Novel autoantibodies to pituitary gland specific factor 1a in patients with rheumatoid arthritis

S. Tanaka, K. Tatsumi, T. Tomita1, M. Kimura, T. Takano, H. Yoshikawa1 and N. Amino

Department of Laboratory Medicine (D2) and
1 Department of Orthopaedic Surgery, Osaka University Graduate School of Medicine, Osaka, Japan


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objective. We recently identified a new protein, pituitary gland specific factor 1a (PGSF1a), that is specifically transcribed in the pituitary gland. In our investigation of anti-PGSF1a antibody for pituitary diseases, we examined it in patients with RA and other autoimmune diseases. We unexpectedly discovered the frequent existence of anti-PGSF1a antibody in patients with RA. We therefore examined the prevalence of this antibody to understand its clinical significance in RA.

Methods. Anti-PGSF1a antibody was detected by radioligand assay using recombinant 35S-labelled PGSF1a protein. Antibody activity is expressed as an index that was obtained by comparison with normal pooled serum.

Results. RA patients had a significantly higher mean anti-PGSF1a antibody index (n=46, 1.28±0.38, P < 0.001) than healthy controls (n=36, 1.04±0.13). Indices greater than the cut-off value (mean+2 S.D. of healthy controls) were found in 43.5% (20/46) and 10.0% (2/20) of patients with RA and osteoarthritis, respectively. There was no correlation between the activities of anti-PGSF1a antibodies and titres of rheumatoid factor (RF) or serum C-reactive protein concentrations, but RA patients with more erosive disease had a higher mean anti-PGSF1a antibody index. Four of eight sera samples obtained from RF-negative RA patients were positive for anti-PGSF1a antibodies.

Conclusion. Anti-PGSF1a antibody is a useful new marker for the diagnosis of RA, especially for RF-negative RA, and may relate to clinical manifestations of RA.

KEY WORDS: Rheumatoid arthritis, Osteoarthritis, Rheumatoid factor, Autoantibody, PGSF1a, Pituitary, RF-negative RA, Seronegative RA.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Rheumatoid arthritis (RA) is the most common disabling autoimmune disease affecting 1% of the population. While there has been progress in defining its aetiology and pathogenesis, these are still incompletely understood.

Pituitary gland specific factor 1a (PGSF1a), which was recently identified in our laboratory, is specifically transcribed in the human pituitary gland [1]. Autoantibodies against tissue-specific proteins have proved to be useful markers for the diagnosis of autoimmune diseases. Therefore, we searched for antibodies to PGSF1a protein by sensitive radioligand assay [2, 3] to support the diagnosis of lymphocytic hypophysitis. In our investigation of the specificity of this antibody for pituitary diseases [4], we examined it in patients with RA and other autoimmune diseases. Unexpectedly, this antibody was frequently detected in patients with RA.

In the present study, we examined the prevalence of this antibody and examined its relationship to clinical parameters.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Serum samples
Serum samples were obtained from 46 patients with RA, 20 with osteoarthritis (OA) and 76 with other autoimmune diseases (14 with systemic lupus erythematosus, six with progressive systemic sclerosis, eight with mixed connective tissue disease, 10 with Hashimoto's thyroiditis, 10 with Graves' disease, 13 with autoimmune hepatitis and 15 with other autoimmune diseases) as well as 36 healthy controls. All RA patients satisfied the 1987 revised diagnostic criteria of the American College of Rheumatology (formerly, the American Rheumatism Association) [5]. Disease activity was classified as the least erosive disease, more erosive disease and mutilating disease, as reported [6]. OA was diagnosed according to the American College of Rheumatology criteria for the classification of OA of the knee [7]. None of the OA patients had any relevant immunological background or history of systemic inflammation. Rheumatoid factor (RF) was measured by nephelometry, and values less than 15.0 IU/ml were considered to be negative. We obtained informed consent from all patients. In all healthy subjects, six kinds of autoantibodies (antinuclear antibody, anti-DNA antibody, RF, anti-mitochondrial antibody, anti-thyroid microsomal antibody and anti-thyroglobulin antibody) were measured as previously described [8], and subjects who had one or more positive antibodies were strictly excluded so as not to include subclinical autoimmune disease. The mean ages of the patient groups were not significantly different from that of the healthy controls, except for those with OA.

