Department of Laboratory Medicine (D2) and
1 Department of Orthopaedic Surgery, Osaka University Graduate School of Medicine, Osaka, Japan
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 (SDSPAGE) (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 MannWhitney 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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. 1). 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.
|
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. 1).
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
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. 1). 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 |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|