Abstract
Objective . Myositis-specific autoantibodies (MSAs) are a useful tool in diagnosis, defining clinical subsets and predicting prognosis of dermatomyositis (DM) and polymyositis (PM). In this study, we identified a novel MSA reactive with 155 and 140 kDa nuclear proteins [anti-155/140 antibody (Ab)] and determined the clinical feature of DM patients positive for this autoantibody (autoAb).
Methods . Sera from 52 Japanese patients with DM, 9 with PM, 48 with systemic lupus erythematosus (SLE), 126 with systemic sclerosis and 18 with idiopathic interstitial pneumonia were examined by immunoprecipitation assays. Positive sera were further characterized by immunodepletion and immunofluorescence staining.
Results . Seven of the 52 (13%) Japanese patients with DM immunoprecipitated 155 and 140 kDa proteins from 35 S-labelled K562 leukaemia cell extract. No patients with SLE, systemic sclerosis or idiopathic interstitial pneumonia as well as healthy controls were positive for this autoAb. Patients with anti-155/140 Ab developed heliotrope rash, Gottron's papules or sign and flagellate erythema significantly more frequently than those negative. Notably, internal malignancy was found at significantly higher frequency in those positive than those negative (71 vs 11%; P < 0.005). In contrast, none of these patients positive for this autoAb had interstitial lung disease.
Conclusions . This novel MSA is associated with cancer-associated DM and may serve as a diagnostic serological marker for this specific subset.
Introduction
Dermatomyositis (DM) and polymyositis (PM) are a group of chronic inflammatory disorders characterized by myogenic changes and/or skin eruptions. DM and PM are often associated with interstitial lung disease and internal malignancy, which considerably determine the severity and prognosis of patients with this clinically heterogenous disorder. While the aetiology of these disorders remains unclear, autoimmunity is considered to play a critical role since the presence of diagnostic autoantibodies (autoAbs), known as myositis-specific autoAbs (MSAs), is a prominent feature [ 1 ]. Importantly, MSAs define subsets of patients who share similar clinical features, responses to therapy, immunogenetics and prognosis [ 2 ]. Autoantigens of MSAs are cytoplasmic and nuclear components, which include aminoacyl-tRNA synthetases, Mi-2 (nuclear helicase), signal recognition particle (SRP) components and recently identified CADM140 [ 3 ]. Antibodies (Abs) to aminoacyl-tRNA synthetases are associated with chronic pulmonary fibrosis and arthritis [ 2 ]. Patients with anti-SRP Abs are distinguished by acute severe muscle weakness, myalgias and cardiac involvement [ 4 ]. Anti-Mi-2 Abs were detected dominantly in DM patients with typical cutaneous lesions, and related with the absence of pulmonary fibrosis and internal malignancy [ 5 , 6 ]. Anti-CADM140 Ab is associated with amyopathic DM and acute progressive interstitial pneumonia [ 3 ]. Furthermore, recent studies have suggested that the autoantigens of MSAs have more direct pathogenic roles in the development of myositis than expected [ 7 , 8 ]. Collectively, identification of MSAs is helpful in defining clinically homogenous patient subsets and in predicting prognosis as well as in clarifying the pathogenesis.
In the current study, we have identified a novel autoAb to 155/140 kDa proteins (anti-155/140 Ab), which is specific for DM and is correlated with internal malignancy and the absence of lung involvement.
