Establishment of the superoxide production assay with human monocytic cell line, U937, for the evaluation of Syk kinase inhibitors

J Immunol Methods. 2002 Oct 15;268(2):123-30. doi: 10.1016/s0022-1759(02)00188-6.


Protein tyrosine kinase Syk is known to play critical roles in the signal transduction from receptors for Fc portion of immunoglobulins (FcRs) and B cell receptor complex (BCR). Its importance was well studied in Fc epsilon RI-induced activation of mast cells; therefore, Syk inhibitors are expected to have anti-allergic effects and to be novel therapy for allergic diseases, such as asthma, allergic rhinitis and atopic dermatitis. We previously developed an enzyme assay of recombinant human Syk kinase for the high throughput screening. In order to evaluate the Syk kinase inhibitors in a human cell system, we have developed an assay with human monocytic cell line, U937, to monitor FcgammaRI-mediated superoxide production. We treated cells with IFN-gamma to enhance the expression of FcgammaRI and to obtain enough production of superoxide. Engagement of FcgammaRI stimulated superoxide production, which was accompanied with Syk phosphorylation. PMA, an activator of protein kinase C, also evoked superoxide production, but Syk was not phosphorylated. Moreover, the treatment of cells with antisense oligonucleotide against syk attenuated Syk protein expression and suppressed superoxide production induced by FcgammaRI-engagement, but not by PMA. These results confirm that Syk is involved in the signal transduction from FcgammaRI upstream of PKC in U937 cells and we can evaluate the efficacy and the selectivity of Syk inhibitors with this assay system.

MeSH terms

  • Enzyme Precursors / antagonists & inhibitors*
  • Humans
  • Interferon-gamma / pharmacology
  • Intracellular Signaling Peptides and Proteins
  • Oligonucleotides, Antisense / pharmacology*
  • Phosphorylation
  • Protein-Tyrosine Kinases / antagonists & inhibitors*
  • Receptors, IgG / physiology
  • Signal Transduction
  • Superoxides / metabolism*
  • Syk Kinase
  • Tyrosine / metabolism
  • U937 Cells


  • Enzyme Precursors
  • Intracellular Signaling Peptides and Proteins
  • Oligonucleotides, Antisense
  • Receptors, IgG
  • Superoxides
  • Tyrosine
  • Interferon-gamma
  • Protein-Tyrosine Kinases
  • SYK protein, human
  • Syk Kinase