglund et al. reported an increased incidence of ANA (26%) in a series of 177 women receiving chloropromazine, compared to 2.4% of 42 contro1s,2g3 and a case of drug-induced LE attributed to treatment with chloropromazine has been described.2Y4 Methyldopa is reportedly capable of producing Coombs’ positive hemolytic anemia, and other immunologic aberrations, including positive LE cell tests and ANA.26g~2g5-300 Patients on other medications such as chlortha1idone,300 sulfonamides g7*26y*301 nitrofurantoin,301 clofibrate,302 penicillin,g7,303 D-penici11amine,304 para-aminosalicylic acid,g7,305 and others have been reported to develop druginduced autoimmunity including ANA, 26gthough the incidence of this reaction is unknown. The appearance of a clinical picture suggestive of SLE has been reported in patients treated with propylthiouracil (PTU).306*307 However, a population of patients treated with PTU did not show increased incidence of ANA over the control popu1ation.308 Treatment with oral contraceptives has been associated with the finding of LE ce11s30g-311and an increased incidence of ANA. 312 In a prospective study of 82 women before and during oral contraceptive use, it was found that 7 women had positive tests for ANA before therapy and all remained positive during drug use; 4 additional women with negative ANA tests prior to treatment developed positive ANA after less than 1 yr of drug use. 312No positive LE cell preparations

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were reported and none of the women developed rheumatic complaints. Though it is true that in a prospectively examined population on oral contraceptives, a statistically significant number of positive tests for ANA developed, it would be of interest to compare these findings with a control population equally examined. Conflicting results were reported by Tarzy et al. in a prospective study. After an average follow-up of 16 mo they found negative ANA tests and no increase in rheumatic complaints in the subjects using oral contraceptives.313 Also, Dubois et a1.314 and McKenna et a1.315 found no evidence of induction of rheumatic complaints, LE cells, or positive ANA by oral contraceptives. From the available evidence it seems that the role of oral contraceptives in inducing ANA is still unproven. Often the presence of ANA associated with the use of certain drugs is difficult to interpret, since in many cases these drugs have been used to treat symptoms which in fact could have been the earliest manifestations of connective tissue syndromes.28” Specificitiesof ANA developed during treatment with drugs have been studied. Serologically, the ANA in drug-induced LE have less diversity and appear to be largely restricted to antibodies with specificity for dDNA and dNP 280,30’though some other specificities are occasionally found. Several reports indicate that patients receiving procainamide hydrochloride develop antibodies to dDNA and dNP, while anti-nDNA antibodies have not been unquestionably demonstrated. 143*280,316 Furthermore, recently Winfield and Davis, using a highly purified preparation of nDNA, reported that asymptomatic patients with ANApositive sera induced by procainamide were negative for anti-nDNA as well as anti-dDNA antibodies.317 Of their symptomatic patients, 50% had anti-dDNA and anti-dNP and all were negative for anti-nDNA. Their findings of significant amounts of dDNA contaminating the DNA preparations from a commercial source are important in evaluating some reports indicating low levels of antinDNA in drug-induced LE. The molecular basis of the spontaneous production of antibodies to nDNA remains a puzzling question, especially in view of the difficulty of their experimental induction and the general absence of these antibodies in drug-induced LE. Antibodies to nuclear antigens developing during treatment with anticonvulsants vary in incidence depending upon the drug being used, though all elicit ANA directed primarily to dNP. Iso Some patients receiving hydantoins have been reported to have anti-Sm antibodies, Iso but these data should be interpreted with caution since the methods used in these studies are not the most appropriate for specificity reactions using partially purified antigens.“5 Other laboratories have not found anti-Sm antibodies in sera from patients with drug-induced LE using precipitation reactions with prototype serum.137.301 The only reported case17* of procainamide-induced ANA with antibodies to MO antigen was found to have precipitins 7 yr after discontinuation of the drug, though this patient remains asymptomatic.6g There are some features common to all ANA induced by drugs. There is usually a decrease in antibody titers soon after the drug has been discontinued. Though most surveys indicate that the majority of the patients have become serologically negative a few months after withdrawal of the drug, in some cases persistance of low titer ANA has been reported up to several years 1ater.280~282Titers of drug-

