Clinical Relevance of Immunologic Findings in Scleroderma STEPHANIA JABLONSKA, MD. MARIA BLASZCZYK, MD TADEUSZ I’. CHORZELSKI, MD MARIA JARZABEK-CHORZELSKA, VIJAY KUMAR, PhD ERNST H. BEUTNER, PhD l
PhD
l
T
he pathogenesis of systemic scleroderma (SSc) still remains unknown, although the immunologic mechanisms appear to be of basic signiticance.’
Cell-Mediated
Immunity
Cellular immunity was shown to be involved in the process of vascular and fibrotic derangements characteristic of the disease.2-6 From the diagnostic point of view, tests for cell-mediated immunity, such as lymphocyte transformation and helper suppressor ratio,‘-lo circulating soluble interleukin 2 (IL2) receptor,” and lymphocyte-induced angiogenesis assay, l* do not contribute to a diagnosis of scleroderma; however, some of them may be helpful in evaluating the activity and severity of the disease, especially the test for natural killer cells (NK),13 and type III collagen aminopropeptide levels in serum.‘4-16 A decrease in NK activity13 and an increased level of IL-2-soluble receptors” correlate to some degree with the severity of SSc. Diminished lymphocyte-induced angiogenesis reflects the derangements in cell-mediated immunity and microvasculature,12 and the modified angiogenesis test with the use of serum is helpful in recognizing limited SSc.”
A Possible Role for Retroviruses in Induction of Autoimmune Responses Of significance from the diagnostic point of view is the identification of circulating antinuclear antibodies From the Department ofDermatology, Warsaw School of Medicine, Warsaw, Poland, and the Department of Microbiology, State University of New York at Buffalo, Buffalo, New York. Address correspondence to Stephania lablonska, MD, Department of Dermatology, Warsaw School of Medicine, ul. Koszykowa 82a, 02 008 Warsaw, Poland.
0 1993 by Elsevier Science Publishing Co., Inc.
l
0738-081x/93/$6.00
(ANAs), some of which were found to be highly characteristic or specific for SSc [eg, antibody to DNA topoisomerase I (Topo I)] and its subsets. The stimulus for autoantibody production in SSc is not yet known. It has been shown that the group-specific antigen of some mammalian retroviruses, p3O@s, has an amino acid sequence nearly identical to that of Topo I and that Topo I antibody may cross-react with viral antigen. Cloned major epitope of Topo I, the marker of SSc, contains a six amino acid match with the p3O@s retroviral protein.ls Although the circulating antibodies may be an epiphenomenon not involved in the pathogenesis of the disease, the role of retroviruses in the induction of immune response appears to be very probable. The role of retroviruses in eliciting autoimmune responses is further substantiated by the presence of anti-human immunodeficiency virus (HIV)-1 p24es antibodies in 25.9% cases of diffuse scleroderma, with no relationship to Topo I (Scl 70) or other antibodies. The significance of this finding is not known, but it is probably due to molecular mimi~ry.‘~ It is of special interest that Topo I antibodies found in the tight skin mouse share with the human Scl70 antibody an interspecies cross-reactive idiotype. This crossreactivity is related to peptide sequence homology also identified in the UL70 protein of cytomegaloviruszO Thus, Topo I is an antigen highly conserved during phylogeny, and the elicitation of antibodies to Topo I may be possible by molecular mimicry of viral antigens.