Materials and methods
Patients
Fifty-two Japanese patients (12 females and 40 males) with DM, 9 with PM, 48 with systemic lupus erythematosus (SLE), 126 with systemic sclerosis and 18 with idiopathic interstitial pneumonia, and 50 healthy Japanese individuals were assessed. Among the 52 DM patients, 46 fulfilled Bohan and Peter's criteria [ 9 , 10 ], while the remaining six did not fulfil Bohan and Peter's criteria but fulfilled Sontheimer's criteria [ 11 ] because of the absence of clinical muscle symptoms and presence of subsistent clinical DM skin eruptions. All patients with SLE or systemic sclerosis fulfilled the American College of Rheumatology (ACR) criteria [ 12 , 13 ]. Patients with idiopathic interstitial pneumonia did not have myositis, skin eruption or other symptoms suggestive of collagen diseases. DM patients were between 13 and 84 yrs of age (mean age 55 yrs). The disease duration of DM patients was 11 ± 16 months. At the first visit, six patients were treated with low-dose corticosteroids (prednisolone 5–20 mg/day). No DM patients had a recent history of other inflammatory diseases. Complete medical histories, physical examinations and laboratory tests were conducted for all patients at the first visit, with limited evaluations during follow-up visits. All patients with DM undertook X-ray examination, CT and gallium scintigraphy. With these examinations, 30 patients with DM were followed up for more than 2 yrs. The follow-up period for 22 patients were within 2 yrs, during which no new malignant tumours had been found. Internal malignancy in DM patients was defined if the malignant disease was diagnosed concurrently with or after diagnosis of DM or if a preceding malignant disease still existed when DM was diagnosed. There were no patients with DM who had a past history of malignant diseases. The protocol was approved by the Kanazawa University School of Medicine and Kanazawa University Hospital. All patients gave written, informed consent for participation.
Immunoprecipitation
Immunoprecipitation assays were performed using extracts of the leukaemia cell line, K562 [ 3 ]. A total of 10 μl of the patient's serum was bound to 2 mg of protein A-Sepharose beads (Amersham Biosciences, Piscataway, NJ, USA) in 500 μl of immunoprecipitation buffer (10 mM Tris–HCl, pH 8.0, 50 mM NaCl, 0.1% Nonidet P40) and incubated for 2 h at 4°C and then washed five times with immunoprecipitation buffer. Ab-coated Sepharose beads were mixed with 100 μl 35 S-methionine-labelled K562 cell extracts derived from 10 6 cells and rotated at 4°C for 2 h. After five washes, the beads were resuspended in sodium dodecyl sulphate sample buffer and the polypeptides were fractionated by 8% sodium dodecyl sulphate-polyacrylamide gel electrophoresis followed by autoradiography. The prototype sera of MSAs except for anti-CADM140 Ab were kindly provided from Dr M. Kuwana (Keio University, Tokyo, Japan). Since the prototype anti-CADM140 serum was not available, anti-CADM140 Ab was determined by the molecular weight and immunodepletion.
Immunodepletion
A 10 μl aliquot of the serum samples positive for autoAb to the 155/140 kDa polypeptide was incubated with protein A-Sepharose beads for 2 h at 4°C. Ab-conjugated Sepharose beads were then mixed with 100 μl 35 S-methionine-labelled K562 cell extracts derived from 10 6 cells, and rotated at 4°C for 2 hrs. Then the supernatant was further incubated with Sepharose beads pre-conjugated with another serum that recognized the 155/140 kDa proteins added. After five washes, the immunoprecipitated proteins were analysed as described above.
Indirect immunofluorescence (IIF)
IIF was performed using HEp-2 cells as substrate and fluorescein isothiocyanate-conjugated anti-human IgG (MBL, Nagoya, Japan). Staining at 40× or higher dilution was considered positive.
Statistical analysis
Fisher's exact probability test was employed for comparison of frequencies and Bonferroni's test for multiple comparisons. A P -value <0.05 was considered statistically significant. All the data were shown as mean ± s . d .