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induced ANA can be quite high. In 44 asymptomatic patients with procainamideinduced LE, ANA was present in all cases in high titers317 The mechanism by which drugs trigger autoimmunity remains obscure and it seems probable that there are different pathways. Several authors have postulated a genetic predisposition which is liable to be triggered by the administration of certain drugs.3’s-320 The high incidence of ANA in patients treated with procainamide makes it unlikely that the drug simply unveils a latent disposition to idiopathic SLE, since the frequency for this disease is, according to some studies, approximately 1 in 80003” to 1 in 25,000.322~323On the contrary, it has been suggested that predisposition might be a factor with hydralazine.31g*320 In one study, 74% of the patients who developed clinical symptoms were found by history or previous laboratory data, to have manifestations of possible underlying lupus diathesis.324 This interesting subject has been a matter of active investigation and much speculation. The finding of serologic abnormalities in a large number of relatives of patients with drug-induced lupus in a few family studies provided some support31”,325,326 but a prospective study did not produce any evidence for this hypothesis.“X2 Studies on the biochemical interaction by which drugs would initiate autoimmunity have yielded interesting information. It has been shown that hydralazine may function as a hapten when combined with proteins,327 and that hydralazine”O’ and INH273 are able to produce physicochemical alterations on soluble dNP. The hydralazine-nucleoprotein complex did not appear to possess antigenic determinants other than those found in native dNP but was different from the latter in that trypsin treatment no longer abolished its antigenicity. It was suggested that in vivo this feature might be a mechanism for enlarging the immunogenic potential of circulating dNP.301 Whether such a mechanism operates in vivo in patients taking hydralazine remains to be determined. The sera of patients with hydralazineinduced LE contain ANA that react with soluble dNP.301 Also, tuberculosis patients receiving INH have ANA to soluble dNP and to INH-altered soluble dNP.273 Nevertheless, the clinical significance of these antibodies is unknown, since antibodies to hydralazine-altered soluble dNP are also found in idiopathic SLE,“73 and it is not clear how the formation of any kind of ANA may activate SLE, even in predisposed individuals. A complex between procainamide and ANA has been produced in vitro by means of photo-oxidation, but antibodies with this specificity have not been described in humans.280 The possibility that the rate of inactivation of drugs such as hydralazine and INH by acetyl transferase enzymes in vivo may be a factor in the induction of autoimmunity by these drugs has been extensively investigated. The phenotypic expression of genes controlling the function of these liver enzymes is represented by two groups, the slow acetylators, which inactivate these circulating drugs more slowly, and the fast acetylators, which inactivate these drugs rapidly.““” An interesting study on hypertensive patients treated with hydralazine showed that at low doses of the drug 60% of the slow acetylators developed ANA, whereas none of the fast acetylators had detectable ANA. However, the development of ANA was not totally dependent on acetyl transferase activity. In patients taking large amounts of hydralazine over prolonged periods of time, the differences in incidence of ANA between slow and fast acetylators gradually disappeared.32g While

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it seems that drug-induced autoimmunity develops more often in subjects of the slow acetylator phenotype treated with hydralazine,26g,32g several studies have found that the presence of ANA in patients being treated with INH is not significantly associated with sex, acetylator phenotype, or drug dose.330,331 The question of whether genetic susceptibility underlies the response to ANAinducing drugs requires further work, although present evidence suggests that genetic background is important in the development of ANA and drug-induced LE. Whatever the mechanism responsible for the appearance of ANA, it seems clear that drugs do not induce anti-nDNA nor do they lead to clinical situations associated with glomerulonephritis and lowered serum complement levels~280,301.317

