Ioannides D, Golden BD, Buyon JP, Bystryn J. Expression of SS-A/Ro and SS-B/La Antigens in Skin Biopsy Specimens of Patients With Photosensitive Forms of Lupus Erythematosus. Arch Dermatol. 2000;136(3):340-346. doi:10.1001/archderm.136.3.340
The reason that only some patients with lupus erythematosus (LE) develop autoantibodies to SS-A/Ro and SS-B/La antigens and photosensitivity is unknown. One hypothesis is that both events are related to the level of expression of these antigens in the skin.
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To test this hypothesis, we measured the expression of the 52-kd SS-A/Ro, 60-kd SS-A/Ro, and 48-kd SS-B/La antigens in normal sun-protected and sun-exposed skin in 14 patients with LE with photosensitivity, 12 patients with LE without photosensitivity, and 4 normal individuals. The presence of circulating antibodies to these antigens was measured in all patients.
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We found that the expression of the 52-kd SS-A/Ro, 60-kd SS-A/Ro, and 48-kd SS-B/La antigens in skin biopsy specimens obtained from the same site was 4- to 10-fold higher in patients with LE with photosensitivity than in those patients with LE without photosensitivity (P<.001). Antigen expression was highly correlated with the presence and titer of circulating anti–SS-A/Ro and anti–SS-B/La antibodies (P<.001).
These findings indicate that photosensitivity and the presence and titer of circulating anti–SS-A/Ro and anti–SS-B/La antibodies are both directly correlated with the expression of accessible and immunoreactive SS-A/Ro and SS-B/La antigens in the skin specimens of patients with LE. Thus, the expression of these antigens in keratinocytes may be an important determinant of the development of both SS-A/Ro and SS-B/La autoantibodies and of photosensitive forms of LE.
PHOTOSENSITIVE cutaneous manifestations of systemic lupus erythematosus (SLE) are associated with the reaction of serum autoantibodies against SS-A/Ro (hereinafter "Ro") and SS-B/La (hereinafter "La") ribonucleoproteins.1,2 The reason for the development of these autoantibodies in some subsets of patients with lupus erythematosus (LE) is not known. However, we recently found that there is a marked variation in the expression of Ro and La antigens in keratinocytes among patients with LE.3 Moreover, it was demonstrated that both antigens are expressed on the surface of cultured human keratinocytes irradiated with UV light,4,5 suggesting that autoantibodies can be formed in response to exposure of sequestered nuclear autoantigens following exposure to sunlight.6 These observations suggest the hypothesis that both Ro and La antibodies and photosensitivity appear preferentially in patients who are high expressors of these antigens in their skin.
To address this hypothesis, we compared the level of expression of Ro and La antigens in sun-exposed and sun-protected skin specimens of patients with photosensitive and nonphotosensitive forms of LE and correlated the results to the presence and level of autoantibodies to these antigens.
Two biopsy specimens of skin were obtained from each of 30 individuals. Of the 30, 4 were normal individuals and 26 were consecutive patients with LE. Of the 26 patients with LE, 7 had photosensitive SLE, 7 had photosensitive subacute cutaneous LE (SCLE), 5 had nonphotosensitive SLE, and 7 had nonphotosensitive discoid LE. Photosensitivity was defined as described below. The diagnoses of SLE, SCLE, and discoid LE were based on clinical, histological, and immunofluorescence criteria. All patients with SLE satisfied the 1982 American Rheumatism Association revised criteria7 for the diagnosis of SLE. The clinical features of the patients and their level of anti-Ro and anti-La antibodies are summarized in Table 1. None of the patients had received systemic therapy for at least 3 weeks at the time the biopsy specimens were obtained. Two biopsy specimens were obtained from each individual: one of clinically normal skin from a sun-exposed site (back of the hand) and another from a sun-protected site (buttock). Two sets of biopsy specimens were obtained on different days in 4 of the patients: 2 with SLE, 1 with SCLE, and 1 with discoid LE. Photosensitivity was evaluated in all patients by irradiating a 5 × 10-cm2 skin patch on the upper part of the back with UV light (peak emission, 360 nm), 3 J/cm2 for 40 minutes, on 2 different occasions. The test sites were examined daily for 3 days after irradiation, and patients were classified as photosensitive if erythema or urticaria was induced.
