期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 103, 期 9, 页码 3316-3321出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0511206103
关键词
autoimmune; cytokine; inflammation; ligand affinity chromatography
资金
- NCI NIH HHS [CA-046934] Funding Source: Medline
- NHLBI NIH HHS [HL-68743, P01 HL068743] Funding Source: Medline
- NIAID NIH HHS [R01 AI015614, AI-15614, R56 AI015614] Funding Source: Medline
IL-32, a recently discovered proinflammatory cytokine with four isoforms, induces IL-1 beta, TNF-alpha, IL-6, and chemokines. Here, we used ligand (IL-32 alpha) affinity chromatography in an attempt to isolate an IL-32a soluble receptor or binding protein. Recombinant IL-32a was covalently immobilized on agarose, and preparations of concentrated crude human urinary proteins were applied for chromatographic separation. A specific 30-kDa protein eluted from the column during acid washing and was identified by mass spectrometry as proteinase 3 (PR3) and confirmed by N-terminal microsequencing. PR3, a neutrophil granule serine protease, exists in a soluble or membrane form and is the major autoantigen for autoantibodies in the systemic vasculitic disease, Wegener's granulomatosis. The affinity of IL-32a to PR3 was determined by surface plasmon resonance. The dissociation constants were 2.65 +/- 0.4 nM for urinary PR3 and 1.2 +/- 0.05 nM for neutrophil-derived PR3. However, irreversible inactivation of PR3 enzymatic activity did not significantly change binding to the cytokine. Nevertheless, limited cleavage of IL-32 yielded products consistent with PR3 enzyme activity. Moreover, after limited cleavage by PR3, IL-32a was more active than intact IL-32a in inducing macrophage inflammatory protein-2 in mouse macrophages and IL-8 in human peripheral blood mononuclear cells. We suggest that PR3 is a specific IL-32a binding protein, independent of its enzymatic activity. However, limited cleavage of IL-32a by PR3 enhances activities of the cytokine. Therefore, specific inhibition of PR3 activity to process IL-32 or neutralization of IL-32 by inactive PR3 or its fragments may reduce the consequences of IL-32 in immune regulated diseases.
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