Endeavors to produce ANA in experimental animals by the administration of these drugs have yielded variable results. Cannat and Seligmann were able to induce ANA in genetically well-defined mice by administration of both INH and hydralazine.332 Ten Veen and Feltkamp also reported the induction of ANA in mice by the administration of the above-mentioned drugs and also by methyldopa, chlorthalidone, and procainamide. 333Very recently Yamauchi et al. have reported the production of anti-nDNA antibodies by immunizing rabbits with hydralazine-human albumin conjugates. 334 Nevertheless, several authors have been unsuccessful in producing serologic abnormalities upon administration of procainamide to several species,335,336 and the drug did not enhance the naturally occurring age-related development of ANA in NZB mice.335 The development of ANA in spontaneous connective tissue syndromes observed in NZB/NZW F, hybrid mice,337,338 in other strains,33g in minks,44v173 and in dogs43 offers the possibility of animal models closely resembling spontaneous SLE. It has been shown that in New Zealand mice polyoma and lymphocytic choriomeningitis viral infections induce an abnormally high incidence of ANA.“” The type C viruses, (immunologically related to the Gross leukemia virus) that are naturally present in NZB mice have been held responsible for the development of the autoimmune diseases observed in this strain,341 though this view is not established. Recently, efforts have been made to induce ANA by inoculation of experimental animals with RNA oncogenic viruses. It has been reported that inoculation with Graffi and Rauscher leukemogenic viruses induced the appearance of ANA in (Balb/c x C57/B16) F, (C x B6) mice. By the indirect IF-ANA test the homogeneous pattern was found and some of these ANA were absorbed completely with dNP. The relationship between RNA viral infection and induction of ANA with dNP specificity is not understood.342 Role of ANA in the lmmunopathogenesis

of CTD

SLE As previously mentioned, the mechanism by which ANA are triggered in this disease is still unknown, but more is understood about their role in the pathogenesis of some of the manifestations of the disease. The discovery of the LE cell factor and the multiplicity of ANA suggested the possibility that these antibodies may be directly responsible for tissue damage in the CTD. However, several lines of evidence indicate that this is not the case. It has been reported that the incidence of IgM ANA in patients with SLE of long duration is similar to that

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in those patients with disease of recent onset. These findings supported the speculation that the continued production of IgM antibody might depend on the persistent presence of antigenic stimulation. Also, the prevalence of IgG antibody at the time of the diagnosis of SLE has suggested that the basic process may be of of much longer duration than suspected clinically. 71 Based on the heterogeneity ANA, Barnett et al. suggested that patients with LE had been extensively immunized by nuclear antigens at the time of the initial diagnosis.7g In agreement with this opinion are the reports of detection of positive Wasserman reaction, not due to syphilis, years before the diagnosis of SLE can be made.343-345 ANA are frequently detected in the absence of clinical disease, and, with the exception of anti-nDNA antibodies, the correlation of their presence with manifestations of active disease is poor. The failure to induce the disease by transfusion of blood or serum from patients with SLE to healthy recipients,346.347 the reports that newborn infants of mothers with SLE are usually unaffected even though anti-dNP is demonstrable in their blood,348,349 and the absence of cytotoxicity of lupus sera on cells grown in tissue culture350,351 are features which make it unlikely that the antibodies themselves cause the disease directly. Indeed, the cell membrane and the complex cytoplasmic organization of the cell seem to prevent access of ANA to the nucleus when the cell is intact. Most studies using the direct IF technique to evaluate the lesions in SLE have not described nuclear localization of Ig. 352p355However, McCoy reported the nuclear localization of IgG and IgM in 6 of 19 patients with lupus nephritis, and this phenomenon was not observed in 225 renal biopsies from patients with other conditions.35” The Ig were not confined to areas of parenchymal destruction, and it was suggested that ANA might gain access to intracellular antigens prior to cell dissolution. Paronetto and Koffler have reported nuclear localization in autopsy kidney specimens from 3 of 16 patients with SLE.357 These findings could possibly represent postmortem diffusion of ANA from serum as the process of autolysis of the specimen begins. 357The observation has been made in biopsies of skin lesions in SLE that the nuclei of some of the cells in the area of inflammation stain directly for gamma globulin, probably indicating that ANA had gained access to the nuclei.““8,35g Similar observations have been made in human kidney360.361 and in kidneys from NZB/NZW mice.34o Since there is little evidence to suggest that Ig normally penetrate the intact cells, it is possible that nuclear localization of ANA may occur under these circumstances in cells injured sublethally during the active phase of the disease process. Since not all the tissues present these findings and there is no correlation with areas affected by the pathologic process, it seems that this phenomenon is not related to the pathogenesis of the disease. One of the pathogenetic mechanisms that might be involved was suggested by studies in experimental animals which showed that antigenantibody complexes can produce renal disease and vasculitis. 33-35 Considerable evidence has accumulated suggesting that SLE nephritis is an immune complex disease and that ANA contribute to the tissue damage in an indirect way. ANA seem to play a distinct role in tissue injury through the formation of circulating antigenantibody complexes. The depression of complement associated with high titers of ANA in severe cases of SLE suggest that ANA may react with circulating nuclear antigens in vivo and fix complement during the course of SLE.110~111~20gEvidence sustaining this view was obtained by demonstrating antigen and its corresponding