Recombinant 35S-labelled PGSF1a
PGSF1a cDNAs were amplified by polymerase chain reaction (PCR) using the human pituitary cDNA, KOD -plus- (TOYOBO, Osaka, Japan) and the following primers to introduce the EcoRI site preceding the initiator ATG and XhoI site after the stop codon: 5'-AGAATTCATGCCGGGAATGAGGCTGGTTTG-3' and 5'-TCTCGAGACATTGTTTCTGCCCTCACGGA-3' (EcoRI and XhoI sites are underlined). After amplification, the PGSF1a cDNAs were digested with EcoRI and XhoI and cloned into the pET28a (+) expression vector (Novagen, Madison, WI). The resulting PGSF1a in vitro transcription vector, pET/PGSF1a, was transcribed and translated in vitro using the TNT coupled reticulocyte lysate system (Promega, Madison, WI) according to the manufacturer's instructions. In brief, 1 µg of pET/PGSF1a was incubated at 30°C for 90 min in 100 µl of TNT coupled reticulocyte lysate system mixture and 2 µl L-[35S]methionine (15 mCi/ml) (Amersham Pharmacia Biotech, Tokyo, Japan). The translation products were run on a Nick chromatography column (Amersham Pharmacia Biotech) to remove free 35S-methionine, analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE) (15% polyacrylamide gel) and autoradiography demonstrated the presence of a 16-kDa band component for PGSF1a. The synthesized 35S-labelled PGSF1a was diluted to 20 000 counts per minute (c.p.m.)/20 µl reaction buffer (NaCl 150 mmol/l, Tris 50 mmol/l, pH 7.4, Tween-20 1 ml/l, bovine serum albumin 4 g/l and NaN3 1 g/l) and was stored at -80°C until use.

Radioligand assay
Four microlitres of serum and 20 000 c.p.m. 35S-labelled PGSF1a were incubated at 4°C overnight in 50 µl of reaction buffer. After incubation, the reaction mixture was transferred into 96-well filtration plates (Multiscreen HVPP, 0.45 µm, Millipore Corp., Bedford, MA) and precipitated with Protein G-Sepharose 4 Fast Flow (Amersham Pharmacia Biotech) that had been blocked with blocking buffer (150 mmol/l NaCl, 50 mmol/l Tris, pH 7.4, 1 ml/l Tween-20, 30 g/l bovine serum albumin and 1 g/l NaN3) for 1 h at 4°C. The complexes were washed 10 times with washing buffer (NaCl 150 mmol/l, Tris 50 mmol/l, pH 7.4, Tween-20 10 ml/l) using a 96-well filtration system (Millipore Corp.). The plates were then dried, and OptiPhase SuperMix (PerkinElmer Life Science, Boston, MA, USA) was added to each well. The quantity of precipitated, 35S-labelled PGSF1a was determined using a 1450 MicroBeta TriLux apparatus (PerkinElmer Life Science). All samples were assayed in duplicate. The results obtained from this assay are presented as an anti-PGSF1a antibody index:c.p.m. of the unknown serum/c.p.m. of normal pooled serum. The intra- and interassay variations ranged between 7.3 and 10.9%.

Recombinant human PGSF1a (rhPGSF1a)
Escherichia coli B834 strain BL21 (DE3) was transformed by the pET/PGSF1a. The inclusion body containing rhPGSF1a was purified using BugBuster Protein Extraction Reagent (Novagen) according to the manufacturer's instructions. The inclusion body was resuspended in phosphate-buffered saline (PBS) containing 8 M urea, and rhPGSF1a was purified using TALON Metal Affinity Resins (CLONTECH Laboratories, Inc., Palo Alto, CA) according to the manufacturer's instructions.

Inhibition experiment
An inhibition study with rhPGSF1a or ovalbumin was carried out to confirm the specificity of the antibody by radioligand assay. Serum and 35S-labelled PGSF1a were incubated with or without 1 µg rhPGSF1a or ovalbumin in reaction buffer with 0.16 M urea, and the radioligand assay was carried out as described above.

