C/iu. exp. Inmmunol. (1979) 36, 145-150.
Antibodies to histones in systemic lupus erythematosus EUGENIA FISHBEIN, D. ALARCON-SEGOVIA & J. M. VEGA Department of Immunology andRheumatology, Instititto Vacrional de la JVntricin, Mexico City, Mexico ( Accepted Jor publication 21 September 1978)
SUMMARY
Antibodies to histones were investigated in the serum of forty-five patients with spontaneously occurring systemic lupus erythematosus (SLE) who were not receiving any form of treatment. Twenty-three had active and twenty-two had inactive disease. Those with active disease were also studied after the initiation of corticosteroid treatment to determine the effect of treatment on anti-histone antibodies. Both a complement fixation method and indirect immunofluorescence of acid-eluted histone-reconstituted tissue sections were used, with excellent correlation between these two methods. Eleven of the forty-five SLE patients, but none of forty-five normal controls had antibodies to histone. Untreated patients with active and inactive disease had a similar incidence of antibodies to histone. They disappeared, however, soon after the initiation of treatment in the patients with active disease. Patients with antibodies to histones had a higher prevalence of cutaneous vasculitis, anaemia, lupus nephropathy and Raynaud's phenomenon, but a lower prevalence of lupus brain involvement than those without such antibodies. Only the latter, however, reached statistical significance. INTRODUCTION The serum of patients with systemic lupus erythematosus (SLE) contains a wide variety of antibodies to nuclear antigens. These include antibodies to DNA, whether directed to its secondary structure, to the sugar backbone, or to the exposed bases (Alarcon-Segovia et al., 1970); to nucleoprotein, both particulate and soluble (Tan, 1967); to the extractable nuclear antigens (Holman, 1965) which include Sm antigen, Tan 1975, ribonucleoprotein (Northway & Tan, 1972), and Asa & Tan, defined antigens (Alspaugh eldfndatgn es well n other te less a,17) INrha riboulort 195, have been histones to Antibodies 1976). & Robitaille, to Robinson 1976); or histones (Stollar, 1971; Tan, the least studied of the antinuclear antibodies (ANA) and there is little information regarding their possible clinical significance. Particular attention has been paid in the past to DNA/anti-DNA immune complexes in the pathogenesis of SLE. More recent data has shown that other systems may also be important (Panem et al., 1976; Izui, Lambert & Miescher, 1977). Our recent finding that antinuclear antibodies can penetrate into viable cells and interact with nuclear antigens (Alarcon-Segovia, Ruiz-Argiielles & Fishbein, 1978) may mean that the formation of immune complexes is only one of the mechanisms of immunologically mediated damage in which ANA may participate in the pathogenesis of SLE. Indeed, intranuclear immunoglobulin may occasionally be found in live circulating Fcy+ monuclear cells from patients with SLE or mixed connective tissue disease (Alarcon-Segovia, Ruiz-Argiielles & Fishbein, 1979). Because the interaction of ANA with different nuclear antigens in viVo could result in different patterns of cellular dysfunction, it seems important to determine the clinical characteristics of SLE patients with different antinuclear antibodies. The purpose of this study of antibodies to histones in SLE patients xras to determine their prevalence, Correspondence: Dr D. Alarc6n-Segovia, Instituto Nacional de la Nutrici6n, Mexico 22, D.F., Mexico. 0099-9104/79/0040-0145$02.00 iCt 1979 Blackwell Scientific Publications K
145
146
Eugenia Fishbein, D. Alarcon-Segovia & J. M. Vega
