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

Journal of Immunological Methods
Publication Date
DOI: 10.1016/s0022-1759(02)00188-6
  • Syk
  • U937
  • Superoxide
  • FcγRi
  • Ifn-γ
  • Antisense
  • Biology
  • Medicine


Abstract 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 FcεRI-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 FcγRI-mediated superoxide production. We treated cells with IFN-γ to enhance the expression of FcγRI and to obtain enough production of superoxide. Engagement of FcγRI 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 FcγRI-engagement, but not by PMA. These results confirm that Syk is involved in the signal transduction from FcγRI upstream of PKC in U937 cells and we can evaluate the efficacy and the selectivity of Syk inhibitors with this assay system.

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