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Abstract

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Objective

To identify novel autoantibodies specific for dermatomyositis (DM), especially those specific for clinically amyopathic DM (C-ADM).

Methods

Autoantibodies were analyzed by immunoprecipitation in 298 serum samples from patients with various connective tissue diseases (CTDs) or idiopathic pulmonary fibrosis (IPF). Antigen specificity of the sera was further examined by immunoblotting and indirect immunofluorescence (IF). The disease specificity and clinical features associated with the antibody of interest were determined.

Results

Eight sera recognized a polypeptide of ∼140 kd (CADM-140 autoantigen) by immunoprecipitation and immunoblotting. Immunoreactivity was detected in the cytoplasm, and indirect IF revealed a granular or reticular pattern. Anti–CADM-140 antibodies were detected in 8 of 42 patients with DM, but not in patients with other CTDs or IPF. Interestingly, all 8 patients with anti–CADM-140 antibodies had C-ADM. Among 42 patients with DM, those with anti–CADM-140 autoantibodies had significantly more rapidly progressive interstitial lung disease (ILD) when compared with patients without anti–CADM-140 autoantibodies (50% versus 6%; P = 0.008).

Conclusion

These results indicate that the presence of anti–CADM-140 autoantibodies may be a novel marker for C-ADM. Further attention should be directed to the detection of rapidly progressive ILD in those patients with anti–CADM-140 autoantibodies.

Polymyositis (PM)/dermatomyositis (DM) is a chronic inflammatory disorder that culminates in injury to the skin and muscle and, sometimes, is associated with interstitial lung disease (ILD) and/or neoplasia (1, 2). A number of autoantibodies are associated with myositis, including those specific for aminoacyl–transfer RNA synthetase (anti-ARS) (3), signal recognition particle (anti-SRP) (4), and Mi-2 (5). These autoantibodies have proven to be clinically useful in the diagnosis and classification of these diseases and are predictive of responses to treatment.

It has been known for some time that certain patients may have the typical skin manifestations of DM but no evidence of myositis, a condition known as amyopathic DM. Recently, Sontheimer proposed the existence of a unique subgroup of patients with DM who have the clinical cutaneous features of DM but no evidence of clinical myositis symptoms for at least 2 years after the onset of skin manifestations (referred to as clinically amyopathic DM [C-ADM]) (6). In other words, C-ADM includes patients with amyopathic DM and patients with hypomyopathic DM (patients with subclinical signs of myositis and DM skin manifestations). Some patients with C-ADM, especially those in Japan (7), have been noted to develop rapidly progressive ILD. This condition in many of these patients is resistant to treatment, and fatal outcomes have been observed.

Because of the severity of ILD accompanying C-ADM, a marker autoantibody would be useful for early diagnosis and treatment monitoring. Potential marker autoantibodies have been described by Targoff et al, who, in a preliminary study, described specificity for a 95-kd Se protein, as well as an unidentified 155-kd protein (8). However, a full survey of the autoantibodies associated with C-ADM has not been performed. In the present study, we examined the sera from 15 Japanese patients with C-ADM to identify additional autoantibodies associated with this disease.

PATIENTS AND METHODS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Patients and sera.

Serum samples were obtained from 255 randomly selected Japanese adult patients with connective tissue diseases (CTDs) who were being followed up in clinics at Keio University in Tokyo and collaborating medical centers. These sera were obtained, prior to therapy, from a cohort of 61 patients with PM, 42 with DM (including 15 with C-ADM), 50 with rheumatoid arthritis, 46 with systemic lupus erythematosus, 27 with mixed CTD/overlap syndrome, 22 with systemic sclerosis, and 7 with Sjögren's syndrome. Sera from 43 patients with idiopathic pulmonary fibrosis (IPF) and 16 normal human sera were used as control sera. The diagnosis of C-ADM was based on diagnostic criteria proposed by Sontheimer (6), i.e., DM patients with no clinical muscle symptoms for more than 2 years after the onset of skin manifestations.

The patients were diagnosed as having ILD according to the results of chest radiography, chest computed tomography (CT), and pulmonary function testing, which included the percent predicted values for forced vital capacity and diffusing capacity for carbon monoxide. A subset of patients with rapidly progressive ILD was defined as those presenting with progressive dyspnea and progressive hypoxemia, and a worsening of interstitial change on the chest radiograph within 1 month from the onset of respiratory symptoms.

Immunoprecipitation.

The immunoprecipitation assay was performed using extracts of the leukemia cell line, K562, as previously described (9). A total of 10 μl of patient serum was mixed with 2 mg of polypeptide A–Sepharose CL-4B (Pharmacia Biotech AB, Uppsala, Sweden) in 500 μl of immunoprecipitation buffer (10 mM Tris HCl, pH 8.0, 500 mM NaCl, 0.1% Nonidet P40) and incubated for 2 hours at 4°C, and then washed 3 times with immunoprecipitation buffer.