their relationship to disease activity or inactivity, their changes in presence or titres as the result of treatment and their possible clinical significance. PATIENTS AND METHODS Patients. We studied sera from forty-five patients who fulfilled the preliminary criteria for the classification of systemic lupus erythemato3us (SLE) of the American Rheumatism Association (Cohen et al., 1971) who were neither receiving corticosteroid or immunosuppressive treatment at the time of the study, nor had received it for at least 3 months. Patients included had also not taken salicylatse or non-steroidal anti-inflammatory agents for at least 2 weeks. None of the patients included had drug-induced SLE. Twenty-three of these forty-five patients had active disease as defined previously (Rivero et al., 1977) and were therefore started on corticosteroid treatment after blood was drawn for this study. When possible, subsequent blood samples were drawn in these patients at 15, 30, 60, 120 and 180 days after the initiation of corticosteroid treatment. The other twenty-two patients had inactive disease (Rivero et al., 1977) and therefore received no treatment. We studied one serum from each of these twenty-two patients. A total of ninety sera were studied. Sera from forty-five normal subjects were studied as controls. Antibodies to histones. We sought antibodies to histones by two methods: complement fixation and immunofluorescence. Complement fixation was done using the micromethod as described previously (Alarc6n-Segovia & Fishbein, 1972) using calf thymus histone (Sigma Chemicals, Saint Louis, Missouri) as antigen at a concentration of 25 pg/ml. This concentration was not found to cause lysis of erythrocytes as suggested by Hekman & Sluyser (1973). Indeed, only 16% haemolysis was observed at a histone concentration of 10 mg/ml. An immunofluorescence study of anti-histone antibodies was carried out as described by Tan et al. (1976). Briefly, acetonefixed 4 p thick mouse kidney sections were treated with 0 1 N HCl to elute histone. Some of the acid-eluted sections were reconstituted by incubation with calf thymus histone at a concentration of 25 pg/ml. A fresh solution was prepared for each assay to prevent aggregation of the histones. Untreated, acid-eluted, and reconstituted sections were used as substrate for indirect immunofluorescent studies using commercial anti-Ig antibody (Behringwerke AG Marburg/Lahn). Sera were diluted 1:10 for initial screening and those found positive were diluted further to determine their titre. Antinuclear antibodies. Antibodies to other nuclear antigens were studied by micro-complement fixation (AlarconSegovia et al., 1970). Nuclear antigens included whole calf thymus nuclei, particulate nucleoprotein, soluble nucleoprotein, native DNA, heat-denatured DNA, Sm antigen, calf thymus RNA, synthetic double stranded RNA (PolyA PolyU), synthetic single stranded RNA (PolyU) and nuclear ribonucleoprotein. The sources of these antigens and their mode of utilization in the study of antinuclear antibodies by complement fixation methods were the same as described previously (Alarcon-Segovia et al., 1970; 1975; 1976; Alarcon-Segovia & Fishbein, 1972; 1975). -
RESULTS Eleven of the forty-five SLE patients were found to have serum antibodies to histone and in all eleven, these were found in titres ranging from 1: 2 to 1: 16 by the complement fixation method, and from 1:10 to 1:80 by immunofluorescence testing (Tables 1 and 2). None of the controls had detectable antibodies to histone. There was little discrepancy between both methods. One serum was positive by complement fixation and negative by immunofluorescence (patient 9, serum 2) and two sera that were anti-complementary on complement fixation (patient 1, sera 2 and 4), were positive by immunofluorescence. These slight differences occurred in patients who had other positive sera. There was no difference in the TABLE 1. Serum antibodies to histones in SLE
Number positive
Complement fixation SLE Patients (45) Sera (90) Active disease, untreated (23) Inactive disease, untreated (22) Controls (45)
11 14 6 5 0
Both Immunofluorescence methods 11 15 6 5 0
11 13 6 5 0
Antibodies to hisiones in SLE
147
TABLE 2. Titres of antibodies to histones in eleven patients with SLE. Comparison between complement fixation and immunofluorescence
Titre
Patient 1
2 3 4 5 6 7 8
9
10
11
Serum number
Complement fixation
1 2 3 4 5 1 1 1 1 1 1 1 2 3 4 1 2 3 4 5 1 2 3 4 5 1
1:4 Ac(1:4)* 1:4 Ac(1:2) Ac(1:2) 1:16 1:4 1:2 1:2 1:2 1:2 1:2 Neg. Neg. Neg. 1:2 1:2 Neg. Neg. Neg. 1:8 1:4 Ac(1 :2) Neg. Neg. 1:2
Immunofluorescence 1:10 1:10 1:10 1:10
Neg.t 1:80 1:10 1:10 1:10 1:20 1:10 1:10 Neg. Neg. Neg. 1:10 Neg. Neg. Neg. Neg. 1:20 1:20 Neg. Neg. Neg. 1:10
* Ac = Anti-complementary; parentheses indicate titre.
t Neg. = Negative.
