The presence of membrane Proteinase 3 in neutrophil lipid rafts and its colocalization with FcγRIIIb and cytochrome b558

Alina David, Ram Fridlich, Irit Aviram

Research output: Contribution to journalArticlepeer-review

Abstract

Proteinase 3 (PR3), the target autoantigen of antineutrophil cytoplasmic antibodies in the autoimmune vasculitis, Wegener's granulomatosis, is a serine proteinase stored in granules of human neutrophils. PR3 is expressed also on the plasma membrane of unactivated neutrophils, and this expression increases in primed or stimulated cells. In the current study, we demonstrate the presence of PR3, FcγRIIIb, and cytochrome b558 of the NADPH oxidase in neutrophil lipid rafts. Activation of neutrophils with PMA, fmet-leu-phe, or TNFα known to increase the membrane expression of PR3 did not affect the amount of PR3 in rafts. Unexpectedly, the cytosolic subunits of the NADPH oxidase, p67phox and p47phox, the recruitment of which to the membrane requires cell stimulation, were detected in the rafts of unstimulated neutrophils. Treatment of neutrophils with the cholesterol-sequestering agent methyl-β-cyclodextrin (MβCD) reduced raft p22phox and PR3. MβCD diminished membrane FcγRIIIb upregulating membrane PR3 (mPR3) and CD11b/CD18. In addition, MβCD significantly reduced PMA-induced activity of the NADPH oxidase without altering fmet-leu-phe-elicited activity. Antibody-mediated cross-linking of membrane PR3 caused activation of ERK and JNK kinases and their translocation to rafts. Confocal analysis revealed colocalization of mPR3, FcγRIIIb, and p22phox in the membrane, confirmed by their coimmunoprecipitation. Cleavage of neutrophil GPI-anchors by PI-PLC reduced mPR3 and FcγRIIIb, implicating a GPI-protein, possibly FcγRIIIb, in the attachment of PR3 to the membrane.

Original languageEnglish
Pages (from-to)156-165
Number of pages10
JournalExperimental Cell Research
Volume308
Issue number1
DOIs
StatePublished - 1 Aug 2005

Keywords

  • FcγRIIIb
  • NADPH oxidase
  • Neutrophil
  • Proteinase 3
  • Rafts

Fingerprint

Dive into the research topics of 'The presence of membrane Proteinase 3 in neutrophil lipid rafts and its colocalization with FcγRIIIb and cytochrome b558'. Together they form a unique fingerprint.

Cite this