For polypeptide studies, antibody-coated Sepharose beads were mixed with 100 μl of 35S-methionine–labeled K562 cell extracts derived from 2 × 105 cells, and rotated at 4°C for 2 hours. After 6 washes, the Sepharose beads were resuspended in sodium dodecyl sulfate (SDS) sample buffer and the polypeptides were fractionated by 6% SDS–polyacrylamide electrophoresis gels. Radiolabeled polypeptide components were analyzed by autoradiography.

For analysis of RNA, the antigen-bound Sepharose beads were incubated with 100 μl of K562 cell extracts (6 × 106 cell equivalents per sample) for 2 hours at 4°C. To extract bound RNA, 30 μl of 3.0M sodium acetate, 30 μl of 10% SDS, 2 μl of carrier yeast transfer RNA (10 mg/ml; Sigma, St. Louis, MO), and 300 μl of phenol:chloroform:isoamyl alcohol (50:50:1, containing 0.1% 8-hydroxyquinoline) were added. After ethanol precipitation, the RNA was resolved using a 7M urea–10% polyacrylamide gel, which was subsequently silver-stained (Bio-Rad, Hercules, CA).

Immunoblotting.

Immunoblotting analysis was performed using K562 cell extracts in a modification of the procedure described by Towbin et al (10).

Immunodepletion.

A 10-μl aliquot of the prototype serum of autoantibodies to the 140-kd polypeptide was mixed with 2 mg of Sepharose beads and incubated for 2 hours at 4°C, followed by 3 washes with immunoprecipitation buffer. Another serum that recognized the 140-kd polypeptide was added in a dose-dependent manner (0 μl, 10 μl, 25 μl, and 50 μl) and then incubated. After 3 washes, immunoprecipitation for polypeptide analysis was performed as described above.

Indirect immunofluorescence (IF).

Indirect IF was performed using HEp-2 cells and fluorescein-labeled anti-human immunoglobulin (Inova Diagnostics, San Diego, CA).

Clinical studies.

The patients whose sera immunoprecipitated a 140-kd polypeptide were examined for their clinical symptoms, clinical course, muscle enzyme levels (creatine kinase [CK] and aldolase), results on chest radiographic and CT scans, and findings of skin pathology. An assessment of muscle weakness was performed using a manual muscle test (11). Some patients were also examined by electromyogram and muscle magnetic resonance imaging (MRI), and by pathologic analysis of the muscle.

Statistical analysis.

The 2 groups of DM patients with or without autoantibodies to the 140-kd polypeptide were compared. The results of comparisons between groups were analyzed using the chi-square test, with Yates' correction where appropriate.

RESULTS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Detection of anti–140-kd polypeptide antibodies in patients with C-ADM.

We screened 298 patient sera and 16 normal human sera by immunoprecipitation. Sera from 8 (19%) of 42 patients with DM immunoprecipitated a polypeptide of ∼140 kd from 35S-methionine–labeled K562 cell extracts (Figure 1A, lanes 1–8). All 8 patients were diagnosed as having C-ADM, a subtype of DM. In the analysis of RNA specificity, these sera did not immunoprecipitate any nucleic acid band, except for 1 patient's serum, which precipitated hYRNA of SSA/Ro components.

thumbnail image

Figure 1. A, Immunoprecipitation of polypeptides with sera from patients with clinically amyopathic dermatomyositis (C-ADM), using 35S-methionine–labeled K562 cell extracts. Lanes 1–8, Sera from C-ADM patients; lane 9, anti–PL-12 serum; lane 10, control normal human serum (NHS). A 140-kd protein was recognized by 8 sera from C-ADM patients (lanes 1–8). B, Immunoprecipitation of polypeptides by the prototype serum and by other known autoantibodies. Lane 1, The prototype anti–CADM-140 serum; lane 2, anti-MJ serum; lane 3, anti–RNA helicase A (RHA) serum; lane 4, anti–PL-12 (alanyl–transfer RNA synthetase) serum; lane 5, anti–RNA polymerase I, II, and III (RNAP I, II, and III) serum; lane 6, control NHS. Anti–CADM-140 serum immunoprecipitated an ∼140-kd polypeptide that was easily distinguished from that of other known antibodies. C, Immunodepletion studies. Sera used for immunoprecipitation were as follows: lane 1, anti–CADM-140; lane 2, anti–PL-12; lane 3, control NHS; lanes 4–7, immunoprecipitation with anti–CADM-140 serum after absorption by another anti–CADM-140–positive serum in a dose-dependent manner. Arrows in A and C denote the 140-kd polypeptide. The sizes of the molecular weight markers are indicated to the left in A–C. D, Immunofluorescence pattern of HEp-2 cells stained with anti–CADM-140 serum. A granular or reticular cytoplasmic staining pattern on HEp-2 cells was observed. (Original magnification × 400.)