prevalence of antibodies to histones in the serum of patients with either active or inactive disease who were not receiving treatment (Table 1). However, when patients with active disease were started on ,orticosteroid treatment, serum antibodies to histones disappeared promptly, as shown in Fig. 1. Only one patient had positive sera for anti-histone antibodies by immunofluorescence and was anti-complementary in the complement fixation test after 60 days of corticosteroid treatment. Subsequent sera from this patient remained anti-complementary, but became negative by immunofluorescence. The patterns seen in immunofluorescence testing on untreated mouse kidney sections in the fifteen sera :ound to have anti-histone antibodies are shown in Table 3. The results with acid-treated sections, )efore and after reconstitution with histones, are also shown. Positive sera on histone-reconstituted -nouse kidney sections gave a gross nuclear lumpy pattern. No cytoplasmic or membrane staining occurred. Xcid-treated sections were all negative in these fifteen sera, irrespective of their original pattern. Sera vithout antibodies to histones gave widely different patterns with untreated, acid-treated and histoneeconstituted sections. Four examples with their probable specificity are presented in Table 4. Two sera from lupus patients had antibodies to histones as the only detectable ANA when studied against the panel of nuclear antigens. There was a significant correlation (P< 0 005) between the reacivity with histones and with soluble nucleoprotein. We found no other correlation between the anti)odies to histones and other ANA. Clinical differences between patients with and without antibodies to histones are presented in Table 5.
148
Eugenia Fislibein, D. Alarco'n-Segovia & J. M. Vega 1:16
-
A-*
1:8
0
1:4
~0* te*
C.
0
u
1:2
E
0o _
A,
And
U
4{o
1
0
15
30
60
120
180
Days on corticosteroid treatment
FIG. 1. Serum antibodies to histones in six patients with active SLE. Effect of corticosteroid treatment on presence and titres is shown in four patients. * Positive by immunofluorescence. t Anti-complementary. Each symbol indicates one patient.
TABLE 3. Serum antibodies to histones in SLE. Patterns in immunofluorescent testing with untreated, acid-treated and acid-treated, histone-reconstituted mouse kidney sections, in fifteen sera with antibodies to histones
Acid-treated sections
Untreated sections, pattern
Acid-treated histone-reconstituted sections Number
Homogeneous
-
+-
Rim
-
Speckled Homogeneous and rim Speckled and rim Homogeneous, speckled, rim and nucleolar
-
+ + + + +
-
-
6 3 1 3 1 1
TABLE 4. Serum antibodies to histones in SLE. Example of pattern in immunofluorescent testing with untreated, acid-treated and acid-treated, histone-reconstituted, mouse kidney sections in four sera without
antibodies to histones
Untreated sections, pattern
Acid-treated sections
Acid-treated histone-reconstituted sections
Antibodies to non-histone nuclear
Speckled Homologous and rim Speckled and rim Homogeneous and speckled
Probable interpretation
Rim Rim
Rim Rim
Homogeneous
Homogeneous
proteins Antibodies to DNA Antibodies to non-histone nuclear proteins and to DNA Antibodies to non-histone nuclear proteins and to DNA
149
Antibodies to histones in SLE TABLE 5. Summary of clinical manifestations in SLE patients with and without antibodies to histones Percentage antibodies to histones
Manifestation
Vasculitis Anaemia Nephropathy Raynaud's phenomenon Neuropsychiatric
Positive (11) 91 91 64 54 5 9
Negative (34)
65 65 32 38 50
Difference P n.s. n.s. n.s. n.s.