Results
Detection of anti-155/140 Abs using immunoprecipitation
During our routine assessment of Ab profiles in patients with DM and other collagen diseases, we noticed that several sera from DM patients immunoprecipitated doublet bands of 155 and 140 kDa. Therefore, we screened 253 patient sera, including 52 with DM and 50 normal control by immunoprecipitation. Sera from 7 (13%) of the 52 DM patients immunoprecipitated polypeptides of 155 kDa and 140 kDa simultaneously from 35 S-methionine-labelled K562 cell extracts ( Fig. 1 A, Patients A–G). Patient E's serum also precipitated U1RNP antigens at 32, 28 and 20 kDa and SS-A antigen at 60 kDa. Also, Patient B's serum precipitated undetermined 66 and 85 kDa protein. No sera from patients with other diseases or healthy controls precipitated these 155 or 140 kDa polypeptides ( Fig. 1 A and data not shown). In the analysis of RNA specificity, these seven sera, except for one patient's serum (Patient E), did not immunoprecipitate any nucleic band, which precipitated U1RNP and SS-A components (data not shown). Among the 52 DM patients, anti-Jo-1 Ab was positive in six, anti-EJ Ab in nine, anti-OJ Ab in one, anti-PL-7 Ab in three, anti-PL-12 Ab in five, anti-Mi-2 Ab in six, anti-SRP Ab in one, and anti-CADM140 Ab in three. None of the seven sera were positive for these MSAs.
( A ) Immunoprecipitation with anti-155/140 Abs. Immunoprecipitates from 35S-labelled K562 cell extract were subjected to 8% sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Lanes 1–7 show the results with anti-155/140 positive sera from DM patients (A–G). The results of the prototype sera of autoAbs to PL-12 and EJ are also shown (lanes 8 and 9). An autoAb considered as anti-CADM140 Ab is shown in lane 10. A negative control using a normal human subject (NHS) serum is shown in lane 11. ( B ) Immunodepletion studies. Serum samples from Patients G, C, D and two patients considered to have anti-CADM140 Ab (CADM140 #1 and #2) were incubated with protein A-Sepharose beads, and Ab-conjugated beads were subjected to incubation with 35 S-methionine-labelled K562 cell extracts (lanes 2, 3, 4, 8 and 10). Then the supernatant from Patient G's sample was further incubated with Sepharose beads pre-conjugated with Patients C, D and E and CADM140 #1 and #2 (lanes 5, 6, 7, 9 and 11). Immunoprecipitated proteins were analysed as in (A). A negative control using a NHS serum is shown in lane 2.
To confirm that these sera recognized the same antigens, K562 cell extracts were first absorbed using a prototype serum, and then immunoprecipitation using other patients’ sera was performed. The prototype serum depleted extracts of both 155 and 140 kDa proteins ( Fig. 1 B). This depletion appeared specific since it did not affect U1RNP and SS-A antigens in Patient E's sample ( Fig. 1 B). Additionally, depletion with the same serum did not affect the levels of other autoAbs including anti-CADM140 Ab ( Fig. 1 B).
The IIF staining pattern was also assessed using HEp-2 cells as substrate. All sera stained the nuclei in fine-speckled pattern with negative nucleolar staining (data not shown). Except for one patient who was also positive for anti-U1RNP Ab (Patient E), IIF titre was relatively low at the maximum dilution of 40–160×.
Clinical and laboratory features of DM patients with anti-155/140 Abs
To examine if patients with anti-155/140 Ab form a distinct disease subset, the clinical and laboratory features were further assessed. In comparison with anti-155/140-negative DM patients, anti-155/140-positive DM patients had heliotrope rash and Gottron's papules or sign more frequently (86 vs 38%, and 100 vs 58%, respectively, P < 0.05; Table 1 ). The most significant difference among skin eruptions was found in flagellate erythema, which was observed in 85.7% of those positive for anti-155/140 Ab but only 20% of those negative ( P < 0.005; Table 1 ). The frequencies of perionychia erythema and nailfold bleeding were slightly higher in patients who were positive than those who were negative, although they were not significant. Notably, lung involvement was completely absent in anti-155/140 Ab-positive DM patients, and thus the prevalence was significantly lower than that in anti-155/140 Ab-negative DM patients (0 vs 64.4%, P < 0.005, Table 1 ). Serum creatine kinase (CK) level was elevated in five patients (318–3967 IU/l). The other two patients showed normal CK levels, while one clinically showed muscle weakness.