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The C-ADM sera that immunoprecipitated the 140-kd polypeptide were also used to immunoblot K562 cell extracts. These sera from C-ADM patients displayed a similar reaction on immunoblot, with a polypeptide band of similar molecular weight (results not shown).

For identification of novel autoantibodies recognizing the 140-kd molecule, the polypeptide immunoprecipitated by the prototype serum was compared with antigens of similar molecular weight recognized by other known autoantibodies (Figure 1B). The protein recognized by the prototype serum migrated slightly faster than the 140-kd protein recognized by anti-MJ antibody (Figure 1B, lane 2) and faster than that recognized by anti–RNA helicase A antibody (Figure 1B, lane 3), but more slowly than the 120-kd protein precipitated by anti–PL-12 antibody (Figure 1B, lane 4). These results clearly indicate that the 140-kd polypeptide immunoprecipitated by the prototype serum was different from the proteins immunoprecipitated by these other known antibodies. We designated this new autoantibody specificity as anti–CADM-140.

The prototype serum depleted extracts of the 140-kd polypeptide in a dose-dependent manner (Figure 1C, lanes 4–7), and the polypeptide recognized by the prototype serum was no longer immunoprecipitated in these extracts (Figure 1C, lane 7). In contrast, the depletion of radiolabeled K562 cell extracts with the use of autoantibodies of different immunologic specificities did not affect the levels of the anti–CADM-140–specific antigen (results not shown). When sera positive for anti–CADM-140 antibodies were assessed in indirect IF studies, a granular or reticular cytoplasmic staining pattern was observed (Figure 1D).

Disease specificity of the anti–CADM-140 antibodies.

The frequencies of myositis-specific antibodies, myositis-associated antibodies, and anti–CADM-140 antibodies are summarized in Table 1. Myositis-specific antibodies are found in most patients with myositis, whereas myositis-associated antibodies are frequently found in patients without myositis (12). Among the patients with CTDs or IPF, myositis-specific antibodies (33 with anti-ARS, 5 with anti-SRP, 2 with anti–Mi-2) and myositis-associated antibodies (44 with anti-SSA/Ro, 48 with anti–U1 RNP, none with anti-PM/Scl or other myositis-associated antibodies) were detected. Anti–CADM-140 autoantibodies were found in 19% of sera from patients with DM (especially in 53% with the C-ADM subtype), but were not detected in patients with other CTDs or IPF.

Table 1. The frequencies of myositis-specific, myositis-associated, and anti–CADM-140 antibodies in patients with connective tissue diseases and IPF*
AutoantibodiesPM (n = 61)DM (n = 42)RA (n = 50)SLE (n = 46)MCTD/OL (n = 27)Systemic sclerosis (n = 22)Sjögren's syndrome (n = 7)IPF (n = 43)
Classic DM (n = 27)C-ADM (n = 15)
  • *

    Values are the number (%) of patients. Anti-PM/Scl and other myositis-associated autoantibodies were not detected in any of the sera tested. PM = polymyositis; DM = dermatomyositis; C-ADM = clinically amyopathic dermatomyositis; RA = rheumatoid arthritis; SLE = systemic lupus erythematosus; MCTD/OL = mixed connective tissue disease/overlap syndrome; IPF = idiopathic pulmonary fibrosis; anti-ARS = anti–aminoacyl–transfer RNA synthetase; anti-SRP = anti–signal recognition particle.

Myositis-specific         
 Anti-ARS (anti–Jo-1)10 (16)6 (22)0000000
 Anti-ARS (non–anti–Jo-1)10 (16)2 (7)0001 (4)004 (9)
 Anti-SRP5 (8)00000000
 Anti–Mi-202 (7)0000000
Myositis-associated         
 Anti-SSA/Ro3 (5)3 (11)2 (14)8 (16)15 (33)6 (22)1 (5)5 (71)1 (2)
 Anti–U1 RNP2 (3)2 (7)01 (2)18 (39)23 (85)2 (9)00
Anti–CADM-140008 (53)000000

Clinical features of C-ADM patients with anti–CADM-140.

