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Rheumatology Advance Access published online on January 25, 2007
Rheumatology, doi:10.1093/rheumatology/kel435
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© The Author 2007. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Clinical characteristics of Japanese patients with anti-OJ (anti-isoleucyl-tRNA synthetase) autoantibodies

Shinji Sato, Masataka Kuwana and Michito Hirakata

Department of Internal Medicine, Keio University, School of Medicine, Tokyo, Japan

Correspondence to: Shinji Sato, Department of Internal Medicine, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. E-mail: shins{at}sc.itc.keio.ac.jp


   Abstract
Top
 Abstract
Introduction
 Patients, materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Objectives. The clinical and laboratory characteristics of seven patients with anti-aminoacyl-tRNA synthetase (ARS) autoantibodies, specifically anti-OJ (anti-isoleucyl-tRNA synthetase), were examined and compared with previously published findings.

Methods. Serum samples from 1135 Japanese patients with various autoimmune diseases and 48 normal individuals were screened for anti-OJ antibodies using RNA and protein immunoprecipitation assays. The patients whose sera contained anti-OJ antibodies were assessed regarding clinical symptoms, clinical course, laboratory findings, chest radiography and chest computed tomography.

Results. Sera from seven patients were found to contain anti-OJ antibodies. These autoantibodies were associated with interstitial lung disease (ILD) and myositis. The diagnoses of the seven patients were idiopathic interstitial pneumonias (IIPs) in three, polymyositis (PM) in three and PM-rheumatoid arthritis (RA) overlap in the remaining one. All patients had ILD, but muscle weakness and polyarthritis were seen only in four. Raynaud's phenomenon and sclerodactyly were absent in all patients.

Conclusions. These results indicate that the presence of anti-OJ autoantibodies may distinguish a subtype of anti-ARS syndrome that is more closely associated with ILD than myositis or Raynaud's phenomenon.

KEY WORDS: Interstitial lung disease (ILD), Polymyositis/dermatomyositis (PM/DM), Anti-aminoacyl-tRNA synthetase (ARS) antibodies


   Introduction
Top
 Abstract
 Introduction
Patients, materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Anti-aminoacyl-tRNA synthetase (anti-ARS) autoantibodies have been found in patients with polymyositis/dermatomyositis (PM/DM) [1, 2]. Six anti-ARS autoantibodies have been described, as follows: anti-histidyl (anti-Jo-1), anti-threonyl (anti-PL-7), anti-alanyl (anti-PL-12), anti-glycyl (anti-EJ), anti-isoleucyl (anti-OJ), and anti-asparaginyl (anti-KS) tRNA synthetases [1–7]. The most common anti-ARS antibodies, anti-Jo-1, are found in approximately 20–30% of PM/DM patients. Anti-OJ antibodies are also found in PM/DM patients, although the frequency is low [1]. In previous studies, anti-OJ antibodies were found in less than 2% of all patients with PM/DM [8]. These anti-ARS antibodies have been reported to be associated with a similar syndrome characterized by myositis with a high frequency of interstitial lung disease (ILD) and arthritis, as well as increased fever, Raynaud's phenomenon, and mechanic's hands compared to the overall myositis population [9]. Although anti-ARS syndromes have common clinical symptoms, further observations have distinguished certain differences in clinical features associated with each of the different anti-ARS antibodies. It has been reported that anti-Jo-1 antibodies are closely associated with myositis [1, 3], whereas patients with anti-PL-12 and anti-KS antibodies are more likely to have ILD without clinical evidence of myositis [7]. On the other hand, we previously observed that the presence of anti-PL-7 antibodies is closely associated with PM/DM-SSc overlap as well as ILD in Japanese patients [10].

There have been two reports on the clinical significance of anti-OJ antibodies in patients in North America [11, 12], and two case reports of anti-OJ antibodies in Japan [8, 13]. However, the clinical characteristics of the Japanese patients with anti-OJ antibodies have not been examined in detail and their clinical significance remains uncertain. Here, we analyse the clinical and laboratory characteristics of Japanese patients with antibodies against anti-OJ and review published reports from elsewhere.


