Anti-U11/U12 RNP antibodies in systemic sclerosis: a new serologic marker associated with pulmonary fibrosis

Abstract

Objective: To characterize a new serum autoantibody in patients with systemic sclerosis (SSc) directed against U11/U12 RNP and to identify the clinical features associated with this autoantibody.

Methods: We identified autoantibodies directed against the U11/U12 RNP complex in sera of patients with SSc and confirmed antibody specificity by immunoprecipitation, reverse transcriptase-polymerase chain reaction, and Southern blotting. We determined the prevalence of these antibodies in SSc and their specificity for SSc. We compared anti-U11/U12 RNP autoantibody-positive and negative SSc patients on demographic, disease classification, clinical variables, and survival.

Results: We identified 33 patients with anti-U11/U12 RNP antibodies. In 2 consecutive series of SSc patients first seen at 10-year intervals (1994-1995 and 2004-2005), the prevalence of anti-U11/U12 RNP antibody-positive patients was 15 of 462 (3.2%). Seventeen (52%) of these 33 patients had limited cutaneous involvement. All patients had Raynaud's phenomenon and 82% had gastrointestinal (GI) involvement. None had "intrinsic" pulmonary arterial hypertension. The most significant clinical difference between anti-U11/U12 antibody-positive and negative cohorts was the prevalence of lung fibrosis, which occurred in 79% of the anti-U11/U12 RNP antibody-positive patients versus 37% of the anti-U11/U12 RNP antibody-negative patients (P < 0.0001). GI involvement was also significantly increased in the anti-U11/U12 RNP antibody-positive group. Patients with anti-U11/U12 RNP antibodies and pulmonary fibrosis had a 2.25-fold greater risk of death than anti-U11/U12 RNP negative patients with pulmonary fibrosis.

Conclusion: Anti-U11/U12 RNP antibodies are present in the sera of approximately 3% of patients with SSc and are a marker for lung fibrosis, which is often severe.

Figure 1

Immunoprecipitation of U11/U12 snRNP complex. (A) K562 cells extracts were used for immunoprecipitation assays using sera of SSc patients. (B) Western blot analysis of proteins precipitated by a monoclonal anti-TMG antibody and probed with human serum. A monoclonal anti-TMG antibody (mTMG) was used to precipitate ribonucleoprotein complexes containing TMG-capped U RNAs. Precipitated samples were analyzed by western blot using sera from a healthy individual (NS) or from patients positive for anti-Sm (Sm), anti-U1 RNP, anti-U11/U12 RNP, or anti-TMG antibodies. A protein of approximately 65 kD is shared by anti-U11/U12 RNP and anti-TMG complexes.

Figure 2

Molecular confirmation of U11 and U12 snRNA. (A) Sera from a healthy donor (NS) or patients positive for anti-U11/U12 RNP (U11/U12), anti-Sm (Sm), anti-TMG (TMG), anti-U3 RNP (U3) antibodies, or a monoclonal anti-TMG antibody (mTMG) were used in immunoprecipitation reactions. Immunoprecipitated complexes were used in RT-PCR with U11 (left panel) or U12 (right panel) specific primers. Amplified products were visualized by electrophoresis on polyacrylamide gels and ethidium product staining. (B) An aliquot of the amplified DNA was electrophoresed on a 2% agarose gel and used in southern blotting with a radiolabeled cDNA probe corresponding to U11 (left panel) or U12 (right panel). Anti-U11/U12 patient sera precipitate the U11/U12 RNP complex containing the corresponding U RNA. U11 and U12 RNAs can be reverse transcribed and amplified using the designed primers.

Figure 3

Identification of anti-U11/U12 RNP antibodies in additional SSc patients. Ribonucleoprotein complexes were immunoprecipitated from K562 cell extracts. Sera from a healthy donor (normal serum; NS), or patient sera positive for anti-Ro, anti-Sm (Sm), anti-TMG (TMG), anti-U4/U6 RNP (U4/U6), anti-U11/U12 RNP (Pt 1–8), anti-U1 RNP, anti-U1 and U2 RNP (U1/U2), or anti-U5 RNP antibodies are shown. Serial serum samples from patients 1–3 were obtained at the indicated years. A marker of all U RNAs is shown (Total RNA). The negative control (Control) represents protein agarose beads incubated with extracts in the absence of human serum. Immunoglobulins in sera of patients 1–8 precipitate U RNAs of similar size to U11 and U12 RNAs.