Prevalence of Various Types of Antinuclear Antibodies in Systemic Scleroderma In our earlier study’ on various tissue substrates, we detected ANAs in approximately 54% cases of limited scleroderma (LS) and in approximately 72% of diffuse scleroderma (DS); however when the studies were repeated many times, at different intervals, the results were ulti-
407
408
JABLONSKA
Clinics in Dermatology 1993;10:407-421
ET AL
Table 1. Prevalence of Various Antibodies in Systemic Sclerosis Antibody* SC170 ACA Nucleolar Homogeneous (PM, Scl) Clumpy (fibrillarin) Speckled (RNA polymerase I) RNP Ro/La Nonidentified lines in DID ANA (+), DID (-) ANA (-), DID (-) NSP I
Table 2. Prevalence of Various Antibodies in Diffuse and Limited Systemic Scleroderma
Total (n = 394) 260 (65.9%)t 53 (13.5%)$ 21 (5.3%) 7 (2.0%) 9 (2.1%) 5 (1.3%) 14 (3.6%) 8 (2.0%)6 5 (1.3%) 21 (5.3%) 11 (2.8%) 1 (0.3%)”
l ACA, anticentromereantibody; JZNP,ribonucleoprotein; ANA, antinuclear antibody;DID, double immunodifision. t In 17 cases,coexistedother antibodies(in IO AC&. # In nine cases,coexistedother antibodiesin addition to lOcaseswith coexistentScl 70. s In two cases,coexistedother antibodies. ‘I In this case,coexistedmitochondtial antibodies.
mately positive in almost all cases of DS, and in LS, the positive results increased to approximately 75%. Overall frequency of positive ANA findings was 71%. In our recent studies, we detected ANAs by indirect immunofluorescence (IIF) using monkey esophagus and HEp 2 cells as substrates, by double immunodiffusion (DID), and, in some cases, by immunoblot (IB) with the recombinant antigens Topo I, kinetochore, and La.21 Our series comprised 394 cases, and antibodies were not found in only 11 (2.8%). In 29 patients, more than one antibody was present (Table 1). As seen in Table 1, various antibodies can be associated with SSc; however, the vast majority of cases were found to be positive for Scl70 or anticentromere antibody (ACA), with a high prevalence of Scl 70 (approximately 66%). Nucleolar antibodies were detected in a lower percentage than reported by some investigators22*23 who recognized as a nucleolar pattern dotted nucleoli within the fine, grainy fluorescence of the nucleoplasm (ie, the characteristic pattern of Scl 70). It should be stressed that some antibodies are still unidentified (eg, sera negative by DID but positive on HEp 2 cells or positive on HEp 2 cells but producing unidentified lines by DID (26 cases, 6.6%). In 29 cases we found coexistence of various ANAs, and of special importance was the concomitant occurrence in 10 patients of Scl70 and ACA, regarded as being mutually exclusive. When our series is classified according to the major subtypes of SSc: DS and LS (acrosclerosis), there is a high prevalence of Scl70 in DS, whereas in LS, both antibodies
Antibody SC170 ACA Nucleolar Homogeneous (PM, Scl) Clumpy (fibrillarin) Speckled (RNA polymerase I) RNP Ro/La Nonidentified lines in DID ANA (+), DID (-) ANA (-), DID (-) NSP I
Diffuse SSc* (n = 162)
Limited 5% (n = 232)
133 (82.1%) 0
127 (54.7%) 53 (22.9%)
0
7 (3.0%)
9 (5.6%)
0
0
5 (2.2%)
2 (1.2%) 2 (1.2%) 1 (0.6%)
12 (5.1%) 6 (2.6%) 4 (1.7%)
9 (5.6%) 6 (3.7%) 0
12 (5.1%) 5 (2.2%) 1 (0.4%)
* SSc,systemicscleroderma;other abbreviationsPSin Table 1.
characteristic of SSc (Scl 70 and ACA) were disclosed (Table 2). As seen in Table 2, Scl 70 antibodies were found in 82.1% of patients with DS and in over 50% of patients with LS. As a single antibody, ACA was not detected in any case of DS, and in over 20% of patients, it was associated with LS (see below Anticentromere Antibody). There was an evident difference in the types of antinucleolar antibodies in DS and LS. No homogeneous [polymyositis (PM)-Scl antibody] and speckled (RNApolymerase I antibody) patterns were found in DS, whereas a clumpy pattern (fibrillarin antibody) was associated exclusively with this variety.