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antibody in serum,362 and in renal lesions36 of patients with SLE. Denatured DNA363 and nDNA have also -been eluted from the kidney deposits of SLE nephritis.36,364 Several authors have reported circulating free DNA in the serum of patients with active SLE, and also after the disappearance of anti-nDNA antibodies.365 The origin of this DNA is probably tissue breakdown since it has been detected in a number of diseases associated with tissue destruction, e.g., hepatitis, metastatic carcinoma, and miliary tuberculosis. 362In reporting free DNA in human sera it is important to rule out that this has not been released from leukocyte nuclei during clotting. It seems that more reliable data could be obtained in plasma determinations.366 Nevertheless, these findings raised the possibility that the appearance of circulating antigen in vivo may result in the formation of immune complexes followed by their entrapment in the kidneys. Indirect evidence for the existence of circulating immune complexes in SLE patients has been reported. 367,368 Recently, several investigators have demonstrated the presence of circulating immune complexes in patients with SLE. Harbeck et al. reported on an assay to detect the presence of circulating DNA-anti-DNA complexes. Essentially, they measured the level of antibody to DNA before and after treatment of sera with DNase I. They reported that 11 of 15 cases of SLE with active renal disease presented a significant rise in DNA binding after digestion with DNase I, indicating that DNA had bound in vivo to the anti-DNA antibodies in the sera. 36g,370These authors could not demonstrate these complexes in any of their inactive SLE patients. This study suggested that there is often a correlation between the amount of the nDNA-anti-nDNA complexes and renal disease activity. Because of the inhibitory effect on the DNA-anti-DNA reaction that anti-ENA antibodies seem to possess, a protective role of these antibodies in preventing the development of nephritis has been postulated. 177,178This effect has been observed in vitro371*372and in laboratory animals372 and future experiments should define the important role of the ENA-anti-ENA system with certainty. Though the importance of the DNA-anti-DNA system in the pathogenesis of the kidney lesions in SLE has been extensively documented, I7 it should be noted that some sera of patients with active lupus nephritis showed no evidence of nDNA-anti-nDNA systems might be involved complexes,“6g suggesting that other antigenantibody as well. Besides quantitative differences, there is evidence indicating that qualitative differences in the antibody population may have important implications in the First, the association of complementpathogenesis of the renal lesion. 125.143,373-375 fixing anti-nDNA antibodies and lupus nephritis has been well demonstrated.17 The presence of non-complement-fixing antibody could possibly explain the lo%-20% of cases of patients with anti-nDNA antibodies that do not develop significant renal disease.‘53,*56 It has been suggested that while complement-fixing ANA are mainly directed against DNA, non-complement-fixing ANA combine with other nuclear antigens.157 However, it has been reported that ANA with One clear example is the RNAother specificities’6s’5’ may also fix complement. protein (MO) system. Sera from patients with SLE may possess very high titers of complement-fixing antibodies to MO antigen in vitro, and usually have normal complement levels even during exacerbations of their disease.16 These observa-

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tions suggest that other factors such as size and availability of the antigen in the circulation might play a role in determining which immune complexes are pathogenic. Dorsch and Barnett reported the presence of precipitating anti-DNA antibodies by CIE.143 They suggested that methods of antibody measurement which, even if less sensitive, detect qualitatively different subpopulations of antibodies may have potential value in the diagnosis and prognosis of CTD. Furthermore, Gershwin and Steinberg recently studied the different characteristics of anti-nDNA in their reaction with nDNA.374 Thirty-eight sera were classified into three categories: precipitating, nonprecipitating, and a mixture of both. Their results indicate that only patients with precipitating antibodies or with a mixture of precipitating and nonprecipitating antibodies had renal disease. Patients with nonprecipitating antiDNA antibodies did not have renal disease. Preliminary data seem to indicate that the avidity of the anti-nDNA molecule for the antigen could also have a role in the development of nephritis.374-3i6 Investigating the possible participation of other antigenantibody complexes in the pathogenesis of lupus nephritis, Robitaille and Tan used a sensitive binding assay to detect antibodies to both soluble dNP and nDNA.‘“’ Serial studies on four patients with SLE showed that high serum binding to radioactive soluble dNP coincided with proteinuria and low serum complement values. Serum antibodies of both specificities disappeared rapidly with treatment of the condition. The authors suggested that in addition to the previously demonstrated role of antibody to nDNA, antibody to soluble dNP might also be implicated in the pathogenesis of immune complex nephritis. This interesting suggestion should be further investigated in a larger series of patients with active SLE. Rheumatoid