Three serum samples with antibodies reacting predominantly with the 52-kd Ro antigen (serum sample 1), the 60-kd Ro antigen (serum sample 2), or the 48-kd La antigen (serum sample 3) were selected from several hundred serum samples that were positive for antinuclear antibodies (ANA) of patients seen at the Division of Rheumatology, New York University Medical Center, New York. The reactivity profile of the 3 sera is summarized in Table 2. All 3 were positive for ANAs (titer, ≥1280 on human Hep-2 cells. The specificity of the antibodies was established using immunodiffusion, enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis. Serum samples 1 and 2 produced only 1 precipitin line of identity with the anti-Ro reference serum; and serum sample 3 produced 1 precipitin line of identity with the anti-La reference serum by double immunodiffusion using mammalian spleen and thymus extract (Zeus Scientific, Raritan, NJ). Serum samples 1 and 2 with anti-Ro reactivity showed a strong positive reaction in the results of ELISA using bovine spleen as the source of the Ro antigen (Immunovision, Springdale, Ariz) (>30 SDs above control) and showed a negative reaction using rabbit thymus as the source of the La antigen (Immunovision). Results of immunoblot analysis using lysates of MOLT-4 cells showed that serum sample 1 reacted strongly with the 52-kd Ro antigen and weakly with the 60-kd Ro antigen, while serum sample 2 reacted strongly with the 60-kd Ro antigen and weakly with the 52-kd Ro antigen. The MOLT-4 cells were specifically chosen because previous studies3,8,9 demonstrated that these cells provide an excellent source of the Ro and La protein components that are reliably recognized in immunoblot analysis in serum samples containing the respective antibodies. Serum sample 3 with anti-La reactivity showed a strong positive reaction in the results of ELISA using rabbit thymus as the source of the La antigen (>30 SDs above control) and showed a negative reaction using bovine spleen as the source of the Ro antigen. The MOLT-4 cells reacted only with the 48-kd La antigen using immunoblot analysis.
All 3 serum samples were negative for antibodies to double-stranded nuclear DNA using the Crithidia luciliae method, for antibodies to nRNP (nuclear ribonucleoprotein), and Sm antigens using hemagglutination and ELISA (Diamedix Corporation, Miami, Fla), and for antibodies to epidermal cytoplasmic, cell surface, or basement membrane zone antigens using indirect immunofluorescence against guinea pig and monkey esophagus and normal human skin. Four normal human serum samples that were negative for ANAs were used as controls.
The expression of Ro and La antigens in skin specimens was determined by indirect immunofluorescence from the end-point titer of the Ro- or La-specific antiserum tested with skin specimens, as described previously.10,11 Briefly, skin biopsy specimens were mounted in optimal cutting temperature medium (Miles Laboratory, Ames Division, Elkhart, Ind) and cut in 4-µm serial sections on a cryostat. Indirect immunofluorescence was performed as previously described10 by incubating the skin sections in serial dilutions of Ro- or La-specific antiserum or normal human serum for 30 minutes, washing, incubating with fluorescein-conjugated antihuman IgG for 30 minutes, washing, and mounting with glycerin-phosphate-buffered saline solution. The level of expression of Ro and/or La antigen(s) was determined from the highest serum sample dilution giving a positive reaction (end-point titer). All specimens were read by the same investigator (D.I.), who did not have knowledge of the patients' diagnosis or antibody profile. To exclude the possibility that positive assay results were due to in vivo binding of ANA, direct immunofluorescence was performed on all tissue specimens using fluorescein-conjugated antihuman IgG, IgM, IgA, and C3. In no case was staining of the epidermal cell surface, cytoplasm, or nucleus observed.
