Phosphatidylinositol 3-kinase controls antineutrophil cytoplasmic antibodies-induced respiratory burst in human neutrophils

J Am Soc Nephrol. 2002 Jul;13(7):1740-9. doi: 10.1097/01.asn.0000019411.36000.06.


Antineutrophil cytoplasmic antibodies (ANCA) activate human polymorphonuclear neutrophils (PMN) primed with tumor necrosis factor alpha (TNF-alpha) in vitro. Phosphatidylinositol 3-kinase (PI3-K) and the protein-serine/threonine kinase Akt have been implicated in the control of the phagocyte respiratory burst. The hypothesis that PI3-K controls the ANCA-induced respiratory burst was tested. TNF-alpha-primed PMN were stimulated with a monoclonal antibody to myeloperoxidase (MPO) and with PR3- and MPO-ANCA, respectively. Akt activation was assessed with phospho-specific antibodies. Superoxide release was measured with ferricytochrome. ANCA antigen translocation was assessed by fluorescence-activated cell sorter. The effect of TNF-alpha and MPO-ANCA on Akt signaling was studied with immunoprecipitation and glutathione S-transferase pull-down assays. Western blotting revealed rapid transient Akt phosphorylation during TNF-alpha priming and a second phosphorylation after ANCA. PI3-K inhibition by LY294002 blocked both Akt phosphorylation and superoxide generation. A total of 20 +/- 3 nmol O(2)(-)/0.75 x 10(6) PMN/45 min was released after stimulation with PR3-ANCA. LY294002 (5 microM) decreased this amount to 0.3 +/- 2.6 nmol (n = 10, P < 0.05); the MPO-ANCA values were 23 +/- 3 versus 1.6 +/- 3.6 (n = 10, P < 0.05). p38 MAPK inhibition with 10 microM SB202190 that also decreased ANCA-induced superoxide generation prevented S473 phosphorylation of Akt in response to TNF-alpha and to ANCA. However, SB202190 but not LY294002 abrogated TNF-alpha-mediated ANCA antigen surface translocation, demonstrating that superoxide generation and ANCA antigen translocation proceed by separate mechanisms. Akt, PAK1, and Rac1 existed as cytosolic complex in resting PMN. TNF-alpha stimulation increased association of PAK1 with Akt. An MPO monoclonal antibody did not alter the Akt signaling complex further. The data demonstrate the importance of PI3-K for the ANCA-induced PMN oxidant production.

MeSH terms

  • Antibodies, Antineutrophil Cytoplasmic / immunology
  • Antibodies, Antineutrophil Cytoplasmic / physiology*
  • Antigens / metabolism
  • Biological Transport / physiology
  • Chromones / pharmacology
  • Enzyme Inhibitors / pharmacology
  • Humans
  • Mitogen-Activated Protein Kinases / physiology
  • Morpholines / pharmacology
  • Neutrophils / physiology*
  • Phosphatidylinositol 3-Kinases / physiology*
  • Phosphoinositide-3 Kinase Inhibitors
  • Protein Serine-Threonine Kinases*
  • Proto-Oncogene Proteins / physiology
  • Proto-Oncogene Proteins c-akt
  • Respiratory Burst / physiology*
  • Signal Transduction / physiology
  • Tumor Necrosis Factor-alpha / pharmacology
  • p38 Mitogen-Activated Protein Kinases


  • Antibodies, Antineutrophil Cytoplasmic
  • Antigens
  • Chromones
  • Enzyme Inhibitors
  • Morpholines
  • Phosphoinositide-3 Kinase Inhibitors
  • Proto-Oncogene Proteins
  • Tumor Necrosis Factor-alpha
  • 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
  • AKT1 protein, human
  • Protein Serine-Threonine Kinases
  • Proto-Oncogene Proteins c-akt
  • Mitogen-Activated Protein Kinases
  • p38 Mitogen-Activated Protein Kinases