Synovitis

The concept of immune complex disease with consequent complementdependent immunologic damage so firmly established for serum sickness, experimental glomerulonephritis, and SLE nephritis’7,33-3”.3”” has been extended to remove the rheumatoid joint.377 Indeed, several studies have suggested that immune mechanisms are operative in the rheumatoid joint. These findings include: Ig complexes precipitated by IgM RF or Clq,37x RF,37g and low levels of complement in the synovial fluid; 380-3*2 Ig and complement within synovial leukocytes;383-3”5 and immune complexes consisting of antiglobulins and IgG in the synovial fluidZjTx and in eluates from the rheumatoid synovial membrane.“86 Also RF, Ig, and complement components have been detected in the rheumatoid synovial membrane.3X7~3X” These studies have led to the concept that the deposition of immune complexes within the synovial membrane may initiate or perpetuate the inflammatory process3” and prompted the search for antigenantibody systems which might be involved in the process. The presence of nuclear antigens has been reported in synovial fluids from patients with RA.163.3XSSeveral laboratories have reported that ANA may be present in the synovial fluid of patients with RA,1X.lR3,391,-3!12 For example, a sensitive radioimmunoassay using ““I-soluble dNP has been employed to detect either soluble dNP antigen or anti-soluble dNP antibody in the synovial fluid.3g3 In this study, anti-soluble dNP antibody was almost restricted to synovial fluids from patients with RA. It was suggested that the

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finding of free soluble dNP antigen or homologous antibody in synovial fluids of patients with RA indicates that immune complexes might form and contribute to synovial injury.3g3 In support of this possibility, Cracchiolo and Goldberg were able to show the presence of ANA in eluates from the synovial membrane in 7 of 18 patients with RA 3g4 However, apparently, discrepant results were reported by Munthe and Natvig.3Q5 Initially, they were not able to detect ANA in eluates from the synovial membrane and in subsequent work they were able to find ANA in only 2 of 97 patients with RA. 386Different experimental conditions could possibly account for these results. For instance, Cracchiolo and Goldberg used higher molarity buffers and lo-hr elution time, while Munthe and Natvig used only I-hr elution time. Also it has been reported that the synovial fluid may contain DNA or other substances which may interfere with the detection of ANA in vitro.386 Though little is known about the antigen or antigens responsible for the immune reaction in the rheumatoid synovial membrane, these studies suggest the possibility that both nuclear antigens and ANA may be involved in the inflammation of the rheumatoid joint. 19.384.386 SUMMARY

The methods currently used for the detection of ANA have been analyzed, with emphasis on their practical application to the diagnosis of the CTD. The use of the indirect IF-ANA test was recommended as a screening procedure to detect ANA. The need to standardize the technique using a single substrate and fluorescent conjugates with uniform F/P ratios was stressed. Most importantly, the value of titrating ANA for the diagnosis of the CTD was discussed. ANA titers higher than l/500 are usually very significant clinically, often found in spontaneous or drug-induced SLE and few other CTD. The immunologic aspects of ANA and their potential value as aids in the diagnosis and management of the CTD were discussed. Anti-nDNA antibodies have been found to have a high degree of specificity for SLE and high titers of these antibodies correlate well with low levels of serum complement and severity of kidney involvement. The spectrum of ANA in the sera from patients with SLE has been expanded with the finding of anti-Sm antibodies which, when detected by gel precipitation with prototype serum, have been found so far only in SLE. Some of these antibodies have been found to have prognostic significance. Patients with MCTD and a group of patients with SLE have high titers of serum ANA with specificity for an RNase-sensitive component of ENA. The group of SLE patients defined by the presence of these antibodies (anti-MO) have a better prognosis and in general develop only mild nephritis or have no kidney involvement at all. High titers of pure antinucleolar antibodies probably are found almost exclusively in the sera of patients with scleroderma. Some ANA have organ specificity, and GS-ANA have been found in all patients with Felty’s syndrome and in a large proportion of patients with RA. One of the great advances in the field has been the recognition that ANA can be induced in the human and in experimental animals by the use of a number of therapeutic agents. Some of these agents can also induce a clinical picture resembling spontaneous SLE, though kidney involvement does not occur or is extremely mild. It is interesting that the whole spectrum of ANA can be found in

