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Clinical Significance of Anti-Filaggrin Antibody Recognizing Uncitrullinated Filaggrin in Rheumatoid Arthritis

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Clinical Significance of Anti-Filaggrin Antibody Recognizing Uncitrullinated Filaggrin in Rheumatoid Arthritis EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 37, No. 6, 546-552, December 2005 Clinical significance of anti-filaggrin antibody recognizing uncitrullinated filaggrin in rheumatoid arthritis Kyung-Ho Choi1*, Eun Bong Lee2*, patients with osteoarthritis, ankylosing spondylitis Chang Dal Yoo2, Han Joo Baek2, or systemic lupus erythematosus. IgG anti-filaggrin Seong Wook Kang2, Ki Chul Shin2, antibodies were more frequently found in patients with rheumatoid arthritis compared to normal Yun Jong Lee2, Hyun Ah Kim2, 1 1 controls (12.3% vs 1.4% respectively, P = 0.04). An Ju-Hong Jeon , Chai-Wan Kim , anti-filaggrin antibody titer was correlated with visual 1 1,3 Dong-Myung Shin , In-Gyu Kim analogue scale of pain, tender joint count, Ritchie and Yeong Wook Song2 articular index or C-reactive protein, but not with anti-nuclear antibody or rheumatoid factor. These 1Department of Biochemistry and Molecular Biology results suggest that anti-filaggrin antibody recog- Biology/Aging and Apoptosis Research Center (AARC) nizes the uncitrullinated filaggrin as an antigen and 2Department of Internal Medicine and Clinical Reasearch Institute its titer correlates with clinical parameters, explain- Seoul National University College of Medicine ing the variable sensitivity of anti-filaggrin antibody Seoul 110-799, Korea test. 3Corresponding author: Tel, 82-2-740-8248; Fax, 82-2-744-4534; E-mail, [email protected] Keywords: autoimmune; cyclin citrullinated peptide; *These authors contributed equally to this work. diseases; filaggrin; immunoenzyme techniques; rheu- matoid arthritis Accepted 29 September 2005 Abbreviations: ADL, activity of daily living; AFA, anti-filaggrin anti- Introduction body; AKA, anti-keratin antibody; ANA, anti-nuclear antibody; APF, anti-perinuclear factor; AS, ankylosing spondylitis; CRP, C-reactive Rheumatoid arthritis (RA) is a chronic inflammatory protein; ESR, erythrocyte sedimentation ratio; OA, osteoarthritis; joint disease affecting around 1% of the general RA, rheumatoid arthritis; RF, rheumatoid factor; SLE, systemic lupus population (Kellgren, 1996). Although rheumatoid erythematosus factor (RF) is helpful in diagnosis of RA (Arnett et al., 1998), it can be found in patients with other rheu- matic diseases or even healthy persons and is ne- gative in 20-30% of RA patients (Schrohenloher et Abstract al., 1997). Filaggrin is expressed in the cornified layer of Various serologic markers were suggested to overcome the low specificity of the RF, including Sa, epidermis and known to be one of the antigenic kalpastatin, antikeratin antibody (AKA) and antiperi- targets in rheumatoid arthritis. Although the citrul- nuclear factor (APF) (Menard et al., 1998). The AKA line residue in filaggrin is thought to be an antigenic and APF, which are the antibodies against the determinant recognized by autoantibodies, the cornified epithelium of rat esophagus and human diagnostic sensitivity of synthetic citrullinated pep- buccal mucosa respectively, are known to be tide is variable. To investigate the implication of specific for RA (Nienhuis et al., 1964; Young et al., anti-filaggrin antibodies recognizing uncitrullinated 1979). The antigen targets of both antibodies were filaggrin in rheumatoid arthritis, we assayed anti- suggested to be the same protein which is filaggrin body titers using unmodified recombinant filaggrin in the human epidermis (Hoet et al., 1991; Simon et in the sera from 73 patients with rheumatoid arthritis, al., 1993; Sebbag et al., 1995). Filaggrin is synthe- 150 patients with other connective tissue diseases sized in the stratum granulosum as a large repeated and 70 normal controls. We also performed the (10-12 repeat of filaggrin monomers) and heavily correlation analysis between antibody titers and the phosphorylated precursor, profilaggrin, which is clinical variables in patients with rheumatoid arthri- stored in the keratohyaline granules. After dephos- tis. Titers of IgG anti-filaggrin antibodies were signi- phorylation and proteolysis, filaggrin monomer is ficantly higher in rheumatoid arthritis patients released from profilaggrin and modified by peptidyl- compared to normal controls (P = 0.02), but not in arginine deiminase which converts the arginine resi- Antifilaggrin antibody in rheumatoid arthritis 547 dues found in filaggrin to the citrulline. Modified pET15b expression vector after digestion with Nde I filaggrin is thought to be interacted and aggregated and Xho I. The recombinant plasmid was introduced with the keratin intermediate filaments in keratino- into the expression host, BL21 (DE3). cytes, facilitating and guiding their alignment (Ber- The recombinant human filaggrin was prepared by thelot et al., 1995). pET bacterial expression system (Novagen, Ma- Since the molecular identity of target antigen was dison, WI). Induction and purification of the recom- elucidated, several approaches have been made to binant protein was performed according to manu- detect antifilaggrin antibody (AFA) using human facturer's instructions. Briefly, transformed cells were filaggrin. Immunoblot tests using the filaggrin extract cultured in LB medium plus 100 µg/ml ampicillin at from human epidermis showed variable sensitivity 37oC. Filaggrin production was induced in the ranging from 12.0-67.9%, with the specificity of presence of 1 mM isopropyl β-D-thiogalactopyrano- 92.0-95.4% (Vincent et al., 1998; Slack et al., 1998). side (IPTG). The washed cells from 1-liter culture ELISA using filaggrin purified from human epidermis were resuspended in 10 ml of binding buffer (20 mM detected IgG AFA in 47% of RA patients (Palosuo et Tris-Cl, 0.5 M NaCl, 5 mM imidazole, pH 7.9) and al., 1998). Recently, ELISA using a cyclic citrul- lysed by sonication. The suspension was centrifuged linated synthetic peptide derived from the sequence at 39,000 × g for 20 minutes at 4oC. The super- of human filaggrin showed a higher specificity than natant was filtered through a 0.45 µm membrane other methods (Schellekens et al., 2000; Girelli et and loaded on 6 ml HisBind metal chelation resin al., 2004). However, the heterogeneity of extracted (Novagen, Madison, WI) pre-charged with 50 mM filaggrin or synthesized peptide sequence makes it NiSO4 and equilibrated with binding buffer. The difficult to get consistent results, and thus to eval- column was washed with 60 ml binding buffer and uate the correlations between the presence of then with 30 ml each of washing buffer containing 60 autoantibody and clinical data related to RA activity. mM or 100 mM imidazole (20 mM Tris-Cl, 0.5 M In this study, we report that ELISA using unmodified NaCl, 60 mM or 100 mM imidazole, pH 7.9). The recombinant filaggrin showed a 12.3% diagnostic recombinant protein was eluted with 30 ml elution sensitivity at a specificity of 95% which was cor- buffer (20 mM Tris-Cl, 0.5 M NaCl, 1 M imidazole, related with visual analogue scale of pain, tender pH 7.9). The eluate was concentrated by ultrafiltra- joint count, Ritchie articular index or C-reactive tion (Ultrfree-15: Millipore, Bedford, MA). protein in RA patients. For the immunoblot analysis, protein samples were electrophoresed under denaturing condition, and transferred to a nitrocellulose membrane. The mem- Materials and Methods brane was blocked with 5% skim milk in Tris-buf- fered saline (TBS) for an hour and then incubated Serum samples with mouse antifilaggrin monoclonal antibody (Bio- Serum samples were obtained from 73 patients medical Technologies, Stoughton, MA) for an hour. diagnosed as RA according to the revised criteria by Peroxidase-labelled anti-mouse Ig antibody (Dako, American Rheumatism Association (Arnett et al., Glostrup, Denmark) was used as a secondary probe 1998), 70 healthy controls and 150 patients with and visualization was performed with enhanced che- other connective tissue diseases including osteoar- miluminescence (Amersham, Piscataway, NJ). thritis (OA), ankylosing spondylitis (AS) and sys- temic lupus erythematosus (SLE) at Rheumatology Immunoblotting with human sera Clinic, Seoul National University Hospital. Serum o One microgram of recombinant filaggrin was run on samples were stored at -70 C until assayed. SDS-polyacrylamide gel and transferred to a nitro- cellulose membrane. The membrane was cut into Expression and purification of the recombinant strips and blocked with 5% skim milk in TBS. The filaggrin monomer strips were incubated with the patient sera diluted 1:100 for an hour at room temperature. After wash- Human filaggrin monomer cDNA was obtained by ing several times, the strips were treated with pero- PCR amplification of the partial cDNA clone of xidase-labelled anti-human IgG antibody (Dako, Glo- λ profilaggrin ( HF11 provided by Dr. P. Steinert; Gan strup, Denmark) for an hour at room temperature. et al., 1990). PCR was performed with sense primer The color reaction was developed by 3,3-diamino- (FilP) 5'-CATATGTTCCTCTACCAGGTGAGC-3' and benzidine substrate (Sigma, St. Louis, MO). antisense primer (FilN) 5'-TCTGGACATTCAGGAT- CTTAACTCGAG-3'. The amplified product was veri- fied by sequencing (Sequenase kit; USB, Cleveland, ELISA OH) and subcloned into the corresponding sites of The recombinant filaggrin (1 µg/well) was coated on 548 Exp. Mol. Med. Vol. 37(6), 546-552, 2005 a 96-well immunoassay plate (Maxisorp; Nunc, knees, feet, cervical spine, lumbosacral spine and Rockside, Denmark) at 4oC, overnight. After washing pelvis in all patients. with phosphate buffered saline-0.05% Tween-20 (PBST), blocking was done with 1% bovine serum Statistical analysis
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