Immunoblotting analysis
An aliquot of 100 ng of rhPGSF1a was electrophoresed through denaturing 15% acrylamide gel, and was transferred to Hybond-P (Amersham Pharmacia Biotech). After blocking with skimmed milk, the membranes were incubated with sera, which were preincubated with strips of Hybond-P impregnated with 50 µg of rhPGSF1a or bovine serum albumin as a control, for 60 min at room temperature (RT). After washing three times for 10 min with Tris-buffered saline (TBS) (500 mmol/l NaCl, 20 mmol/l Tris, pH 7.4)-0.05% Tween-20 (TBST), the membranes were incubated with a 1:5000 dilution of HRP-Labelled Protein A (Amersham Pharmacia Biotech) in 5% skimmed milk/TBST for 60 min at RT. Subsequently, the proteins were visualized with TMB Membrane Peroxidase Substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD).

Statistical analysis
The Mann–Whitney U-test was used to compare the anti-PGSF1a antibody indices between the patient groups and the healthy controls. Spearman's correlation coefficient test was used to analyse the relationship among the anti-PGSF1a antibody index, the activity of RF (IU/ml) and C-reactive protein (CRP) concentration (mg/dl) in the patients with RA. A P < 0.05 was considered to be significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Validation of the radioligand assay
Sera obtained from three patients with RA were diluted in reaction buffer. The dilution curve was linear in the dilution range between 6.25- and 50-fold (data not shown). We therefore decided to use a 1:12.5 dilution in the subsequent experiments.

Specificity and prevalence of autoantibodies against PGSF1a
To confirm the specificity of the radioligand assay, an inhibition study with rhPGSF1a was performed. The anti-PGSF1a antibody index was decreased when incubated with rhPGSF1a, and was unchanged when incubated with ovalbumin (data not shown). Using an immunoblotting method, anti-PGSF1a antibody was detected, and the signal was absorbed when the serum was preincubated with rhPGSF1a (data not shown).

Using a radioligand assay, we examined sera obtained from patients with various autoimmune diseases and compared them with that from healthy controls (Fig. 1Go). The mean anti-PGSF1a antibody index was significantly higher in patients with RA (P < 0.001) than in healthy controls. With a cut-off value above 1.29 (mean+2 S.D. of healthy controls), the specificity was 100% and the sensitivities were 43.5% (20/46), 10.0% (2/20) and 5.2% (4/76) for patients with RA, OA and other autoimmune diseases, respectively.



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FIG. 1. Anti-PGSF1a indices in control subjects, patients with RA, OA and other autoimmune diseases by radioligand assay. The horizontal dotted line indicates the cut-off value (1.29; mean+2 S.D. of healthy controls). (inset) The distribution of anti-PGSF1a indices in control subjects, and three subgroups of patients with RA according to the classification of Ochi et al. [6].

 

Correlation between anti-PGSF1a antibody and disease activity in RA
When RA patients were divided into three subgroups according to the classification of Ochi et al. [6], the mean antibody index in patients with more erosive disease was higher than that in patients with the least erosive disease (Fig. 1Go).

Relationship among anti-PGSF1a antibody, RF and CRP
No correlation was found between anti-PGSF1a antibody indices and RF titres or CRP concentrations (data not shown), but RF did correlate with CRP (r=0.313, P < 0.05). Interestingly, four of eight patients with RF-negative RA had positive anti-PGSF1a antibodies (data not shown).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We identified PGSF1a protein as a novel autoantigen in the patients with RA. Although its function is unknown and the amino acid sequence of PGSF1a protein has no homology with other mammalian proteins, it has a weak homology with the soybean nodulin 26B-carboxy terminal domain [1]. It has one sequence (EKRTA) similar to the RA severity-associated ‘shared epitope’ sequence of HLA-DR (QK/RRAA) molecules [9, 10] (Fig. 2Go). Patients with a double dose of the shared sequence tend to have more serious disease manifestations. Thus it may be speculated that the presence of anti-PGSF1a antibodies has a casual relationship to RA.



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FIG. 2. Sequence homology between PGSF1a and the RA severity-associated ‘shared epitope’ of HLA-DR molecules. Amino acids for PGSF1a 33–61 and HLA-DR-beta-1*0405 58–70 are indicated. The identical and conserved amino acids are indicated by bold and underlined type, respectively.