   Patients, materials and methods
Top
 Abstract
 Introduction
 Patients, materials and methods
Results
 Discussion
 Acknowledgements
 References
 
Patients and sera
Serum samples were obtained from 1135 Japanese patients who had or were suspected of having connective tissue diseases (CTDs) seen at the Keio University Hospital and collaborating centers between 1990 and 2000. These included 120 with PM/DM, 400 with systemic lupus erythematosus, 192 with systemic sclerosis, 58 with rheumatoid arthritis (RA), 101 with overlap syndrome including mixed connective tissue disease, 114 with ILD and 150 patients with arthritis or erythema who were suspected to have CTDs. These included three patients with anti-OJ antibodies previously reported by our study group [8, 13]. We also examined 48 sera from normal individuals. Blood samples were obtained after the patients and normal controls had provided written informed consent approved by the Keio University Institutional Review Board.

Immunoprecipitation (IPP)
The IPP assay with HeLa cell extracts was performed as previously described [6]. For the analysis of RNAs, antibodies bound to protein A-Sepharose CL-4B beads were incubated with extracts of HeLa cells. They were then washed with NET-2 buffer (50 mM Tris-HCL, pH 7.5, 150 mM NaCl, 0.05% Nonidet P-40). After ethanol precipitation, RNAs were dissolved in electrophoresis sample buffer composed of 10 M urea, 0.025% bromophenol blue, and 0.025% xylene cyanol-FF in TBE buffer (90 mM Tris-HCl, pH 8.6, 90 mM boric acid and 1 mM EDTA). The RNA samples were resolved in 7 M urea-10% polyacrylamide gels, which were then silver stained (Bio-Rad Laboratories, Hercules, CA, USA). For protein studies, antibody-coated Sepharose beads were mixed with [35S] methionine-labelled HeLa extracts. After washing, the Sepharose beads were resuspended in SDS–sample buffer (2% SDS, 10% glycerol, 62.5 mM Tris-HCl, pH 6.8, 0.005% bromophenol blue). The proteins were then fractionated by 10% SDS-PAGE gels and dried. Radiolabelled protein components were analysed by autoradiography. With these assays, myositis-specific or -associated autoantibodies such as anti-ARS, anti-signal recognition particle, anti-Mi-2, anti-SSA, anti-SSB, anti-U1-RNP and anti-Ku autoantibodies are distinguishable, compared with corresponding standard sera [2]. The identification of anti-OJ antibodies was accomplished by comparing IPP patterns for both RNA and protein with standard anti-OJ serum as described previously [13].

Clinical features
The clinical symptoms, clinical course, laboratory findings, the results of chest radiography and chest computed tomography (CT) were retrospectively assessed from medical records in all patients positive for anti-OJ antibodies. Some patients were also assessed for electromyogram (EMG), muscle biopsy, and pathological findings from video-assisted thoracoscopic surgery (VATS) at first evaluation. The assessment of muscle weakness was performed using a manual muscle test (MMT) [14].

The diagnosis of PM/DM was based on criteria proposed by Bohan and Peter [15]. ILD was defined according to the results of chest radiography, chest CT, lung function testing (percentage predicted forced vital capacity: FVC and carbon monoxide diffusing capacity: DLCO) and the diagnosis of IIPs was based on consensus classification of IIPs [16]. The resolution of myositis symptoms was defined as both improved muscle strength on a manual muscle test and normalization of the serum CK value. Pulmonary symptoms were considered improved when shown by both chest CT and pulmonary function testing.

Statistical analysis
All comparisons between the two patient groups were performed using Fisher's 2-tailed exact test or Student's t-test.


