In the JournalsPerspective

Antibodies to 14-3-3 may be a biomarker for large vessel vasculitis

Around 78% of patients with large vessel vasculitis may have antibodies to 14-3-3 proteins, the presence of which may serve as a novel biomarker for the disease, according to researchers at the Cleveland Clinic and University Hospitals Case Medical Center.

Analyses were performed on 11 aorta samples from 11 patients with large vessel vasculitis (LVV) and 11 control participants matched for age, sex and race. Western blot and immunoprecipitation studies were performed on aortic samples from 23 patients with aortitis undergoing thoracic aortic reconstructive surgery, 23 matched control participants with noninflammatory aortic aneurysm, and 41 patients with vasculitis or other diseases. Sera from 23 patients with LVV, 26 matched control participants, 48 control participants with other diseases and 11 healthy participants were analyzed by ELISA-based colorimetric assays developed by the researchers with 100 µg/mL of bovine serum albumen on a 96-well plate.

A significant difference was seen in sera from patients with LVV and immunoglobulin G (IgG) antibodies, which had higher absorbance compared to other samples from patients with other diseases or healthy donor samples.

In 22 frozen aortic samples from patients with LVV, light and heavy chains of IgG were seen and a third band of positive reactivity was observed in 78% of patients with LVV at around 30 kd, which was absent from 89% of matched samples from patients with noninflammatory diseases. The band was also absent from 80% of samples from patients with granulomatosis with polyangiitis and was not seen in samples from healthy participants. Overall, 6.7% of the samples from participants other than patients with LVV showed the band at 30 kd. No differences were observed in patients treated with prednisone.

Mass spectroscopy of the 30-kd region with in-gel trypsin digestion was used to identify the proteins present. Of the 33 proteins identified, 14-3-3 was the most abundant in the lysates from patients with LVV.

Three independent experiments with eight aorta lysates and 12 samples of serum were performed to determine the auto-antigenicity of 14-3-3, the presence of anti-14-3-3 antibodies with antigen pull-down assays. Immunoprecipitation of 14-3-3 from the tissue from patients with LVV was observed on Western blot and mass spectroscopy.

Detailed mass spectroscopy techniques revealed three 14-3-3 protein isoforms of γ, ζ and ε in samples from patients with LVV in two independent experiments.

After additional testing, the researchers wrote that immunoreactivity was confirmed with each of the three isoforms.

“While our findings will need to be confirmed, they do raise important questions for further studies that may address whether targeted 14-3-3 isoforms are native or modified antigens, whether there is a relevant T cell immune response to 14-3-3, and whether circulating antigen concentrations and/or antibodies to these proteins may be useful surrogates for diagnosis and monitoring disease activity,” the researchers wrote. – by Shirley Pulawski

Disclosure: The research is supported by the Vasculitis Foundation (grant VF1301), the Clinical and Translational Science Collaborative of Cleveland (CTSC Cleveland grant T53250), the American Heart Association (Scientist Development grant 23080025 to Chakravarti), and the Konigsberg Family Fund for Vasculitis Research. The acquisition of the Orbitrap Elite instrument was made possible by the NIH Shared Instrumentation Grant Program (grant NIH-1S10-RR-031537-01).

Around 78% of patients with large vessel vasculitis may have antibodies to 14-3-3 proteins, the presence of which may serve as a novel biomarker for the disease, according to researchers at the Cleveland Clinic and University Hospitals Case Medical Center.

Analyses were performed on 11 aorta samples from 11 patients with large vessel vasculitis (LVV) and 11 control participants matched for age, sex and race. Western blot and immunoprecipitation studies were performed on aortic samples from 23 patients with aortitis undergoing thoracic aortic reconstructive surgery, 23 matched control participants with noninflammatory aortic aneurysm, and 41 patients with vasculitis or other diseases. Sera from 23 patients with LVV, 26 matched control participants, 48 control participants with other diseases and 11 healthy participants were analyzed by ELISA-based colorimetric assays developed by the researchers with 100 µg/mL of bovine serum albumen on a 96-well plate.

A significant difference was seen in sera from patients with LVV and immunoglobulin G (IgG) antibodies, which had higher absorbance compared to other samples from patients with other diseases or healthy donor samples.