The 2-tailed Mann-Whitney U test was used for comparison of unpaired nonparametric data from the patient groups. For estimation of correlation between variables in individual patients, the 2-tailed nonparametric Spearman rank correlation coefficient was used. All calculations were performed using statistical software (GraphPad Instat; GraphPad Software Inc, San Diego, Calif).
The expression of Ro and La antigens in sun-exposed and sun-protected skin was measured in 30 individuals using as probes 3 human serum samples containing antibodies directed predominantly to the 52-kd Ro, the 60-kd Ro, or the 48-kd La antigen. The results are summarized in Table 3.
All 3 antigens were expressed in the nuclei of epidermal keratinocytes and showed a staining pattern consisting of closely packed fine speckles. All skin specimens expressed all 3 antigens. However, the expression of each antigen varied widely among different individuals, as evidenced by a 60- to 512-fold difference in the end-point titer when the same antiserum sample was tested with the skin specimens obtained from the same site in different individuals. In sun-protected skin specimens, the expression of the 52-kd Ro antigen varied by more than 512-fold (from an end-point titer of 10-5120), the 60-kd Ro antigen by 60-fold (from an end-point titer of 20-1280), and the La antigen by 128-fold (end-point titer of 20-2560). Variation in the expression of these antigens was even greater in skin specimens obtained from sun-exposed sites (Table 3). Similar results were obtained when the skin specimens were tested with another set of antiserum samples to these 3 antigens (data not shown).
To exclude the possibility that the variability in antigen expression was due to poor reproducibility of the assay or to antigen degradation during biopsy or processing, skin specimens from the same sun-exposed and sun-protected sites were obtained on 2 different occasions in 4 patients. The end-point titer was similar (within a factor of 1 dilution) in all instances (Table 4). To further exclude antigen loss due to degradation during the assay procedure, 4 of the skin specimens were preincubated in a saline solution for 5, 10, 15, 30, 45, 60, 90, or 120 minutes prior to assay for antigen expression. Again, there was no difference in the end-point titers (data not shown). To exclude the possibility that the variations in staining were due to the presence of autoantibodies in the skin specimens, the specimens were tested with phosphate-buffered saline solution instead of the test serum samples and showed no nuclear staining. Finally, some skin specimens of patients expressed more 60-kd Ro than 52-kd Ro antigen (for example, patients 1, 3, and 11), while the reverse was true for skin specimens of other patients (patients 8, 13, and 24). These observations indicate that the reactivity of the anti–52-kd Ro antiserum sample was not due to the low level of anti–60-kd Ro that it contained.
As opposed to the wide interindividual variations observed, all 3 antigens in skin specimens of the same patient were generally expressed in a comparable level (within a factor of 1 dilution). However, some skin specimens expressed one antigen more strongly than the other. For example, patients 16 and 20 expressed much more Ro than La antigen, whereas the reverse was true for patients 17 and 24.
The studies described herein were conducted on skin specimens obtained from 14 patients with photosensitive LE (7 with SLE and 7 with SCLE), 12 with nonphotosensitive LE (5 with SLE and 7 with discoid LE), and 4 normal individuals. The results are presented in Table 3 and summarized in Table 5. The expression of all 3 antigens was much higher in patients with photosensitivity than in those without photosensitivity both in sun-exposed and in sun-protected skin specimens. The expression of the 52-kd Ro antigen (expressed as a mean end-point titer) in sun-exposed skin specimens of patients with (n=14) or without (n=16) photosensitivity was 1091 compared with 141 (P<.001), the expression of the 60-kd Ro antigen was 1806 compared with 212 (P<.001), and the expression of the 48-kd La antigen was 1246 compared with 281 (P<.001), respectively. There was a similar highly statistically significant (P<.001) increase in the expression of the 3 antigens in patients with photosensitivity when the comparison was made using skin specimens obtained from sun-protected sites. The expression of the 3 antigens in the skin specimens of patients with nonphotosensitive LE did not differ from the expression in the skin specimens of normal individuals (P<.10).