Clinical findings were compared between DM patients (including those with C-ADM) with anti–CADM-140 autoantibodies and those without anti–CADM-140 autoantibodies (Table 2). There were no significant differences in the frequencies of skin symptoms. However, the frequency of rapidly progressive ILD was significantly increased in anti–CADM-140–positive patients compared with that in anti–CADM-140–negative patients (50% versus 6%; P = 0.008). No myositis-specific antibodies were found in patients with anti–CADM-140; nevertheless, there was no significant difference in the frequency of these autoantibodies in comparison with the anti–CADM-140–negative group.

Table 2. Comparison of clinical features in anti–CADM-140–positive versus anti–CADM-140–negative patients with dermatomyositis
FeatureAnti–CADM-140–positive (n = 8)Anti–CADM-140–negative (n = 34)P
  1. * Except where indicated otherwise, values are the percent of patients. NS = not significant; CK = creatine kinase; ILD = interstitial lung disease; MSAs = myositis-specific autoantibodies; MAAs = myositis-associated autoantibodies.

Age at onset, mean ± SD years44.5 ± 12.746.5 ± 15.7NS
No. male/no. female2/68/26NS
Gottron's sign or papules7588NS
Heliotrope rash5053NS
Muscle weakness0760.02
Elevation of CK25740.03
Fever2550NS
Raynaud's phenomenon1324NS
Arthritis5071NS
Rapidly progressive ILD5060.008
Malignancy018NS
MSAs029NS
MAAs1318NS

None of the 8 patients with anti–CADM-140–positive sera were treated with steroids or other immunosuppressive medications prior to being assessed for C-ADM. All of these patients had Gottron's sign or papules, or periorbital heliotrope erythema and skin biopsy specimens yielding results compatible with DM. None of these patients had muscle weakness. CK levels were in the normal range in 6 patients (75%) and slightly elevated in the remaining 2 patients. Of the 6 patients assessed for the muscle enzyme aldolase, levels were normal in 2 patients. Of the 2 patients who underwent muscle MRI, neither showed findings compatible with a diagnosis of myopathy. Four patients had a muscle biopsy, and 2 of the muscle specimens exhibited mild infiltration of inflammatory cells, but there was no evidence of necrosis of muscle fibers, variation in fiber size, regeneration, or phagocytosis. Of the 7 patients with ILD (88%), 4 developed rapidly progressive disease.

DISCUSSION

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

We have identified novel autoantibodies (anti–CADM-140 autoantibodies) to an ∼140-kd polypeptide in patients with DM. Anti–CADM-140 antibodies were detected specifically in patients with DM, especially those with C-ADM. In addition, anti–CADM-140 antibodies were associated with rapidly progressive ILD.

It has been reported that amyopathic DM may be accompanied by rapidly progressive ILD, especially in Japanese patients and other Asian patients (7). In contrast, rapidly progressive ILD was shown to be rare in patients with amyopathic DM in a North American population (13). In our series, 5 of 15 patients with C-ADM (33%) (4 of whom had anti–CADM-140 antibodies) had rapidly progressive ILD during their clinical course. Rapidly progressive ILD was more frequent in our series compared with that reported previously in North American populations (13). Although the number of patients that we studied was very limited, it remains possible that racial differences are the reason for this discrepancy, because other clinical studies of Japanese patients also demonstrated findings similar to ours (7).

Furthermore, in a recent preliminary report, using immunoprecipitation and immunoblotting of HeLa cell extracts, Targoff et al documented the presence of antibodies to a 155-kd protein and/or Se protein in patients with C-ADM (8). Thirteen of 18 C-ADM sera possessed an anti–155-kd polypeptide antibody, and 6 also immunoprecipitated a 95-kd polypeptide (anti-Se antibody). In contrast, Oddis et al identified the anti-MJ antibody, which was also found to recognize a 140-kd polypeptide, in patients with juvenile DM (14, 15). We have been able to conclude that anti–CADM-140 is distinctively different from anti-MJ, because the molecular weights of the immunoprecipitated polypeptides are different. Moreover, the clinical features of anti-MJ are quite different from those associated with anti–CADM-140. Anti-MJ is detected mainly in juvenile DM, has been observed in the US and Argentina, and is clinically characterized by severe DM with a chronic and polycyclic course, sometimes accompanied by vasculitis (14). In order to elucidate the racial differences in the frequency of these antibodies, the examination of a larger number of patients from several different populations is required.

Our results have thus demonstrated the presence of anti–CADM-140 autoantibodies in patients with C-ADM, and these were found to be associated with rapidly progressive ILD. Further studies of this novel autoantibody specificity may provide insight into the pathogenic mechanisms of C-ADM accompanied by rapidly progressive ILD.

Acknowledgements

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

We thank Ms N. Fertig for preparing the prototype anti-MJ serum, and Ms Mutsuko Ishida for assisting in RNA immunoprecipitation assays.

REFERENCES

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES
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