In 22 frozen aortic samples from patients with LVV, light and heavy chains of IgG were seen and a third band of positive reactivity was observed in 78% of patients with LVV at around 30 kd, which was absent from 89% of matched samples from patients with noninflammatory diseases. The band was also absent from 80% of samples from patients with granulomatosis with polyangiitis and was not seen in samples from healthy participants. Overall, 6.7% of the samples from participants other than patients with LVV showed the band at 30 kd. No differences were observed in patients treated with prednisone.

Mass spectroscopy of the 30-kd region with in-gel trypsin digestion was used to identify the proteins present. Of the 33 proteins identified, 14-3-3 was the most abundant in the lysates from patients with LVV.

Three independent experiments with eight aorta lysates and 12 samples of serum were performed to determine the auto-antigenicity of 14-3-3, the presence of anti-14-3-3 antibodies with antigen pull-down assays. Immunoprecipitation of 14-3-3 from the tissue from patients with LVV was observed on Western blot and mass spectroscopy.

Detailed mass spectroscopy techniques revealed three 14-3-3 protein isoforms of γ, ζ and ε in samples from patients with LVV in two independent experiments.

After additional testing, the researchers wrote that immunoreactivity was confirmed with each of the three isoforms.

“While our findings will need to be confirmed, they do raise important questions for further studies that may address whether targeted 14-3-3 isoforms are native or modified antigens, whether there is a relevant T cell immune response to 14-3-3, and whether circulating antigen concentrations and/or antibodies to these proteins may be useful surrogates for diagnosis and monitoring disease activity,” the researchers wrote. – by Shirley Pulawski

Disclosure: The research is supported by the Vasculitis Foundation (grant VF1301), the Clinical and Translational Science Collaborative of Cleveland (CTSC Cleveland grant T53250), the American Heart Association (Scientist Development grant 23080025 to Chakravarti), and the Konigsberg Family Fund for Vasculitis Research. The acquisition of the Orbitrap Elite instrument was made possible by the NIH Shared Instrumentation Grant Program (grant NIH-1S10-RR-031537-01).

    Perspective
    Leonard H. Calabrese

    Leonard H. Calabrese

    Among the vasculitides, large vessel vasculitis remains one of the most difficult to classify and understand. It occurs in two phenotypic forms – Takayasu arteritis and giant cell arteritis – and the differences between the two is often difficult to define.

    Many attempts to identify a causal factor include numerous infectious agents, such as parainfluenza influenza virus, herpes simplex virus, varicella-zoster virus, Chlamydia and Burkholderia among others and none with conclusive evidence. From an “autoimmunity” perspective, the diseases continue to confound because of a lack of a defined autoantigen.

    Researchers at the Cleveland Clinic have published a provocative study incriminating antibodies to 14-3-3 proteins in 78% of patients with thoracic aortic aneurysms, a condition and site favoring vasculitis, but in only in 6.7% of controls. This study is novel in that used sterile snap frozen specimens and not only demonstrated autoantibody in tissue and serum, but also up regulation of 14-3-3 family proteins in tissue. Is this a new biomarker?

    14-3-3 proteins are coming on fast and are both novel and complex. There are seven isoforms that homodimerize and heterodimerize, and they appear to play central roles in hundreds of protein-protein interactions involved with cell proliferation, differentiation and migration. Furthermore, these proteins are known to elicit autoantibody formation as demonstrated in rheumatoid arthritis. Why we would make antibodies to these endogenous and key regulatory proteins is hard to understand but they may be vulnerable to post-translational modification similar to other proteins. Given the strong interest in the 14-3-3 field, I predict a flurry of studies on the role of this family in autoimmune diseases. Stay tuned.

    References:

    Grayson PC, et al. Ann Rheum Dis. 2012; doi:10.1136/annrheumdis-2011-200795.

    Maksymowych WP, et al. Novel autoantibodies to 14-3-3 Eta are highly specific for RA. Arthritis and Rheumatism Abstract Supplement; 2012;64.

    • Leonard H. Calabrese, DO
    • Consulting Medical Editor, Healio.com/Rheumatology Professor of Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University RJ Fasenmyer Chair of Clinical Immunology Theodore F. Classen, DO Chair of Osteopathic Research and Education Vice Chairman, Department of Rheumatic and Immunologic Diseases Cleveland Clinic Cleveland

    Disclosures: Calabrese reports he is a consultant for Genentech, Pfizer, Bristol-Myers Squibb, GlaxoSmithKline, Sanofi, Jansen and Abbvie; and is on the speakers bureau for Genentech, Abbvie and Bristol-Myers Squibb and Crescendo Bioscience.