Since skin specimens were available from sun-exposed skin (back of the hand) and sun-protected skin (buttock) in all patients, we further examined whether sun exposure influenced the expression of Ro and La antigens. The results are shown in Table 3 and summarized in Table 5. The expression of the 60-kd Ro antigen was always much higher in sun-exposed than in sun-protected skin specimens in all 14 patients with photosensitive LE (mean end-point titer of 1806 compared with 509, respectively). By contrast, the expression of this antigen in skin specimens of 16 patients with nonphotosensitive LE or normal individuals was similar in both sites (mean end-point titer of 212 compared with 151, respectively), as was the expression of the 52-kd Ro and 48-kd La antigens.
Table 6 shows a strong correlation between the expression of Ro and La antigens in skin, the titer of circulating antibody to the respective antigens, and photosensitivity. For example, all 8 patients with a high level of the 52-kd Ro antigen in sun-protected skin specimens were photosensitive and showed circulating anti-Ro antibodies. By contrast, photosensitivity and anti-Ro antibodies were present in only 2 (15%) and 3 (23%), respectively, of the skin specimens of 13 patients who were low expressors of this antigen.
The correlation coefficient between circulating anti-Ro titer (as shown in Table 1) and 52-kd Ro expression in sun-exposed skin (as shown in Table 3) among all 30 patients was 0.82 (95% confidence interval [CI], 0.65-0.91; P<.001). The correlation coefficients between anti-Ro titer and 60-kd Ro expression in sun-exposed and sun-protected sites were 0.75 (95% CI, 0.53-0.88; P<.001) and 0.60 (95% CI, 0.29-0.79; P<.001), respectively. The correlation coefficients between anti-La and expression of the 48-kd La antigen in both sites were 0.65 (95% CI, 0.37-0.82; P<.001) and 0.66 (95% CI, 0.38-0.83; P<.001), respectively.
The results of this study indicate that the expression of immunoreactive Ro and La antigens is higher in the skin of patients with photosensitive forms of LE than in patients with nonphotosensitive forms of LE and normal individuals and that patients who are high antigen expressors are more likely to have circulating anti-Ro or anti-La antibodies than those who are low antigen expressors.
The study confirms earlier observations3 that the expression of the 52-kd Ro, 60-kd Ro, and 48-kd La antigens varies markedly the skin taken from the same site in different individuals with LE. There was a 60- to 512-fold difference in the expression of these antigens in skin obtained from the same sun-protected site (buttock) in different individuals. A similar variation in expression was present in sun-exposed skin (back of the hand). These differences in expression were so large that they could easily be detected by indirect immunofluorescence. These differences were real, as they were much greater than the day-to-day variation in the reproducibility of the assay, which varied by no more than 50% when performed on different days on the same specimen of skin in different individuals. These differences in expression were much greater than those we observed previously for other epidermal antigens, such as the bullous pemphigoid, epidermolysis bullosa acquisita, pemphigus vulgaris, and pemphigus foliaceus antigens whose expression in skin of the same anatomical region in different individuals varies by no more than 50%.10- 12 The reasons for these differences in antigen expression are not known. It does not seem to be related to age or sex.3 It is not due to differences in the specificity or binding affinity of the Ro or La antibodies, since the same serum samples were used to test all skin specimens, or to regional variations in antigen expression, since the specimens of skin in all cases were obtained from the same anatomical location. However, the possibility that the changes in antigen level reflect changes in the accessibility of the antigen to antibody, rather than to the actual amount of antigen, has not been excluded. In addition, the changes in antigen reactivity that we observed also might reflect changes in the conformation, posttranslational modification, or epitope availability of the antigens. Whatever the agents that determine the expression of immunoreactive Ro and La antigens, they modulate the expression of these antigens independently, as reflected by the dissociation in the expression of these antigens between different individuals in some instances.