   Results
Top
 Abstract
 Introduction
 Patients, materials and methods
 Results
Discussion
 Acknowledgements
 References
 
Identification of anti-OJ antibodies
Of the 1183 sera tested, seven immunoprecipitated a characteristic identical nucleic acid band of tRNA of a size identical to anti-isoleucyl tRNA synthetase (anti-OJ). Representative examples are shown in Fig. 1A. Two bands of RNA in the tRNA size range were immunoprecipitated and are clearly distinguishable from the pattern of tRNAs precipitated by the other anti-ARS antibodies. The same sera also immunoprecipitated several protein bands corresponding to polypeptides precipitated by anti-OJ standard serum. These included at least three protein bands that were more intense than the other seven bands of the OJ complex. These proteins were easily distinguishable from those immunoprecipitated by sera reactive with the other described anti-ARS antibodies (Fig. 1B). Thus, it is concluded that they contained anti-OJ antibodies.


Figure 1
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  		FIG. 1. (A) Immunoprecipitation (IPP) of nucleic acids with representative anti-OJ sera and controls. Urea (7M) and 10% PAGE of phenol-extracted immunoprecipitates from HeLa cell extracts were developed with silver stain. TNA, total nucleic acids, with the 5.8 and 5.0 S small ribosomal RNAs and the tRNA region indicated. Sera used for IPP include: lanes 1-5, anti-synthetase sera indicated, with antibodies to Jo-1 (histidyl-tRNA synthetase), PL-7 (threonyl-tRNA synthetase), PL-12 (alanyl-tRNA synthetase), EJ (glycyl-tRNA synthetase), OJ (isoleucyl-tRNA synthetase); lanes 6-10, anti-OJ sera as indicated; and lane 11, control serum indicated (NHS, normal human serum). The tRNA pattern with anti-OJ sera is easily distinguishable from that of the other anti-synthetases. (B) IPP of proteins with anti-OJ sera and controls. Autoradiogram of 10% SDS-PAGE of immunoprecipitates from [35S] methionine-labelled HeLa cell extracts. Mr, molecular weight markers of the sizes indicated to the left (kDa). The sera used for immunoprecipitation are the same as those in Fig. 1A. The same characteristic pattern of protein bands including at least three that were thought to be glutamine, isoleucine and leucine tRNA synthetases (arrows) was seen with each of the five anti-OJ sera. And the pattern was easily different from the bands immunoprecipitated by sera containing the other anti-synthetases.

 
Clinical features in patients with anti-OJ antibodies
In this study, anti-OJ antibodies were detected in four of 120 PM/DM patients (3.3%) and three of 112 ILD patients (2.7%). However, anti-OJ antibodies were not detected in other CTDs or normal human sera.

Clinical features in the seven patients with anti-OJ autoantibodies are summarized in Table 1. Of these seven, four were female. All seven patients had ILD, although none of them progressed to severe acute respiratory failure. Four patients had muscle weakness that was graded as 4/5 based on MMT and serum CK elevation. From the results of EMG and muscle biopsy, four patients were diagnosed as definite or probable PM. Two of these manifested symptoms of ILD preceding their myositis symptoms. One of the four PM patients had destructive changes in joint radiography compatible with RA. The other three patients had no muscle or skin manifestations but interstitial changes in the lung at chest high resolution CT and/or histopathlogical change in VATS resulted in a diagnosis of IIPs. VATS was done in all patients with IIPs and the results were compatible with cryptogenic organizing pneumonia (patient #5), usual interstitial pneumonia (patient #6) and non-specific interstitial pneumonia (patient #7). No Raynaud's phenomenon or sclerodactyly was present at any time in any of the seven patients. Treatment of the myositis with prednisolone and/or other immunosupressants resulted in improvement of muscle strength assessed by MMT and reduction of serum CK level in three PM patients. Three patients with IIPs had also received prednisolone that had then been gradually tapered and discontinued; improvement of chest CT and pulmonary function was achieved in two of them.


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  		TABLE 1. Clinical features of patients with anti-OJ antibodies

 
Comparison with the clinical features of patients with anti-OJ in the literature
The clinical features of patients with anti-OJ antibodies reported in the English literature were previously reviewed [11, 12]. Targoff et al. [11] reported on nine patients with anti-OJ and Gelpi et al. [12] described one patient with co-existing anti-Jo-1 and anti-OJ antibodies.