Scl70 Antibody as a Marker for Systemic Scleroderma Scl 70 antibody, despite low detectability (6 of 26 cases, 23%) in the original report was regarded as a marker for DS.24 In the same series, this antibody was also found in 13% of patients with LS (acrosclerosis). The frequency of its detection in SSc varies considerably, which might be due to different, not well-standardized techniques of antigen preparation for DID25 and, in part, to the divergent clinical classification of the disease. Enzyme-linked immunosorbent assay (ELISA) with purified antigens, especially with smaller active components (70-60 kd), was reported to give 75% positive results in DS, whereas DID was positive only in 25% of cases.26 Some discordant results between ELISA with Topo I antigen and IB with lOO-kd antigen were reported
Clinics in Dermatology 1993;10:407-421
CLINICAL
RELEVANCE
OF IMMUNOLOGIC
FINDINGS
JABLONSKA ET AL IN SCLERODERMA
409
Table 3. Clinical and Immunologic Findings in Diffuse SclerodermaPositive c-k)and Negative (-J for Scl 70 Antibodu SC1 70+
(n = 112) Mean age (years) Gender, F/M (96 F)’
Mean duration of R (years) Mean duration of S (years) Mean period between R and S (years) Indurative edema Contractures of the fingers Digital scars
Figure 2.
IIF on HEp 2 cells. Characteristic pattern of Scl 70 (diffusely grainy nucleoplasm with clearly visible dotted nucleoli); (upper left) strongly stained metaphasechromatin.
Calcinosis Telangiectasia Central involvement
by Weiner et al. 27 Verheijen et a12* detected Topo I antibody in IB assay in 70% of SSc cases using recombinant Topo I and Topo I antigen from HeLa cell extract. A high detectability of Topo I antibody was reported by McNeilage et a129.Of note, in Australia Topo I was found in 77% of Oriental patients and in only 26% of white patients. This points to the significance of genetic factors in specific autoimmune responses. Different sensitivities of IB and ELISA for detection of Topo I IgG class antibodies were reported by Weiner et a12’ For IgG class antibodies, IB was more sensitive and even more so for IgM class antibodies. For IgA antibodies, the results of both methods were essentially the same. Although the most simple and sensitive method appears to be ELISA, until the antigen is fully characterized, purified or cloned, one should not rely on a single method, and the use of all available techniques (ELISA, IB, DID, and IIF) may be helpful. The IIF pattern on HEp 2 cells is highly characteristic of Scl70 antibodies, and IIF study should be used for screening in every assay for Topo I (Fig 1). Unfortunately, many researchers do not use IIF for the detection of Topo I antibodies. For example, sera negative for Topo I antibodies in ELISA and positive for lOO-kd antigen in IB could be directed against other antigens with the same molecular weight, and some, such as antigen To, could be easily recognized by the characteristic immunofluorescence (IF) pattern. In our series, DS was classified according to preliminary criteria for the classification of SSc30and LS according to the criteria of Le Roy et a1.31In Table 3 we present the major clinical characteristics and laboratory findings in cases of DS found to be positive or negative for Scl70
Arthritis Visceral involvement Esophagus Heart Lung Kidney Muscle ANA ACA Nucleolar (clumpy) RNP Ro/La Other ANA negative
46.8 100/12 (89.3%) 9.1 4.8 4.3
SC170(n = 24) 39.9 17/7 (70.8%) 2.7 2.4 0.3 22 (91.6%)
(89458%) 63/111 (56.7%) 76/111 (68.5%) 32/108 (29.6%) 67/110 (60.9%) 95/111 (85.5%) 95/108 (87.9%) 74/111 (66.6%) 55/108 (50.9%) 60/110 (54.5%) 17/108 (15.7%) 44/100 (44%) 112/112 (100%) P 0 0 0 0 0
13 (54.1%) 11 (45.8%)t 4 (16.7%) 4 (16.7%)$ 24 (loo%)t 18 (75%)
16 (66.6%) 15 (62.5%) 9 (37.5%) 3 (12.5%) 14 (58.3%) 18 (75%)$ 0 8 2 1 7$ 6 (25%)$
F, female;A$ male; other abbreviationsas in Table 1. f P < .05. $P
PhD
l
T
he pathogenesis of systemic scleroderma (SSc) still remains unknown, although the immunologic mechanisms appear to be of basic signiticance.’