Clinical and laboratory profile of DM patients with autoAb to 155/140 kDa proteins
| Positive anti-155/155 | Negative anti-155/140 | |
|---|---|---|
| Ab a ( n = 7) | Ab ( n = 45) | |
| Age at onset, years, mean ± s . d . | 71 ± 10 | 53 ± 16 |
| Sex, male:female | 2:5 | 10:35 |
| Duration, months, mean ± s . d . | 10 ± 12 | 11.8 ± 16.2 |
| Skin eruption | ||
| Heliotrope rash | 86 * | 38 |
| Gottron's papules or sign | 100 * | 58 |
| Flagellate erythema | 86 ** | 20 |
| Perionychia erythema | 71 | 49 |
| Nailfold bleeding | 71 | 53 |
| Organ involvement | ||
| Internal malignancy | 71 ** | 11 |
| Interstitial pneumonia | 0 ** | 64 |
| Joint | 14 | 33 |
| Laboratory findings | ||
| Elevated CK | 71 | 60 |
| Positive ANA | 100 * | 53 |
| Positive anti-155/155 | Negative anti-155/140 | |
|---|---|---|
| Ab a ( n = 7) | Ab ( n = 45) | |
| Age at onset, years, mean ± s . d . | 71 ± 10 | 53 ± 16 |
| Sex, male:female | 2:5 | 10:35 |
| Duration, months, mean ± s . d . | 10 ± 12 | 11.8 ± 16.2 |
| Skin eruption | ||
| Heliotrope rash | 86 * | 38 |
| Gottron's papules or sign | 100 * | 58 |
| Flagellate erythema | 86 ** | 20 |
| Perionychia erythema | 71 | 49 |
| Nailfold bleeding | 71 | 53 |
| Organ involvement | ||
| Internal malignancy | 71 ** | 11 |
| Interstitial pneumonia | 0 ** | 64 |
| Joint | 14 | 33 |
| Laboratory findings | ||
| Elevated CK | 71 | 60 |
| Positive ANA | 100 * | 53 |
a Unless noted, otherwise, values are percentages.
* P < 0.05 vs DM patients without anti-155/140 Ab.
** P < 0.005 vs DM patients without anti-155/140 Ab.
Clinical and laboratory profile of DM patients with autoAb to 155/140 kDa proteins
| Positive anti-155/155 | Negative anti-155/140 | |
|---|---|---|
| Ab a ( n = 7) | Ab ( n = 45) | |
| Age at onset, years, mean ± s . d . | 71 ± 10 | 53 ± 16 |
| Sex, male:female | 2:5 | 10:35 |
| Duration, months, mean ± s . d . | 10 ± 12 | 11.8 ± 16.2 |
| Skin eruption | ||
| Heliotrope rash | 86 * | 38 |
| Gottron's papules or sign | 100 * | 58 |
| Flagellate erythema | 86 ** | 20 |
| Perionychia erythema | 71 | 49 |
| Nailfold bleeding | 71 | 53 |
| Organ involvement | ||
| Internal malignancy | 71 ** | 11 |
| Interstitial pneumonia | 0 ** | 64 |
| Joint | 14 | 33 |
| Laboratory findings | ||
| Elevated CK | 71 | 60 |
| Positive ANA | 100 * | 53 |
| Positive anti-155/155 | Negative anti-155/140 | |
|---|---|---|
| Ab a ( n = 7) | Ab ( n = 45) | |
| Age at onset, years, mean ± s . d . | 71 ± 10 | 53 ± 16 |
| Sex, male:female | 2:5 | 10:35 |
| Duration, months, mean ± s . d . | 10 ± 12 | 11.8 ± 16.2 |
| Skin eruption | ||
| Heliotrope rash | 86 * | 38 |
| Gottron's papules or sign | 100 * | 58 |
| Flagellate erythema | 86 ** | 20 |
| Perionychia erythema | 71 | 49 |
| Nailfold bleeding | 71 | 53 |
| Organ involvement | ||
| Internal malignancy | 71 ** | 11 |
| Interstitial pneumonia | 0 ** | 64 |
| Joint | 14 | 33 |
| Laboratory findings | ||
| Elevated CK | 71 | 60 |
| Positive ANA | 100 * | 53 |
a Unless noted, otherwise, values are percentages.
