Possible roles of protein kinases in neutrophil chemotactic factor production by leucocytes in allergic inflammation in rats

Abstract

1 In an air pouch-type allergic inflammation model in rats, leucocytes that had infiltrated into the pouch fluid collected 4 h after the antigen challenge produced proteinaceous chemotactic factors for neutrophils when they were incubated in the medium. 2 To clarify the mechanism of activation of the infiltrated leucocytes in producing these factors, the effects of protein kinase inhibitors on neutrophil chemotactic factor production were examined. 3 When the infiltrated leucocytes were incubated for 4h in medium containing the non-selective protein kinase inhibitor K-252a (1–100 ng ml⁻¹, 2.14–214 nm), the tyrosine kinase inhibitor genistein (1–50 μg ml⁻¹, 3.7–185 μm), and the more selective protein kinase C inhibitor H-7 (5–100 μg ml⁻¹, 13.7–274 μm); neutrophil chemotactic activity in the conditioned medium was decreased in a concentration-dependent manner, but the adenosine 3′:5′-cyclic monophosphate (cAMP)-dependent protein kinase inhibitor H-89 (1–1000 ng ml⁻¹, 2.24–2240 nm) showed no effect. 4 Isoelectric focusing of the conditioned medium revealed that the leucocytes produced two neutrophil chemotactic factors, leucocyte-derived neutrophil chemotactic factor (LDNCF) 1 and LDNCF-2. Treatment of the leucocytes with K-252a, genistein, and H-7, but not H-89, inhibited production of both LDNCF-1 and LDNCF-2. 5 These results suggest that activation of tyrosine kinase and protein kinase C., but not cAMP-dependent protein kinase, is responsible for the production of LDNCF-1 and LDNCF-2. 6 The steroidal anti-inflammatory drug dexamethasone and the protein synthesis inhibitor cyclohex-imide inhibited neutrophil chemotactic factor production in a concentration-dependent manner. Time-course experiments showed that the inhibitory effect by dexamethasone was apparent even 30min after the incubation. 7 Mechanism for inhibiting the production of LDNCF-1 and LDNCF-2 by dexamethasone is also discussed.