Cell-Mediated
Immunity
Cellular immunity was shown to be involved in the process of vascular and fibrotic derangements characteristic of the disease.2-6 From the diagnostic point of view, tests for cell-mediated immunity, such as lymphocyte transformation and helper suppressor ratio,‘-lo circulating soluble interleukin 2 (IL2) receptor,” and lymphocyte-induced angiogenesis assay, l* do not contribute to a diagnosis of scleroderma; however, some of them may be helpful in evaluating the activity and severity of the disease, especially the test for natural killer cells (NK),13 and type III collagen aminopropeptide levels in serum.‘4-16 A decrease in NK activity13 and an increased level of IL-2-soluble receptors” correlate to some degree with the severity of SSc. Diminished lymphocyte-induced angiogenesis reflects the derangements in cell-mediated immunity and microvasculature,12 and the modified angiogenesis test with the use of serum is helpful in recognizing limited SSc.”
A Possible Role for Retroviruses in Induction of Autoimmune Responses Of significance from the diagnostic point of view is the identification of circulating antinuclear antibodies From the Department ofDermatology, Warsaw School of Medicine, Warsaw, Poland, and the Department of Microbiology, State University of New York at Buffalo, Buffalo, New York. Address correspondence to Stephania lablonska, MD, Department of Dermatology, Warsaw School of Medicine, ul. Koszykowa 82a, 02 008 Warsaw, Poland.
0 1993 by Elsevier Science Publishing Co., Inc.
l
0738-081x/93/$6.00
(ANAs), some of which were found to be highly characteristic or specific for SSc [eg, antibody to DNA topoisomerase I (Topo I)] and its subsets. The stimulus for autoantibody production in SSc is not yet known. It has been shown that the group-specific antigen of some mammalian retroviruses, p3O@s, has an amino acid sequence nearly identical to that of Topo I and that Topo I antibody may cross-react with viral antigen. Cloned major epitope of Topo I, the marker of SSc, contains a six amino acid match with the p3O@s retroviral protein.ls Although the circulating antibodies may be an epiphenomenon not involved in the pathogenesis of the disease, the role of retroviruses in the induction of immune response appears to be very probable. The role of retroviruses in eliciting autoimmune responses is further substantiated by the presence of anti-human immunodeficiency virus (HIV)-1 p24es antibodies in 25.9% cases of diffuse scleroderma, with no relationship to Topo I (Scl 70) or other antibodies. The significance of this finding is not known, but it is probably due to molecular mimi~ry.‘~ It is of special interest that Topo I antibodies found in the tight skin mouse share with the human Scl70 antibody an interspecies cross-reactive idiotype. This crossreactivity is related to peptide sequence homology also identified in the UL70 protein of cytomegaloviruszO Thus, Topo I is an antigen highly conserved during phylogeny, and the elicitation of antibodies to Topo I may be possible by molecular mimicry of viral antigens.