* P < 0.05 vs DM patients without anti-155/140 Ab.
** P < 0.005 vs DM patients without anti-155/140 Ab.
Most intriguingly, internal malignancy was found in 71% (5/7) of patients with anti-155/140, while only 11% of patients without anti-155/140 Ab had malignancy ( P < 0.005, Table 1 ). All the malignancies in patients positive for anti-155/140 Ab were concurrently found in the initial examination of DM. The malignancies found included gastric cancer in two patients, and lung, breast and gallbladder cancer in one each of the others. Thus, DM patients with anti-155/140 Ab were characterized by a high rate of internal malignancy and the absence of interstitial lung disease.
Discussion
The current study has identified a novel autoAb that is highly specific for DM. This autoAb simultaneously reacted with two proteins of 155 and 140 kDa. In all cases, the doublet bands were depleted together in the immunoabsorption study, suggesting that these two are related proteins and may form a complex in nuclei. Known autoantigens of similar molecular weights recognized by other autoAbs include anti-CADM140 Ab [ 3 ], although negative results of immunodepletion studies suggest that they were different. Intriguingly, another Ab recognizing a 155 kDa protein, which has a molecular weight similar to the other band of the doublet, has been reported by Targoff et al . [ 14 ]. Since the 155 kDa band always associated with an unreported 140 kDa band in our study, the autoAb that Targoff described appeared different from ours. Alternatively, ethnical differences may result in different patterns of antigen recognition. As for sensitivity, anti-155/140 Ab was found in 13% of DM patients. This frequency was slightly lower than anti-EJ Ab but was comparable or higher than other MSAs examined, including anti-Jo-1, anti-OJ, anti-PL-7, anti-PL-12, anti-Mi-2, anti-SRP and anti-CADM140 Abs. Importantly, anti-155/140 Ab did not co-exist with other known MSAs. As for specificity, in a series of surveying of patients with SLE, systemic sclerosis and idiopatic interstitial pneumonia, none was positive for anti-155/140 Ab, suggesting high specificity of this Ab for DM. Thus, anti-155/140 Ab can be classified as an MSA.
Clinical characteristics of the patients positive for anti-155/140 Ab were remarkably distinct. Increased risk of cancer in patients with DM/PM is well-known [ 15 , 16 ], while there have been no MSAs associated with the presence of malignancy. Rather, the presence of all the previously described MSAs decreases the likelihood of cancer [ 17 ]. In this study, a significantly higher association with internal malignancy was demonstrated in patients positive for anti-155/140 Ab. While the follow-up periods varied among patients, the presence or absence of malignancy was determined prior to this study, and thus the positivity of anti-155/140 Ab did not influence the clinical correlations. Therefore, this Ab may be a potential serological marker for cancer-associated myositis. In contrast, none of the patients with anti-155/140 Ab exhibited interstitial lung disease during any of the follow-ups. This is consistent with the fact that interstitial lung disease and malignancy do not usually co-exist [ 17 ]. Skin manifestations in the patients positive for anti-155/140 Ab were also homogenous and typical for DM. They presented high frequencies of typical DM rashes including heliotrope, Gottron's papules or sign and flagellate erythema. Most patients exhibited muscle weakness or elevation of serum CK levels. Collectively, they presented typical manifestations of classic DM. It can be hypothesized that the 155/140 kDa antigen is highly expressed in cancer, skin (keratinocytes) and muscle, but is absent or low in lung, which may determine the phenotype.
Collectively, the current study demonstrates novel autoAbs frequently found in patients with DM associated with malignancy, and suggests the possibility of anti-155/140 Ab as a serological marker for a subset with classic DM with cancer. Identification of target antigen will be required for further analysis of potential pathogenic roles and the development of therapy.
Acknowledgements
Funding to pay the Open Access publication charges for this article was provided by …
The authors have declared no conflicts of interest.
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