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drug-induced LE except anti-nDNA antibodies which have been associated to the pathogenesis of immune complex nephritis in spontaneous SLE. There is no doubt that research on ANA has contributed a great deal to the understanding of the CTD and will continue to be a valuable tool for the clinician and the investigator. REFERENCES 1. Hargraves MM, Richmond H, Morton R: Presentation of two bone marrow elements: The “tart” cell and “L.E.” cell. Proc Staff Meet Mayo Clin 23:25, 1948 2. Haserick J, Lewis L, Bortz DW: Blood factor in acute disseminated lupus erythematosus; Determination of gamma globulin as specific plasma fraction. Am J Med Sci 219:660, 1950 3. Miescher P, Straessle R: New serological methods for the detection of the L.E. factor. VOX Sang 2:283, 1957 4. Friou GJ, Finch SC, Detre KD: Interaction of nuclei and globulin from lupus erythematosus serum demonstrated with fluorescent antibody. J Immunol80:324, 1958 5. Bardawil WA, Toy BL, Galins N, et al: Disseminated lupus erythematosus, scleroderma and dermatomyositis as manifestations of sensitization to DNA-protein. Am J Pathol 34:607, 1958 6. Holborow EJ, Weir DM, Johnson CD: A serum factor in lupus erythematosus with affinity for tissue nuclei. Br Med J 2:732, 1957 7. Talbott JH, Ferrandis RM: Collagen Diseases, Including Systemic Lupus Erythematosus, Polyarteritis, Dermatoymyosits, Systemic Scleroderma, Thrombotic Thrombocytopenic Purpura. New York, Grune & Stratton, 1956 8. Goodman HC, Fahey JL, Malmgren RA: Serum factors in lupus erythematosus and other diseases reacting with cell nuclei and nucleoprotein extracts: Electrophoretic, ultracentrifugal and chromatographic studies. J Clin Invest 39:1595, 1960 9. Berman L, Axelrod AR, Goodman HL, et al: So-called “lupus erythematosus inclusion phenomenon” of bone marrow and blood; Morphologic and serologic studies. Am J Clin Pathol 20:403, 1950 IO. Lee SL, Michael SR, Vural IL: The L.E. (lupus erythematosus) cell; Clinical and chemical studies. Am J Med 10:446, 1951 I I. Fallet GH, Lospalluto J, Ziff M: Chromatographic and electrophoretic studies of the L.E. factor. Arthritis Rheum 1:419, 1958 12. Wilkens RF, Drechler M, Larson DL: Partial purification and preparation of lupus erythematosus cell factor. Proc Sot Exp Biol Med 99:645, 1959

13. Friou GJ: The L.E. cell phenomenon and antinuclear antibodies, in Hollander JL (ed): Arthritis and Allied Conditions, ed 7. Philadelphia Lea and Febiger, 197 1,p 99 14. Beck JS: Antinuclear antibodies: Methods of detection and significance. Mayo Clin Proc 44:600, 1969 15. Beutner EH: Defined immunofluorescent staining: Past progress, present status and future prospects for defined conjugates. Ann NY Acad Sci 177:506, 1970 16. Reichlin M, Mattioli M: Antigens and antibodies characteristic of systemic lupus erythematosus. Bull Rheum Dis 24:756, 1973 17. Cochrane CC, Koffler D: Immune complex disease in experimental animals and man. Adv Immunol 16:185, 1973 18. Faber V, Elling P, Norup G, et al: An antinuclear factor specific for leukocytes. Lancet 2~344, 1964 19. Elling P: Reaction of antinuclear factors with polymorphonuclear leukocytes. I. Absorption studies. Acta Pathol Microbial Stand 68:281, 1966 20. Faber V, Elling P: Leukocyte-specific antinuclear factors in patients with Felty’s syndrome, rheumatoid arthritis, systemic lupus erythematosus and other diseases. Acta Med Stand 1791257, 1966 21. Smalley MJ, Mackay IR, Whittingham S: Antinuclear factors and human leukocytes: Reaction with granulocytes and lymphocytes. Aust Ann Med 17:28, 1968 22. Feltkamp TEW: Idiopathic autoimmune disease. Thesis, 1964 (Amsterdam) 23. Albini B, Wick G: Erytbrocyte specific ANA in chickens of the obese strain (OS). Immunology 2 1:957, 1966 24. Cleymaet JE, Nakamura RM: Indirect immunofluorescent antinuclear antibody tests: Comparison of sensitivity and specificity of different substrates. Am J Clin Pathol 58:388, I972 25. Seligmann M, Milgrom F: Mise en evidence par la fixation du complement de la reaction entre acid desoxyribonucleique et serum des malades atteints de lupus erythemateux dissemine. C R Acad Sci (Paris) 245:1472, 1957