Prevalence of Various Types of Antinuclear Antibodies in Systemic Scleroderma In our earlier study’ on various tissue substrates, we detected ANAs in approximately 54% cases of limited scleroderma (LS) and in approximately 72% of diffuse scleroderma (DS); however when the studies were repeated many times, at different intervals, the results were ulti-
407
408
JABLONSKA
Clinics in Dermatology 1993;10:407-421
ET AL
Table 1. Prevalence of Various Antibodies in Systemic Sclerosis Antibody* SC170 ACA Nucleolar Homogeneous (PM, Scl) Clumpy (fibrillarin) Speckled (RNA polymerase I) RNP Ro/La Nonidentified lines in DID ANA (+), DID (-) ANA (-), DID (-) NSP I
Table 2. Prevalence of Various Antibodies in Diffuse and Limited Systemic Scleroderma
Total (n = 394) 260 (65.9%)t 53 (13.5%)$ 21 (5.3%) 7 (2.0%) 9 (2.1%) 5 (1.3%) 14 (3.6%) 8 (2.0%)6 5 (1.3%) 21 (5.3%) 11 (2.8%) 1 (0.3%)”
l ACA, anticentromereantibody; JZNP,ribonucleoprotein; ANA, antinuclear antibody;DID, double immunodifision. t In 17 cases,coexistedother antibodies(in IO AC&. # In nine cases,coexistedother antibodiesin addition to lOcaseswith coexistentScl 70. s In two cases,coexistedother antibodies. ‘I In this case,coexistedmitochondtial antibodies.
mately positive in almost all cases of DS, and in LS, the positive results increased to approximately 75%. Overall frequency of positive ANA findings was 71%. In our recent studies, we detected ANAs by indirect immunofluorescence (IIF) using monkey esophagus and HEp 2 cells as substrates, by double immunodiffusion (DID), and, in some cases, by immunoblot (IB) with the recombinant antigens Topo I, kinetochore, and La.21 Our series comprised 394 cases, and antibodies were not found in only 11 (2.8%). In 29 patients, more than one antibody was present (Table 1). As seen in Table 1, various antibodies can be associated with SSc; however, the vast majority of cases were found to be positive for Scl70 or anticentromere antibody (ACA), with a high prevalence of Scl 70 (approximately 66%). Nucleolar antibodies were detected in a lower percentage than reported by some investigators22*23 who recognized as a nucleolar pattern dotted nucleoli within the fine, grainy fluorescence of the nucleoplasm (ie, the characteristic pattern of Scl 70). It should be stressed that some antibodies are still unidentified (eg, sera negative by DID but positive on HEp 2 cells or positive on HEp 2 cells but producing unidentified lines by DID (26 cases, 6.6%). In 29 cases we found coexistence of various ANAs, and of special importance was the concomitant occurrence in 10 patients of Scl70 and ACA, regarded as being mutually exclusive. When our series is classified according to the major subtypes of SSc: DS and LS (acrosclerosis), there is a high prevalence of Scl70 in DS, whereas in LS, both antibodies
Antibody SC170 ACA Nucleolar Homogeneous (PM, Scl) Clumpy (fibrillarin) Speckled (RNA polymerase I) RNP Ro/La Nonidentified lines in DID ANA (+), DID (-) ANA (-), DID (-) NSP I
Diffuse SSc* (n = 162)
Limited 5% (n = 232)
133 (82.1%) 0
127 (54.7%) 53 (22.9%)
0
7 (3.0%)
9 (5.6%)
0
0
5 (2.2%)
2 (1.2%) 2 (1.2%) 1 (0.6%)
12 (5.1%) 6 (2.6%) 4 (1.7%)
9 (5.6%) 6 (3.7%) 0
12 (5.1%) 5 (2.2%) 1 (0.4%)
* SSc,systemicscleroderma;other abbreviationsPSin Table 1.
characteristic of SSc (Scl 70 and ACA) were disclosed (Table 2). As seen in Table 2, Scl 70 antibodies were found in 82.1% of patients with DS and in over 50% of patients with LS. As a single antibody, ACA was not detected in any case of DS, and in over 20% of patients, it was associated with LS (see below Anticentromere Antibody). There was an evident difference in the types of antinucleolar antibodies in DS and LS. No homogeneous [polymyositis (PM)-Scl antibody] and speckled (RNApolymerase I antibody) patterns were found in DS, whereas a clumpy pattern (fibrillarin antibody) was associated exclusively with this variety.
