Native IL-32 is released from intestinal epithelial cells via a non-classical secretory pathway as a membrane-associated protein

Cytokine. 2011 Jan;53(1):74-83. doi: 10.1016/j.cyto.2010.09.002.

Abstract

Although IL-32 has been shown to be induced under various pathological conditions, a detailed understanding of native IL-32 intracellular distribution and mechanism of release from cells has not been reported. We examined the expression of IL-32 in the intestinal epithelial cell line HT-29 following TNFα and IFNγ co-stimulation. The subcellular localization of induced IL-32 was associated with the membrane of lipid droplet-like structures and vacuolar structures that co-localized with markers of endosomes and lysosomes. Prolonged co-stimulation resulted in cell death and appearance of IL-32 in the culture medium. IL-32 released from co-stimulated HT-29 cells was found in a detergent-sensitive particulate fraction, and in a step density gradient the IL-32 particulate was buoyant, suggesting association with a membrane-bound vesicle. Upon Triton X-114 partitioning, most of the IL-32 partitioned to the detergent phase, suggesting hydrophobic characteristics. When IL-32-containing vesicles were subjected to protease K treatment, a protease resistant ∼12kDa fragment was generated from ∼24kDa IL-32. We propose that under these conditions, native IL-32 is released via a non-classical secretory route perhaps involving multi-vesicular bodies and exosomes. Demonstration of membrane association for both intracellular and released IL-32 suggests this unique cytokine may have a complex biosynthetic pathway and mechanism of action.

MeSH terms

  • Cell Compartmentation / drug effects
  • Detergents / pharmacology
  • Endocytosis / drug effects
  • Endopeptidase K / pharmacology
  • Epithelial Cells / drug effects
  • Epithelial Cells / metabolism*
  • HT29 Cells
  • Humans
  • Hydrophobic and Hydrophilic Interactions / drug effects
  • Interferon-gamma / pharmacology
  • Interleukins / genetics
  • Interleukins / metabolism*
  • Intestines / cytology*
  • Intracellular Membranes / drug effects
  • Intracellular Membranes / metabolism
  • Lipids / chemistry
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Molecular Weight
  • Protein Isoforms / genetics
  • Protein Isoforms / metabolism
  • Protein Transport / drug effects
  • Secretory Pathway* / drug effects
  • Secretory Vesicles / drug effects
  • Secretory Vesicles / metabolism
  • Subcellular Fractions / drug effects
  • Subcellular Fractions / metabolism
  • Surface Properties / drug effects
  • Tumor Necrosis Factor-alpha / pharmacology

Substances

  • Detergents
  • IL32 protein, human
  • Interleukins
  • Lipids
  • Membrane Proteins
  • Protein Isoforms
  • Tumor Necrosis Factor-alpha
  • Interferon-gamma
  • Endopeptidase K