Frequencies of several clinical manifestations can be compared between the anti-OJ-positive patients reported by Targoff et al. and those in the present study. The frequency of myositis in our Japanese patients with anti-OJ antibodies tended to be lower than in the patients of Targoff et al. (57% vs 89%), but this difference did not reach statistical significance. The frequencies of ILD and Raynaud's phenomenon in our series were similar compared with previously reported patients [11].

Comparison with the clinical features of patients with anti-OJ and anti-Jo-1 antibodies
In the present study, anti-Jo-1 antibodies, representative of anti-ARS antibodies, were detected in 22 patients with PM/DM (18% of PM/DM in our study). We compared the frequencies of several clinical manifestations between anti-OJ- and anti-Jo-1-positive patients in our series (Table 2). It was found that the frequency of myositis and Raynaud's phenomenon in patients with anti-OJ was significantly lower than in those with anti-Jo-1 (P = 0.010 and P = 0.002, respectively), whereas the frequency of ILD was similar.


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  		TABLE 2. Comparison of clinical features in anti-OJ-positive vs anti-Jo-1-positive patients

 

   Discussion
Top
 Abstract
 Introduction
 Patients, materials and methods
 Results
 Discussion
Acknowledgements
 References
 
Seven sera from 1135 CTD patients or suspected CTDs were found to contain anti-OJ antibodies. In the present study, these seven patients seemed not to have the typical features characteristic of the anti-ARS syndromes previously described. The most striking differences were that none of them had Raynaud's phenomenon that is common in anti-ARS syndromes in general. In fact, the frequency of Raynaud's phenomenon in our series was significantly lower than in our patients with anti-Jo-1 antibodies that are the representative anti-ARS cases. However, the sample is too small to draw a definitive conclusion in this study, but our results are similar to those in North American patients with anti-OJ antibodies [11]. Thus, the low frequency of Raynaud's phenomenon seems to be a characteristic feature of patients with anti-OJ autoantibodies compared to other anti-ARS syndromes.

Three of the anti-OJ-positive patients had no signs of myositis and were diagnosed as IIPs. This suggested that the presence of anti-OJ is more closely associated with ILD than myositis, as is the case with anti-PL-12 or anti-KS antibodies.

Although three patients received a diagnosis of IIPs at this time, the possibility remains that muscle symptoms may appear in the future, because it is known that pulmonary manifestations can appear before muscle symptoms in PM/DM patients. Another possibility is that existing myositis was underdiagnosed because of the effect of prednisolone treatment; indeed, three patients diagnosed as IIPs had been taking prednisolone for their pulmonary symptoms. Nonetheless, it is unlikely that they had myositis because the duration of prednisolone therapy was relatively short and they never had any clinical symptoms related to myositis throughout their clinical course. However, observation should be continued for identification of any future muscle symptoms.

The mechanisms responsible for these differences in clinical features associated with each of the anti-ARS autoantibodies are unknown and an accumulation of larger numbers of cases will be required to clarify this in the future.

In conclusion, we report seven Japanese patients with anti-OJ antibodies classed as suffering from IIPs or PM accompanied by ILD. These patients lacked any manifestations of Raynaud's phenomenon or sclerodactyly. Anti-OJ autoantibodies are a clinically important marker for a specific subset of anti-ARS syndrome that is more closely associated with ILD than myositis or Raynaud's phenomenon. The measurement of anti-OJ was found to be useful for diagnosis of patients with ILD with or without myositis. Further analysis of these autoantibodies may provide insights into the etiological and pathogenetic mechanisms of ILD and myositis.

Formula


   Acknowledgements
Top
 Abstract
 Introduction
 Patients, materials and methods
 Results
 Discussion
 Acknowledgements
References
 
We thank Ms Mutsuko Ishida for assisting with the RNA immunoprecipitation assay.

The authors have declared no conflict of interest.


