Westbury, NY), as previously described. All steps were per-formed at room temperature.
Immunoaffinity purification of antibodies.
Autoanti-bodies to AUF1 from 1 patient with RA and 2 patients withSLE were affinity-purified by the blot elution technique, aspreviously described (19). Autoantibodies to hnRNP A2 puri-fied by the same method were used as controls. Briefly,nitrocellulose strips containing the blotted antigens were incu-bated overnight at 4°C with sera diluted 1:10 in blocking buffer, washed thoroughly, cut into small pieces, and finally incubatedfor 2 minutes with 0.1
Tris, pH 10.5. The eluate wasimmediately neutralized with 1/10 volume of 1
Tris HCl, pH6.8. Eluted antibodies were concentrated in Centricon-30 tubes(Amicon, Danvers, MA) by centrifugation for 20 minutes at5,000
. To estimate the yield, affinity-purified antibodies wereanalyzed by SDS-PAGE and Coomassie blue staining, usingpurified human IgG (Sigma, St. Louis, MO) as standard.Finally,elutedantibodies(100ng/ml)wereanalyzedbyimmuno-blotting, using either nuclear extracts or purified AUF1 asantigen.
Indirect immunofluorescence microscopy.
Commer-cial acetone-fixed HEp-2 cell slides (ANA HEp-2 slides;Generic Assays, Dahlewitz, Germany) were used in immuno-fluorescence studies. Slides were incubated with serum (di-luted 1:200 in PBS) or affinity-purified anti-AUF1 antibody(100 ng/ml in PBS) for 60 minutes at room temperature, washed 3 times with PBS, and subsequently incubated for 30minutes with fluorescein isothiocyanate–conjugated goat anti-human IgG (Caltag, South San Francisco, CA).
Preparation of rabbit antibodies.
AUF1-specific anti-sera were generated by immunizing rabbits with a keyholelimpet hemocyanin–coupled AUF1-derived peptide (theN-terminal 12–amino acid motif contained in all 4 AUF1isoforms). Animals were immunized subcutaneously with 0.8mg peptide and boosted intravenously with 0.6 mg peptideevery 6 weeks. Antisera were collected and were used eitherunpurified or peptide affinity-purified. Affinity-purified sera were monospecific for AUF1, as tested by immunoblottingusing HeLa cellular extracts.
For immunohisto-chemical analyses, 1–3-
m paraffin sections of synovial tissueobtained from patients with RA, patients with OA, and normalsubjects were mounted on poly-
-lysin–coated slides (Oligene,Berlin, Germany), incubated overnight at 58°C, and deparaf-finized with xylene. The affinity-purified anti-AUF1 rabbitantibody was applied at a 1:1,500 dilution, and anti-AUF1binding was detected using the LSAB/AP kit (Dako, Glostrup,Denmark).
RNA protein crosslink assay.
Equimolar amounts (0.3
P-labeled RNA oligonucleotide and purified recom-binant AUF1p45 or deletion mutants, respectively, were mixedand incubated at room temperature for 20 minutes in 20
l of binding buffer (10 m
Tris HCl, pH 7.5, 1 m
EDTA, 4%glycerol, 0.1% Triton X-100, 10 m
2-mercaptoethanol). Thereaction mixture was transferred to a microtiter plate, put onice, and irradiated with an ultraviolet (UV) lamp (Stratalinker;Stratagene, La Jolla, CA) at 254 nm at a dose of 9.9 mJ/mm
. After the addition of Laemmli sample buffer (50 m
Tris HCl,pH 6.8, 2% weight/volume SDS, 10% volume/volume glycerol,5% w/v 2-mercaptoethanol, 0.01% w/v bromophenol blue), thesamples were boiled for 10 minutes and analyzed on 10%SDS–polyacrylamide gels. The gels were dried and autoradio-graphed. For quantitative analysis, an electronic autoradio-graphy system (InstantImager; Packard, Meriden, CT) wasused.
Gel retardation assay.
The binding and competitionassays were performed essentially as described above exceptthat samples were not UV irradiated but were immediatelytransferred to 5% nondenaturing polyacrylamide gels run inelectrophoresis buffer (25 m
Tris HCl, 192 m
glycine, pH8.3) and separated for 60 minutes at 10 V/cm. For supershiftexperiments, the reactions were initially incubated for 15minutes at 30°C, subsequently 1
l of affinity-purified antibody was added, and incubation continued for an additional 15minutes. Finally, gels were dried and analyzed by autoradiog-raphy.
Fisher’s exact test was used toanalyze the significance of anti-AUF1 antibody prevalencesbetween patient groups and between patients and healthycontrol subjects and for analyzing correlations of anti-AUF1autoantibodies with clinical features.
RESULTS Autoantibodies to hnRNP D (AUF1) in sera frompatients with rheumatic diseases.
In routine immuno-blot analyses of patient sera, in which a preparation of semipurified hnRNP proteins was used as an antigenicsource, prominent autoreactivity to 2 proteins withestimated molecular masses of 45 kd and 42 kd, respec-tively, was repeatedly observed, and was particularlypronounced in sera from patients with SLE. To furtherinvestigate this finding, a total of 266 sera from patients with various rheumatic diseases were selected from theserum bank and analyzed by immunoblotting. In thesestudies,
30% of SLE sera and
20% of RA sera wereshown to be reactive with the 45/42 doublet (Figure 1A).The molecular masses of the 2 proteins suggested thatthey belong to the subgroup of hnRNP D proteins, which comprises 4 members with molecular massesbetween 38 kd and 45 kd generated by alternativesplicing. These proteins are better known as AUF1,because they selectively bind to certain AU-rich ele-ments that are frequently present in the 3
-UTR of short-lived mRNA.Therefore, we expressed the 45-kd variant of AUF1 in
and investigated by immuno-blotting whether sera reactive with the 45/42-kd antigenalso recognized the recombinant protein. In these stud-ies, the majority of positive sera (89%) were indeedreactive with recombinant AUF1. Moreover, antibodiesaffinity-purified from recombinant AUF1 cross-reacted with both the 45-kd and 42-kd antigens (Figure 1B), andantibodies affinity-purified from the natural proteinsclearly recognized the recombinant protein (Figure 1C).The 4 AUF1 isoforms differ by 2 insertions of 19and 49 amino acids, respectively, which are contained in
AUF1 PROTEINS IN SLE AND RELATED AUTOIMMUNE DISORDERS 513