Proteinase 3 is an IL-32 binding protein

Proc Natl Acad Sci U S A. 2006 Feb 28;103(9):3316-21. doi: 10.1073/pnas.0511206103. Epub 2006 Feb 17.


IL-32, a recently discovered proinflammatory cytokine with four isoforms, induces IL-1beta, TNF-alpha, IL-6, and chemokines. Here, we used ligand (IL-32alpha) affinity chromatography in an attempt to isolate an IL-32alpha soluble receptor or binding protein. Recombinant IL-32alpha 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-32alpha 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-32alpha was more active than intact IL-32alpha 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-32alpha binding protein, independent of its enzymatic activity. However, limited cleavage of IL-32alpha 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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Line
  • Humans
  • Interleukins / chemistry
  • Interleukins / isolation & purification
  • Interleukins / metabolism*
  • Interleukins / urine
  • Kinetics
  • Mass Spectrometry
  • Mice
  • Myeloblastin
  • Neutrophils / enzymology
  • Protein Binding
  • Serine Endopeptidases / chemistry
  • Serine Endopeptidases / metabolism*
  • Serine Endopeptidases / pharmacology
  • Serine Endopeptidases / urine


  • IL32 protein, human
  • Interleukins
  • Serine Endopeptidases
  • Myeloblastin