   References
Top
 Abstract
 Introduction
 Patients, materials and methods
 Results
 Discussion
 Acknowledgements
 References
 

  1. Targoff IN. (2004) Laboratory testing in the diagnosis and management of idiopathic inflammatory myopathies. Rheum Dis Clin North Am 28:859–90.
  2. Hirakata M, Mimori T, Akizuki M, Craft J, Hardin JA, Homma M. (1992) Autoantibodies to small nuclear and cytoplasmic ribonucleoproteins in Japanese patients with inflammatory muscle disease. Arthritis Rheum 35:449–56.[Web of Science][Medline]
  3. Yoshida S, Akizuki M, Mimori T, Yamagata H, Inada S, Homma M. (1983) The precipitating antibody to an acidic nuclear protein antigen, the Jo-1, in connective tissue diseases. A maker for a subset of polymyositis with interstitial pulmonary fibrosis. Arthritis Rheum 26:604–11.[Web of Science][Medline]
  4. Mathews MB, Reichlin M, Hughes GR, Bernstein RM. (1984) Anti-threonyl-tRNA synthetase, a second myositis-related autoantibody. J Exp Med 160:420–34.[Abstract/Free Full Text]
  5. Bunn CC, Bernstein RM, Mathews MB. (1986) Autoantibodies against alanyl tRNA synthetase and tRNAAla coexist and are associated with myositis. J Exp Med 163:1281–91.[Abstract/Free Full Text]
  6. Targoff IN. (1990) Autoantibodies to aminoacyl-transfer RNA synthetases for isoleucine and glycine. Two additional synthetases are antigenic in myositis. J Immunol 144:1737–43.[Abstract]
  7. Hirakata M, Suwa A, Nagai S, et al. (1999) Anti-KS: Identification of autoantibodies to asparaginyl-transfer RNA synthetase associated with interstitial lung disease. J Immunol 162:2315–20.[Abstract/Free Full Text]
  8. Suwa A, Hirakata M, Satoh S, Ezaki T, Mimori T, Inada S. (1999) A case of polymyositis with anti-OJ (isoleucyl-transfer RNA synthetase) antibodies. Clin Exp Rheumatol 17:755–6.[Web of Science][Medline]
  9. Bernstein RM and Mathews MB. (1985) Jo-1 and other myositis autoantibodies. In Brooks PM and York JR (Eds.). Rheumatology-85, excerpta medica international congress series(Elsevier Science Publishers, New York) pp. 273–8.
  10. Sato S, Hirakata M, Kuwana M, et al. (2005) Clinical characteristics in Japanese patients with anti-PL-7 (anit-threonyl-tRNA synthetase) autonatibodies. Clin Exp Rheumatol 23:609–15.[Web of Science][Medline]
  11. Targoff IN, Trieu EP, Miller FW. (1993) Reaction of anti-OJ autoantibodies with components of the multi-enzyme complex of aminoacyl-tRNA synthetases in addition to isoleucyl-tRNA synthetase. J Clin Invest 91:2556–64.[Web of Science][Medline]
  12. Gelpi C, Kanterewicz E, Gratacos J, Targoff IN, Rodriguez-Sanchez JL. (1996) Coexistence of two antisynthetases in a patient with the antisynthetase syndrone. Arthritis Rheum 39:692–7.[Medline]
  13. Satoh S, Hirakata M, Nakamura K, et al. (1998) Two cases of polymyositis associated with interstitial pneumonia with interstitial pneumonia with anti-OJ (isoleucyl tRNA synthetase) antibodies. Ryumachi 38:534–41 (in Japanese).[Medline]
  14. Lane RJ, Emslie-Smith A, Mosquera IE, Hudgson P. (1989) Clinical, biochemical and histological responses to treatment in polymyositis: a prospective study. J R Soc Med. 82:333–8.[Abstract]
  15. Bohan A and Peter JB. (1975) Polymyositis and dermatomyositis. N Engl J Med 292:344.[Web of Science][Medline]
  16. . American Thoracic Societym European Respiratory Society. (2002) ATS/ERS International Multidisciplinary Consensus Classification of the Idiopathic Interstitial International Pneumonias Statement. Am J Respir Crit Care Med 165:277–304.[Free Full Text]
Submitted 21 June 2006; revised version accepted 8 December 2006.
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