Differential regulation of CD3-and CD28-induced IL-2 and IFN-gamma production by a novel tyrosine kinase inhibitor XR774 from Cladosporium cf. cladosporioides

Inhibition of CD28 signalling after an immune response impedes T cell activ ation and can lead to immunosuppression. To identify inhibitors of anti-CD2 8 induced IL-2 production, a library of fungal metabolites was screened in a cell-based, high throughput assay. A reduced novel benzofluoranthene, ten tatively named as (6bS, 7R, 8S)-7-methoxy- 4, 8, 9-trihydroxy- 1, 6b, 7, 8- tetrahydro-2 H-benzo[j] fluoranthen-3-one (XR774), from Cladosporium cf. cl adosporioides, was isolated. XR774 inhibited IL-2 mRNA and protein expressi on induced by anti-CD28 and anti-CD3 but had no effect on IL-2 induction by PMA and ionomycin. Moreover, XR774 inhibited the activity of the tyrosine kinases, Fyn, Lck, Abl and epidermal growth factor receptor (EGFR) with nan omolar activity, whereas micromolar concentrations of XR774 were ineffectiv e on the serine-threonine kinase, PKA. Kinetic analysis of Fyn kinase inhib ition was consistent with XR774 as a competitive inhibitor with respect to ATP. In peripheral blood mononuclear cells (PBMC), XR774 inhibited anti-CD3 and anti-CD28 induced IL-2 and IL-2R ru chain (CD25) expression but was consist ently less active for inhibition of IFN-gamma production. On stimulation wi th PMA and anti-CD28, XR774 inhibited IL-2 production but had no effect on CD25 expression and enhanced IFN-gamma production. In contrast, the ansamyc in, geldanamycin, inhibited both IL-2 and IFN-gamma production induced by a nti-CD3 and anti-CD28 or PMA and anti-CD28. No significant associated cytot oxicity or inhibition of protein synthesis was observed at concentrations u p to 14 muM Thus, XR774 represents a novel class of pharmacological agent w ith selective biological activities that distinguish it from other natural product inhibitors, such as the ansamycins. (C) 2001 Elsevier Science B.V. All rights reserved.