 
The measurement of RF is one of the most useful serological tests for the diagnosis of RA, but approximately 20% of patients have RF-negative RA. In the present study, anti-PGSF1a antibody indices did not correlate with RF or CRP, and 50.0% of RF-negative RA patients had positive anti-PGSF1a antibodies. As anti-PGSF1a antibodies were independent of RF, the measurement of anti-PGSF1a antibody will support the diagnosis of RA in RF-negative RA patients.

It is of interest whether the anti-PGSF1a antibodies modify the clinical features of RA. The presence of the anti-PGSF1a antibodies appeared to be linked to severe manifestations of RA, i.e. more erosive disease and mutilating disease conditions [11] (Fig. 1Go). Therefore it may be important to examine the relationship between disease progress and antibody activity.

Because PGSF1a is a pituitary-specific protein, the relationship between RA and pituitary dysfunction may be interesting to pursue. In patients with RA, a number of studies have shown inappropriate cortisol and sex hormone production [12]. However, little is known regarding pituitary dysfunction in RA. In newly diagnosed and untreated patients with RA, growth hormone response to growth hormone-releasing hormone is disturbed [13]. As anti-PGSF1a antibodies were found in patients with pituitary disorders in our recent study [4] and were found frequently in patients with RA in this study. Anti-PGSF1a antibodies might play a role in pituitary dysfunction among patients with RA and may be a possible risk factor for pituitary dysfunction among patients with RA.

In conclusion, anti-PGSF1a antibody is a useful marker for diagnosis of RA and may relate to clinical manifestations of RA.


    Acknowledgments
 
This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology; the Ministry of Health, Labour and Welfare of Japan.


    Notes
 
Correspondence to: K. Tatsumi, Department of Laboratory Medicine, Osaka University Graduate School of Medicine (D2), Suita-shi Yamada-oka 2–2, Osaka 565-0871, Japan. E-mail: tatsumi{at}labo.med.osaka-u.ac.jp Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Tanaka S, Tatsumi K, Okubo K et al. Expression profile of active genes in the human pituitary gland. J Mol Endocrinol 2002;28:33–44.[Abstract/Free Full Text]
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  3. Petersen JS, Hejnaes KR, Moody A et al. Detection of GAD65 antibodies in diabetes and other autoimmune diseases using a simple radioligand assay. Diabetes 1994;43:459–67.[Abstract]
  4. Tanaka S, Tatsumi K, Kimura M et al. Detection of autoantibodies against the pituitary-specific proteins in patients with lymphocytic hypophysitis. Eur J Endocrinol 2002;147:767–75.[ISI][Medline]
  5. Arnett FC, Edworthy SM, Bloch DA et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988;31:315–24.[ISI][Medline]
  6. Ochi T, Iwase R, Yonemasu K et al. Natural course of joint destruction and fluctuation of serum C1q levels in patients with rheumatoid arthritis. Arthritis Rheum 1988;31:37–43.[ISI][Medline]
  7. Altman R, Asch E, Bloch D et al. Development of criteria for the classification and reporting of osteoarthritis. Classification of osteoarthritis of the knee. Diagnostic and Therapeutic Criteria Committee of the American Rheumatism Association. Arthritis Rheum 1986;29:1039–49.[ISI][Medline]
  8. Iijima T, Tada H, Yagoro A et al. Incidence of postpartum onset of disease among patients with rheumatoid arthritis. J Rheumatol 1999;26:755–6.[Medline]
  9. Wakitani S, Murata N, Toda Y et al. The relationship between HLA-DRB1 alleles and disease subsets of rheumatoid arthritis in Japanese. Br J Rheumatol 1997;36:630–6.[CrossRef][ISI][Medline]
  10. Weyand CM, Goronzy JJ. The molecular basis of rheumatoid arthritis. J Mol Med 1997;75:772–85.[CrossRef][ISI][Medline]
  11. Tomita T, Shimaoka Y, Kashiwagi N et al. Enhanced expression of CD14 antigen on myeloid lineage cells derived from the bone marrow of patients with severe rheumatoid arthritis. J Rheumatol 1997;24:465–9.[ISI][Medline]
  12. Straub RH, Cutolo M. Involvement of the hypothalamic–pituitary–adrenal/gonadal axis and the peripheral nervous system in rheumatoid arthritis: viewpoint based on a systemic pathogenetic role. Arthritis Rheum 2001;44:493–507.[CrossRef][ISI][Medline]
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Submitted 3 June 2002; Accepted 5 August 2002





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