0-02
The higher prevalence of cutaneous vasculitis, anaemia, lupus nephropathy and Raynaud's phenomenon in patients with antibodies to histones did not reach statistical significance. Patients with antibodies to histones had a significantly lower prevalence of central nervous system involvement than those without. DISCUSSION One fourth of our SLE patients had antibodies to histone irrespective of whether the disease was active or inactive. These antibodies, however, disappeared promptly after the initiation of corticosteroid therapy. This study confirms the observation of Tan et al. (1976) that antibodies to histones cross-react with antibodies to soluble nucleoprotein. With the immunofluorescent method this could be expected since in the acid-treated mouse kidney section DNA remains and may, upon reconstitution with histone, form complexes of DNA-histone. The similar findings with complement fixation methods could reflect the fact that soluble nucleoprotein is a fragmentary portion of particulate or 'native' nucleoprotein where histone antigenic determinants may be exposed so as to cross-react with antibodies to histone. Although it is unusual in SLE to have only one ANA present in the serum, such monospecific sera may be useful tools for the study of the function of histone, particularly since the capacity of these antibodies to enter living cells has been recognized (Alarc6n-Segovia et al., 1978). The different frequency of various clinical manifestations in SLE patients who have anti-histone antibodies should be considered with caution. In general, the more active the disease, the wider the number of clinical manifestations and the wider the range of autoantibodies in the patient (Alarc6nSegovia et al., 1970). The presence of an antibody that concurs with a decreased frequency of a clinical manifestation may be more meaningful. Anti-RNP antibody has been found to be associated with decreased kidney involvement in patients with SLE (Reichlin & Mattioli, 1972), although its presence may in turn be indicative of a different disease with features of SLE, scleroderma, polymyositis and rheumatoid arthritis known as mixed connective tissue disease (Sharp et al., 1972). It is of interest that patients with drug-induced SLE have recently been found to be particularly prone to develop antibodies to histone (Fritzler & Tan, 1977). This may also indicate that different ANA tend to correlate with different subsets of SLE. Antibody to histone in spontaneously occurring SLE may either be a marker of a subset of SLE having less frequent central nervous system involvement, or may intervene in such a way so as to result in the diminished involvement of the central nervous system in the course of systemic lupus erythematosus. This work was supported in part by Research Grants from the Consejo Nacional de Ciencia y Tecnologia (CONACYT), Mexico (Grant 1485, Programa Nacional de Salud) and the Academia Nacional de Medicina (Eli Lilly Fund), Mexico.
150
Eugenia Fishbein, D. Alarcon-Segovia ' J. M. Vega REFERENCES
ALARC6N-SEGOVIA, D. & FISHBEIN, E. (1972) Complement fixation tests for the detection of antinuclear antibodies. WHO Booklet on Immunological Techniques (ed. by G. Torrigiani), p. 31. WHO, Geneva. ALARC6N-SEGOVIA, D. & FISHBEIN, E. (1975) Immunochemical characterization of the anti-RNA antibodies found in scleroderma and systemic lupus erythematosus. I.
Differences in reactivity with Poly(U) and Poly(A)Poly(U). J. Immunol. 115, 28. ALARc6N-SEGOVIA, D., FISHBEIN, E., ALCALA, H., OLGUfNPALACIOS, E. & ESTRADA-PARRA, S. (1970) The range and specificity of antinuclear antibodies in systemic lupus erythematosus. Clin. exp. Immunol. 6, 557. ALARC6N-SEGOVIA, D., FISHBEIN, E., ESTRADA-PARRA, S. & GARCfA-ORTIGOZA, E. (1976) Inimunochemical characterization of the anti-RNA antibodies found in scleroderma and systemic lupus erythematosus. II. Reactivity with HSA-coupled uridine-containing monophosphoric ribonucleotides. Immunology, 30, 413. ALARC6N-SEGOVIA, D., FISHBEIN, E., GARCfA-ORTIGOZA, E. & ESTRADA-PARRA, S. (1975) Uracil-specific anti-RNA antibodies in scleroderma. Lancet, i, 363. ALARc6N-SEGOVIA, D., RUfZ-ARGOELLES, A. & FISHBEIN, E. (1978) Antibody to ribonucleoprotein can penetrate live human mononuclear cells through Fc receptors. Nature (Lond.), 271, 67. ALARC6N-SEGOVIA, D., RUfZ-ARGUELLES, A. & FISHBEIN, E. (1979) Antibody penetration into living cells. I. Intranuclear immunoglobulin in peripheral blood mononuclear cells in mixed connective tissue disease and systemic lupus erythematosus. Clin. exp. Immunol. 35, 364. ALSPAUGH, M.A. & TAN, E.M. (1975) Antibodies to cellular antigens in Sjogren's syndrome. 7. clin. Invest. 55, 1067. ALSPAUGH, M.A. & TAN, E.M. (1976) Serum antibody in rheumatoid arthritis reactive with a cell associated antigen. Arthr. and Rheum. 19, 711. COHEN, A.S., REYNOLDS, W.E., FRANKLIN, E.C., KULKA, J.P., RoPES, M.W., SHULMAN, L.E. & WALLACE, S.L. (1971) Preliminary criteria for the classification of systemic lupus erythematosus. Bull. Rheum. Dis. 21, 643.