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26. Robbins WC, Holman HR, Deicher HRG, et al: Complement fixation with cell nuclei and DNA in lupus erythematosus. Proc Sot Exp Biol Med 96575, 1957 27. Cepellini R, Polli E, Celada F: A DNAreacting factor in serum of a patient with lupus erythematosus diffusus. Proc Sot Exp Biol Med 96572, 1957 28. Pearson CM, Craddock CG, Simmons N: Complement fixation reaction with DNA and leukocyte material in SLE: Correlation with the L.E. phenomenon and the clinical status. J Lab Clin Med 52:588, 1958 29. Holman HR, Deicher HRG, Kunkel H: The L.E. cell and the L.E. serum factors. Bull NY Acad Med 35:409, 1959 30. Bozicevich J, Nasou JP, Kayhoe DE: Deoxyribonucleic acid (DNA) bentonite flocculation test for lupus erythematosus. Proc Sot Exp Biol Med 103:636, 1960 31. Rothfield NF, Phythyon JM, McEwen C, et al: The role of antinuclear reactions in the diagnosis of systemic lupus erythematosus. A study of 53 cases. Arthritis Rheum 4:223, 1961 32. Dorner M, Enderlin M, Spiegelberg H, et al: Clinical and serological features of visceral lupus erythematosus. Dtsch Med Wochenschr 86:378, 1961 33. Germouth FG Jr, McKinnon GE: Studies on the biological properties of antigen-antibody complexes. 1. Anaphylactic shock induced by in soluble antigen-antibody complexes unsensitized normal guinea pigs. Bull Johns Hopkins Hosp 101:13, 1957 34. Benacerraf B, Potter JC, McClusky RT, et al: The pathologic effect of intravenously administered soluble antigen-antibody complexes. I. Acute glomerulonephritis in rats. J Exp Med II 1:195, 1960 35. Dixon FJ, Feldman JD, Vasquez JJ: Experimental glomerulonephritis. The pathogenesis of a laboratory model resembling the spectrum of human glomerulonephritis. J Exp Med 113:899, 1961 36. Koffler D, Schur PH, Kunkel HG: Immunological studies concerning the nephritis of systemic lupus erythematosus. J Exp Med 126: 60, 1967 37. Koffler D, Carr R, Agnello V, et al: Antibodies to polynucleotides in human sera: Antigenie specificity and relation to disease. J Exp Med 134:294, 1971 38. Cracchiolo A, Barnett EV: The role of immunological tests in routine synovial fluid analysis. J Bone Joint Surg (Am) 54:828, 1972 39. Heiyer, erythematosus