Scl70 Antibody as a Marker for Systemic Scleroderma Scl 70 antibody, despite low detectability (6 of 26 cases, 23%) in the original report was regarded as a marker for DS.24 In the same series, this antibody was also found in 13% of patients with LS (acrosclerosis). The frequency of its detection in SSc varies considerably, which might be due to different, not well-standardized techniques of antigen preparation for DID25 and, in part, to the divergent clinical classification of the disease. Enzyme-linked immunosorbent assay (ELISA) with purified antigens, especially with smaller active components (70-60 kd), was reported to give 75% positive results in DS, whereas DID was positive only in 25% of cases.26 Some discordant results between ELISA with Topo I antigen and IB with lOO-kd antigen were reported
Clinics in Dermatology 1993;10:407-421
CLINICAL
RELEVANCE
OF IMMUNOLOGIC
FINDINGS
JABLONSKA ET AL IN SCLERODERMA
409
Table 3. Clinical and Immunologic Findings in Diffuse SclerodermaPositive c-k)and Negative (-J for Scl 70 Antibodu SC1 70+
(n = 112) Mean age (years) Gender, F/M (96 F)’
Mean duration of R (years) Mean duration of S (years) Mean period between R and S (years) Indurative edema Contractures of the fingers Digital scars
Figure 2.
IIF on HEp 2 cells. Characteristic pattern of Scl 70 (diffusely grainy nucleoplasm with clearly visible dotted nucleoli); (upper left) strongly stained metaphasechromatin.
Calcinosis Telangiectasia Central involvement
by Weiner et al. 27 Verheijen et a12* detected Topo I antibody in IB assay in 70% of SSc cases using recombinant Topo I and Topo I antigen from HeLa cell extract. A high detectability of Topo I antibody was reported by McNeilage et a129.Of note, in Australia Topo I was found in 77% of Oriental patients and in only 26% of white patients. This points to the significance of genetic factors in specific autoimmune responses. Different sensitivities of IB and ELISA for detection of Topo I IgG class antibodies were reported by Weiner et a12’ For IgG class antibodies, IB was more sensitive and even more so for IgM class antibodies. For IgA antibodies, the results of both methods were essentially the same. Although the most simple and sensitive method appears to be ELISA, until the antigen is fully characterized, purified or cloned, one should not rely on a single method, and the use of all available techniques (ELISA, IB, DID, and IIF) may be helpful. The IIF pattern on HEp 2 cells is highly characteristic of Scl70 antibodies, and IIF study should be used for screening in every assay for Topo I (Fig 1). Unfortunately, many researchers do not use IIF for the detection of Topo I antibodies. For example, sera negative for Topo I antibodies in ELISA and positive for lOO-kd antigen in IB could be directed against other antigens with the same molecular weight, and some, such as antigen To, could be easily recognized by the characteristic immunofluorescence (IF) pattern. In our series, DS was classified according to preliminary criteria for the classification of SSc30and LS according to the criteria of Le Roy et a1.31In Table 3 we present the major clinical characteristics and laboratory findings in cases of DS found to be positive or negative for Scl70
Arthritis Visceral involvement Esophagus Heart Lung Kidney Muscle ANA ACA Nucleolar (clumpy) RNP Ro/La Other ANA negative
46.8 100/12 (89.3%) 9.1 4.8 4.3
SC170(n = 24) 39.9 17/7 (70.8%) 2.7 2.4 0.3 22 (91.6%)
(89458%) 63/111 (56.7%) 76/111 (68.5%) 32/108 (29.6%) 67/110 (60.9%) 95/111 (85.5%) 95/108 (87.9%) 74/111 (66.6%) 55/108 (50.9%) 60/110 (54.5%) 17/108 (15.7%) 44/100 (44%) 112/112 (100%) P 0 0 0 0 0
13 (54.1%) 11 (45.8%)t 4 (16.7%) 4 (16.7%)$ 24 (loo%)t 18 (75%)
16 (66.6%) 15 (62.5%) 9 (37.5%) 3 (12.5%) 14 (58.3%) 18 (75%)$ 0 8 2 1 7$ 6 (25%)$
F, female;A$ male; other abbreviationsas in Table 1. f P < .05. $P