FRITZLER, M.J. & TAN, E.M. (1977) Antibodies to histones in sera of patients with systemic lupus erythematosus. Clin. Res. 25, 483A. HEKmAN, A. & SLUYSER, M. (1973) Antigenic determinants on lysine-rich histones. Biochim. biophys. Acta. 295, 613. HOLMAN, H.R. (1965) Partial purification and characterization of an extractable nuclear antigen which reacts with SLE sera. Ann. N.Y. Acad. Sci. 124, 800. IZUI, S., LAMBERT, P.H. & MiEscHER, P.A. (1977) Failure to detect circulating DNA-anti-DNA complexes by four radioimmunological methods in patients with systemic lupus erythematosus. Clin. exp. Immunol. 30, 384. NORTHWAY, J.D. & TAN, E.M. (1972) Differentiations of antinuclear antibodies giving speckled staining patterns in immunofluorescence. Clin. Immunol. Immunopathol. 1,
140. PANEM, S., ORDO&EZ, N.G., KIRSTEIN, W.H., KATZ, A.I. & SPARGO, B.H. (1976) C-type virus expression in systemic lupus erythematosus kidneys. New Engl. ). Med. 295, 470. REICHLIN, M. & MATTIOLI, M. (1972) Correlation of a precipitin reaction to an RNA protein antigen and a low prevalence of nephritis in patients with systemic lupus erythematosus. New Engl. J. Med. 286, 908. RivERo, S.J., LLORENTE, L., DfAZ-JOUANEN, E. & ALARC6NSEGOVIA, D. (1977) T-lymphocyte subpopulation in untreated SLE. Variations with disease activity. Arthr. and Rheum. 20, 1169. SHARP, G.C., IRVIN, W.S., TAN, E.M., GouLD, R.G. & HOLMAN, H.R. (1972) Mixed connective tissue diseasean apparently distinct rheumatic disease syndrome associated with a specific antibody to an extractable nuclear antigen (ENA). Amer. J. Med. 52, 148. STOLLAR, B.D. (1971) Reactions of systemic lupus erythematosus sera with histone fractions and histone-DNA complexes. Arthr. and Rheum. 14, 485. TAN, E.M. (1967) An immunologic precipitin system between soluble nucleoprotein and serum antibody in systemic lupus erythematosus. 7. clin. Invest. 46, 735. TAN, E.M., ROBINSON, J., ROBITAILLE, P. (1976) Studies on antibodies to histones by immunofluorescence. Scand. ]. Immunol. 5, 811.
Antibodies to histones in systemic lupus erythematosus EUGENIA FISHBEIN, D. ALARCON-SEGOVIA & J. M. VEGA Department of Immunology andRheumatology, Instititto Vacrional de la JVntricin, Mexico City, Mexico ( Accepted Jor publication 21 September 1978)
SUMMARY
Antibodies to histones were investigated in the serum of forty-five patients with spontaneously occurring systemic lupus erythematosus (SLE) who were not receiving any form of treatment. Twenty-three had active and twenty-two had inactive disease. Those with active disease were also studied after the initiation of corticosteroid treatment to determine the effect of treatment on anti-histone antibodies. Both a complement fixation method and indirect immunofluorescence of acid-eluted histone-reconstituted tissue sections were used, with excellent correlation between these two methods. Eleven of the forty-five SLE patients, but none of forty-five normal controls had antibodies to histone. Untreated patients with active and inactive disease had a similar incidence of antibodies to histone. They disappeared, however, soon after the initiation of treatment in the patients with active disease. Patients with antibodies to histones had a higher prevalence of cutaneous vasculitis, anaemia, lupus nephropathy and Raynaud's phenomenon, but a lower prevalence of lupus brain involvement than those without such antibodies. Only the latter, however, reached statistical significance. INTRODUCTION The serum of patients with systemic lupus erythematosus (SLE) contains a wide variety of antibodies to nuclear antigens. These include antibodies to DNA, whether directed to its secondary structure, to the sugar backbone, or to the exposed bases (Alarcon-Segovia et al., 1970); to nucleoprotein, both particulate and soluble (Tan, 1967); to the extractable nuclear antigens (Holman, 1965) which include Sm antigen, Tan 1975, ribonucleoprotein (Northway & Tan, 1972), and Asa & Tan, defined antigens (Alspaugh eldfndatgn es well n other te less a,17) INrha riboulort 195, have been histones to Antibodies 1976). & Robitaille, to Robinson 1976); or histones (Stollar, 1971; Tan, the least studied of the antinuclear antibodies (ANA) and