When the results were stratified by the presence of photosensitivity, the expression of all 3 antigens in skin was on average 3- to 8-fold greater in patients with photosensitive SLE and SCLE than in patients with nonphotosensitive LE or normal individuals. These differences were highly significant with P≤.001. These differences in antigen expression were a general phenomenon that occurred in both sun-exposed and sun-protected skin. Since the same serum samples were used to test all skin specimens, all skin specimens were obtained from the same locations on the body, and these differences were observed in sun-protected and sun-exposed sites, these differences were not due to differences in antibody specificity or binding affinity or to regional differences in antigen expression. By contrast, antigen expression in the skin of patients with nonphotosensitive LE was similar to that in normal individuals. These observations taken together indicate that specimens from patients with photosensitive forms of LE generally have high expression of immunoreactive Ro and La antigens.
There was also a strong and highly significant (P<.001) correlation between the level of expression of Ro and La antigens in skin, the presence of circulating antibodies to these antigens, and photosensitivity. For example, anti-Ro and anti-La antibodies and photosensitivity were all present in 100% of 8 patients whose skin expressed high levels of these antigens compared with 3 (23%), 1 (8%), and 2 (15%), respectively, of 13 patients whose skin expressed low levels of antigens.
The 60-kd Ro antigen may be particularly important in the development of photosensitivity, since its expression was 3- to 4-fold greater in sun-exposed than sun-protected skin specimens, but only in patients with photosensitive forms of LE. This contrasts with the expression of this antigen in patients with nonphotosensitive LE and normal individuals and with the expression of the 52-kd Ro and the 48-kd La antigens, which were generally similar in sun-exposed and sun-protected skin. The selective increased expression of the 60-kd Ro antigen in sun-exposed skin of patients with photosensitive forms of LE is not an artifact of the testing procedure; as the phenomenon was selective, it only involved this antigen and only patients who were photosensitive. The finding suggests that the expression of the 60-kd Ro antigen is up-modulated by UV light in vivo, a possibility supported by the ability of UV to modulate the expression of nuclear antigens in vitro4,5; but this modulation occurs only in those patients who are photosensitive. The finding further supports the notion that individuals whose skin shows a high expression of certain nuclear antigens are more likely to become photosensitive. Whether high antigen expression is constitutive or induced by sunlight is presently not known.
An incidental finding is that the 4 patients with LE in whom the disease was inactive at the time this study was conducted (patients 1, 2, 11, and 17) showed much lower expressions of the 52-kd and 60-kd Ro antigens in both sun-exposed and sun-protected skin specimens compared with patients with active disease. This observation suggests the possibility that the level of Ro antigen may play a role not only in the development of photosensitivity and Ro and La antibodies but also in the severity and/or prognosis of the disease. However, the number of patients is too small for this observation to be conclusive.
In summary, the expression of the 52-kd Ro, 60-kd Ro, and 48-kd La antigens in skin is greater in patients with photosensitive cutaneous LE, such as SCLE, than in patients with nonphotosensitive LE, and there is a strong correlation between the expression of these antigens and the presence and titer of circulating autoantibodies against them. These observations suggest that the concentration or accessibility of Ro and La antigens in skin is an important determinant of both the development of Ro and La autoantibodies and photosensitivity in patients with LE.
Accepted for publication August 10, 1999.
This work was supported in part by research grants P30CA16087, AR39749, 5RO1 AR27663, AR07190, and RO1 AM 27663-09 from the US Public Health Service, Rockville, Md, and by grant AR42455-01 from the National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, Md. Dr Ioannides was supported by International Research Fellowship Award F05-TWO4200-01 from the US Public Health Service.
Reprints: Jean-Claude Bystryn, MD, Ronald O. Perelman Department of Dermatology, New York University Medical Center, 550 First Ave, New York, NY 10016 (e-mail: firstname.lastname@example.org).