BJ, Howie JB: Positive lupus tests in a crossbred strain of mice

AND MATTIOLI

NZB/BL-NZY/Bl. Proc Univ Otago Med Sch (New Zealand) 39:3, 1961 40. Friou GJ, Teague PO: Spontaneous autoimmunity in mice: Antibodies to nucleoprotein in strain A/J. Science 143:1333, 1964 41. Shulman LE, Gumpel JM, D’Angelo WA, et al: Antinuclear factor in inbred strains of mice: The possible role of environmental influence. Arthritis Rheum 7:753, 1964 42. Lewis RM, Schwartz RS, Gilmore CE: Autoimmune disease in domestic animals. Ann NY Acad Sci 124: 178, 1965 43. Lewis RM, Schwartz R, Henry WB Jr: Canine systemic lupus erythematosus. Blood 25:143, 1965 44. Barnett EV, Williams RC, Kenyon AJ: Nuclear antigens and antinuclear antibodies in mink sera. Arthritis Rheum 1 l:92, 1968 45. Tan EM, Kunkel HG: Characteristics of a soluble nuclear antigen precipitating with sera of patients with systemic lupus erythematosus. J Immunol96:464, 1966 46. Tan EM: An immunologic precipitin system between soluble nucleoprotein and serum antibody in systemic lupus erythematosus. J Clin Invest 46:735, 1967 47. Friou GJ: Clinical application of lupus serum-nucleoprotein reaction using fluorescent antibody technique. J Clin Invest 36:890, 1957 48. Coons AH, Kaplan MH: Localization of antigen in tissue cells. II. Improvements in a method for detection of antigen by means of a fluorescent antibody. J Exp Med91:1, 1950 49. Holman HR, Kunkel HG: Affinity between the lupus erythematosus serum factor and cell nuclei and nucleoprotein. Science 126: 162, 2957 50. Friou GJ: The LE cell factor and antinuclear antibodies, in Cohen AS (ed): Laboratory Diagnostic Procedures in the Rheumatic Diseases. Boston, Little, Brown, 1967, p 114 51. Beck JS: Variations in the morphological patterns of “autoimmune” nuclear fluorescence. Lancet 1:1203, 1961 52. Bickel YB, Barnett EV, Pearson CM: Immunofluorescent patterns and specificity of human antinuclear antibodies. Clin Exp Immunol 3:641, I968 53. Lachmann PJ, Kunkel HG: Correlation of antinuclear antibodies and nuclear staining patterns. Lancet 2:436, 1961 54. Bergquist NR, Danielsson D: Evaluation of two different antigen preparations in the fluorescent antibody test for antinuclear antibodies (ANA). A comparison between two laboratories. Acta Path01 Microbial Stand 81:446, 1973

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55. Casals SP, Friou GJ, Teague PO: Specific nuclear reaction pattern of antibody to DNA in lupus erythematosus sera. J Lab Clin Med 621625, 1963 56. Dreesen G, Haven Cl, Halbridge N: A modified procedure for the detection of antinuclear factors using human leukocytes. Am J Clin Pathol57:463, 1972 57. Chodirker WB, Bock GN, Vaughan JH: Isolation of human PMN leukocytes and granules: Observations on early blood dilution. J Lab Clin Med 7 1:9, 1968 5X. Burnham TK, Fine G, Neblett TR: Tumor imprints as a source of nuclear substrate for the detection of antinuclear factors. J Invest Dermatol43:7, 1964 59. Amortegui AJ, Frisch CR: Endometrial cell imprints as a source of antigen for antinuclear antibody detection. Am J Clin Pathol 59591, 1973 60. Guss SB, Ugel AR: Methanol-fixed paraffin-embedded bovine and human epidermis for antinuclear antibodies. Arch Dermatol 106:674, 1972 61. Parker MD, Kerby GP: Combined titer and fluorescent pattern of IgG antinuclear antibodies using cultured cell monolayers in evaluating connective tissue diseases. Ann Rheum Dis 33:465, 1974 62. Smith Kline ANA, Clinical Laboratory Reference (ed 3) Medical Laboratory Observer, 1976 63. ANA by IF, Electronucleonics Lab Inc, Clinical Laboratory Reference (ed 3) Medical Laboratory Observer, 1976 64. Beutner EH, Sepulveda MR, Barnett EV: Quantitative studies of immunofluorescent staining. Relationships of characteristics of unabsorbed antihuman IgG conjugates to their specific and non-specific staining properties in an indirect test for antinuclear factor. Bull WHO 39587, 1968 65. Veen JH: Studies on the standardization of antinuclear antibody (ANA) determination. Ann NY Acad Sci 177:346, 1971 66. Sander G: Observer perception of fluorescence and autofluorescence, in Holborow EJ (ed): Standardization of Immunofluorescence. Oxford, Blackwell, 1968 67. Hale WL, Bergquist R: Chessboard analyses with antinuclear antibodies. Ann NY Acad Sci 177:354, 1970 6X. Beutner EH, Holborow EJ, Johnson GD: A new fluorescent antibody method. Mixed antiglobulin immunofluorescence or labeled indirect immunofluorescent staining. Nature 208:353, I965

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