there is little information regarding their possible clinical significance. Particular attention has been paid in the past to DNA/anti-DNA immune complexes in the pathogenesis of SLE. More recent data has shown that other systems may also be important (Panem et al., 1976; Izui, Lambert & Miescher, 1977). Our recent finding that antinuclear antibodies can penetrate into viable cells and interact with nuclear antigens (Alarcon-Segovia, Ruiz-Argiielles & Fishbein, 1978) may mean that the formation of immune complexes is only one of the mechanisms of immunologically mediated damage in which ANA may participate in the pathogenesis of SLE. Indeed, intranuclear immunoglobulin may occasionally be found in live circulating Fcy+ monuclear cells from patients with SLE or mixed connective tissue disease (Alarcon-Segovia, Ruiz-Argiielles & Fishbein, 1979). Because the interaction of ANA with different nuclear antigens in viVo could result in different patterns of cellular dysfunction, it seems important to determine the clinical characteristics of SLE patients with different antinuclear antibodies. The purpose of this study of antibodies to histones in SLE patients xras to determine their prevalence, Correspondence: Dr D. Alarc6n-Segovia, Instituto Nacional de la Nutrici6n, Mexico 22, D.F., Mexico. 0099-9104/79/0040-0145$02.00 iCt 1979 Blackwell Scientific Publications K
145
146
Eugenia Fishbein, D. Alarcon-Segovia & J. M. Vega
their relationship to disease activity or inactivity, their changes in presence or titres as the result of treatment and their possible clinical significance. PATIENTS AND METHODS Patients. We studied sera from forty-five patients who fulfilled the preliminary criteria for the classification of systemic lupus erythemato3us (SLE) of the American Rheumatism Association (Cohen et al., 1971) who were neither receiving corticosteroid or immunosuppressive treatment at the time of the study, nor had received it for at least 3 months. Patients included had also not taken salicylatse or non-steroidal anti-inflammatory agents for at least 2 weeks. None of the patients included had drug-induced SLE. Twenty-three of these forty-five patients had active disease as defined previously (Rivero et al., 1977) and were therefore started on corticosteroid treatment after blood was drawn for this study. When possible, subsequent blood samples were drawn in these patients at 15, 30, 60, 120 and 180 days after the initiation of corticosteroid treatment. The other twenty-two patients had inactive disease (Rivero et al., 1977) and therefore received no treatment. We studied one serum from each of these twenty-two patients. A total of ninety sera were studied. Sera from forty-five normal subjects were studied as controls. Antibodies to histones. We sought antibodies to histones by two methods: complement fixation and immunofluorescence. Complement fixation was done using the micromethod as described previously (Alarc6n-Segovia & Fishbein, 1972) using calf thymus histone (Sigma Chemicals, Saint Louis, Missouri) as antigen at a concentration of 25 pg/ml. This concentration was not found to cause lysis of erythrocytes as suggested by Hekman & Sluyser (1973). Indeed, only 16% haemolysis was observed at a histone concentration of 10 mg/ml. An immunofluorescence study of anti-histone antibodies was carried out as described by Tan et al. (1976). Briefly, acetonefixed 4 p thick mouse kidney sections were treated with 0 1 N HCl to elute histone. Some of the acid-eluted sections were reconstituted by incubation with calf thymus histone at a concentration of 25 pg/ml. A fresh solution was prepared for each assay to prevent aggregation of the histones. Untreated, acid-eluted, and reconstituted sections were used as substrate for indirect immunofluorescent studies using commercial anti-Ig antibody (Behringwerke AG Marburg/Lahn). Sera were diluted 1:10 for initial screening and those found positive were diluted further to determine their titre. Antinuclear antibodies. Antibodies to other nuclear antigens were studied by micro-complement fixation (AlarconSegovia et al., 1970). Nuclear antigens included whole calf thymus nuclei, particulate nucleoprotein, soluble nucleoprotein, native DNA, heat-denatured DNA, Sm antigen, calf thymus RNA, synthetic double stranded RNA (PolyA PolyU), synthetic single stranded RNA (PolyU) and nuclear ribonucleoprotein. The sources of these antigens and their mode of utilization in the study of antinuclear antibodies by complement fixation methods were the same as described previously (Alarcon-Segovia et al., 1970; 1975; 1976; Alarcon-Segovia & Fishbein, 1972; 1975). -
RESULTS Eleven of the forty-five SLE patients were found to have serum antibodies to histone and in all eleven, these were found in titres ranging from 1: 2 to 1: 16 by the complement fixation method, and from 1:10 to 1:80 by immunofluorescence testing (Tables 1 and 2). None of the controls had detectable antibodies to histone. There was little discrepancy between both methods. One serum was positive by complement fixation and negative by immunofluorescence (patient 9, serum 2) and two sera that were anti-complementary on complement fixation (patient 1, sera 2 and 4), were positive by immunofluorescence. These slight differences occurred in patients who had other positive sera. There was no difference in the TABLE 1. Serum antibodies to histones in SLE
Number positive
Complement fixation SLE Patients (45) Sera (90) Active disease, untreated (23) Inactive disease, untreated (22) Controls (45)
11 14 6 5 0
Both Immunofluorescence methods 11 15 6 5 0
11 13 6 5 0
Antibodies to hisiones in SLE
147
TABLE 2. Titres of antibodies to histones in eleven patients with SLE. Comparison between complement fixation and immunofluorescence
Titre
Patient 1
2 3 4 5 6 7 8
9
10
11
Serum number
Complement fixation
1 2 3 4 5 1 1 1 1 1 1 1 2 3 4 1 2 3 4 5 1 2 3 4 5 1
1:4 Ac(1:4)* 1:4 Ac(1:2) Ac(1:2) 1:16 1:4 1:2 1:2 1:2 1:2 1:2 Neg. Neg. Neg. 1:2 1:2 Neg. Neg. Neg. 1:8 1:4 Ac(1 :2) Neg. Neg. 1:2
Immunofluorescence 1:10 1:10 1:10 1:10
Neg.t 1:80 1:10 1:10 1:10 1:20 1:10 1:10 Neg. Neg. Neg. 1:10 Neg. Neg. Neg. Neg. 1:20 1:20 Neg. Neg. Neg. 1:10
* Ac = Anti-complementary; parentheses indicate titre.
t Neg. = Negative.
prevalence of antibodies to histones in the serum of patients with either active or inactive disease who were not receiving treatment (Table 1). However, when patients with active disease were started on ,orticosteroid treatment, serum antibodies to histones disappeared promptly, as shown in Fig. 1. Only one patient had positive sera for anti-histone antibodies by immunofluorescence and was anti-complementary in the complement fixation test after 60 days of corticosteroid treatment. Subsequent sera from this patient remained anti-complementary, but became negative by immunofluorescence. The patterns seen in immunofluorescence testing on untreated mouse kidney sections in the fifteen sera :ound to have anti-histone antibodies are shown in Table 3. The results with acid-treated sections, )efore and after reconstitution with histones, are also shown. Positive sera on histone-reconstituted -nouse kidney sections gave a gross nuclear lumpy pattern. No cytoplasmic or membrane staining occurred. Xcid-treated sections were all negative in these fifteen sera, irrespective of their original pattern. Sera vithout antibodies to histones gave widely different patterns with untreated, acid-treated and histoneeconstituted sections. Four examples with their probable specificity are presented in Table 4. Two sera from lupus patients had antibodies to histones as the only detectable ANA when studied against the panel of nuclear antigens. There was a significant correlation (P< 0 005) between the reacivity with histones and with soluble nucleoprotein. We found no other correlation between the anti)odies to histones and other ANA. Clinical differences between patients with and without antibodies to histones are presented in Table 5.
148
Eugenia Fislibein, D. Alarco'n-Segovia & J. M. Vega 1:16
-
A-*
1:8
0
1:4
~0* te*
C.
0
u
1:2
E
0o _
A,
And
U
4{o
1
0
15
30
60
120
180
Days on corticosteroid treatment
FIG. 1. Serum antibodies to histones in six patients with active SLE. Effect of corticosteroid treatment on presence and titres is shown in four patients. * Positive by immunofluorescence. t Anti-complementary. Each symbol indicates one patient.
TABLE 3. Serum antibodies to histones in SLE. Patterns in immunofluorescent testing with untreated, acid-treated and acid-treated, histone-reconstituted mouse kidney sections, in fifteen sera with antibodies to histones
Acid-treated sections
Untreated sections, pattern
Acid-treated histone-reconstituted sections Number
Homogeneous
-
+-
Rim
-
Speckled Homogeneous and rim Speckled and rim Homogeneous, speckled, rim and nucleolar
-
+ + + + +
-
-
6 3 1 3 1 1
TABLE 4. Serum antibodies to histones in SLE. Example of pattern in immunofluorescent testing with untreated, acid-treated and acid-treated, histone-reconstituted, mouse kidney sections in four sera without
antibodies to histones
Untreated sections, pattern
Acid-treated sections
Acid-treated histone-reconstituted sections
Antibodies to non-histone nuclear
Speckled Homologous and rim Speckled and rim Homogeneous and speckled
Probable interpretation
Rim Rim
Rim Rim
Homogeneous
Homogeneous
proteins Antibodies to DNA Antibodies to non-histone nuclear proteins and to DNA Antibodies to non-histone nuclear proteins and to DNA
149
Antibodies to histones in SLE TABLE 5. Summary of clinical manifestations in SLE patients with and without antibodies to histones Percentage antibodies to histones
Manifestation
Vasculitis Anaemia Nephropathy Raynaud's phenomenon Neuropsychiatric
Positive (11) 91 91 64 54 5 9
Negative (34)
65 65 32 38 50
Difference P n.s. n.s. n.s. n.s.
0-02
The higher prevalence of cutaneous vasculitis, anaemia, lupus nephropathy and Raynaud's phenomenon in patients with antibodies to histones did not reach statistical significance. Patients with antibodies to histones had a significantly lower prevalence of central nervous system involvement than those without. DISCUSSION One fourth of our SLE patients had antibodies to histone irrespective of whether the disease was active or inactive. These antibodies, however, disappeared promptly after the initiation of corticosteroid therapy. This study confirms the observation of Tan et al. (1976) that antibodies to histones cross-react with antibodies to soluble nucleoprotein. With the immunofluorescent method this could be expected since in the acid-treated mouse kidney section DNA remains and may, upon reconstitution with histone, form complexes of DNA-histone. The similar findings with complement fixation methods could reflect the fact that soluble nucleoprotein is a fragmentary portion of particulate or 'native' nucleoprotein where histone antigenic determinants may be exposed so as to cross-react with antibodies to histone. Although it is unusual in SLE to have only one ANA present in the serum, such monospecific sera may be useful tools for the study of the function of histone, particularly since the capacity of these antibodies to enter living cells has been recognized (Alarc6n-Segovia et al., 1978). The different frequency of various clinical manifestations in SLE patients who have anti-histone antibodies should be considered with caution. In general, the more active the disease, the wider the number of clinical manifestations and the wider the range of autoantibodies in the patient (Alarc6nSegovia et al., 1970). The presence of an antibody that concurs with a decreased frequency of a clinical manifestation may be more meaningful. Anti-RNP antibody has been found to be associated with decreased kidney involvement in patients with SLE (Reichlin & Mattioli, 1972), although its presence may in turn be indicative of a different disease with features of SLE, scleroderma, polymyositis and rheumatoid arthritis known as mixed connective tissue disease (Sharp et al., 1972). It is of interest that patients with drug-induced SLE have recently been found to be particularly prone to develop antibodies to histone (Fritzler & Tan, 1977). This may also indicate that different ANA tend to correlate with different subsets of SLE. Antibody to histone in spontaneously occurring SLE may either be a marker of a subset of SLE having less frequent central nervous system involvement, or may intervene in such a way so as to result in the diminished involvement of the central nervous system in the course of systemic lupus erythematosus. This work was supported in part by Research Grants from the Consejo Nacional de Ciencia y Tecnologia (CONACYT), Mexico (Grant 1485, Programa Nacional de Salud) and the Academia Nacional de Medicina (Eli Lilly Fund), Mexico.
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Eugenia Fishbein, D. Alarcon-Segovia ' J. M. Vega REFERENCES
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