Down-regulation of Th2 cytokine expression by glucocorticoids in cultivated human lung mast cells

Abstract

VOLUME 111, NUMBER 2 using dot blot analysis with the specific antibody, BB1. Ca 2+ fluxes were measured using Fluo-3. Inositol 1,4,5-trisphosphate (IP3) was measured by radio-receptor binding assay. RESULTS: Thrombin (250 U/ml) and trypsin (10 lag/ml) elevated intracetlular Ca 2§ ([Ca2+]i) in KU812 cells. Selective agonist peptides, to PAR-I (TFLLR, 100 gM) and PAR4 (AYPGKF, 300 ~tM) elevated [Ca2+]i but PAR2 selective peptides (SLIGRL, 50 gM and SLIGKV, 100 gM) caused no stimulation. PAR-I and PAR-4 selective peptides transiently increased IP 3 levels in KU812 cells, mRNA for PAR-I and PAR-4 was detected in KU812 cells and purified human basophils (n=3). Additionally mRNA for PAR-2 and -3 was faintly detected in one donor's basopbils. Agonist peptides failed to stimulate basogranulin release from basophils in four out of five experiments. CONCLUSIONS: These data suggest the expression of PAR-1 and PAR4 by human basophils and KU812 cells. PAR-2 and PAR-3 may be expressed by basophils in some individuals. Selective stimulation of PAR1 or PAR-4 is generally insufficient to induce degranulation. Funding: National Asthma Campaign 4 7 7 Down-regulation of Th2 Cytokine Expression by Glucocorticoids in Cultivated Human Lung Mast Cells J. G. Mohanty, J. M. Abboud, E. S. Schulman; Pulmonary and Critical Care Medicine, Drexel University College of Medicine, Philadelphia, PA. RATIONALE: In allergen-induced asthma, mast cells play a critical role through the release of various mediators including Th2 cytokines. Glucocorticoids are used for treatment of allergic disorders; however their effects on human lung mast cell (HLMC) function have not been ftdly studied. Recently, we developed techniques to culture partially purified HLMC over months using stem cell factor and interleukin (IL)-4, in which HLMC become homogeneous, proliferate and maintain a functional phenotype equivalent to freshly isolated cells. METHODS: HLMC were cultured overnight with or without triamcinolone (10-6 M) to examine its effects on the regulation ofTh2 cytokines expression. HLMC were stimulated by antibody cross-linking of the highaffinity IgE receptor (FceRI). At 20 min post stimulation, percent histamine release (HR) was measured; at 2 hour, total cellular RNA was isolated, and reverse transcriptase/polymererase chain reaction (RT-PCR) was performed to study cytokine gene expression. RESULTS: Spontaneous HR was 20-fold, with concurrent increases in IL-6 mRNA levels and DNA binding activity for key transcriptional regulators of the IL-6 promoter, NF-kB and AP-I. Comparisons of conditioned media and eosinophilfibroblasts co-culture using transwell barriers, indicated cell contact is unnecessary and the soluble nature of the mediators. Antibody neutralization of TGF-13 in the conditioned media produced only a 20% inhibition of fibroblast IL-6 secretion. Interestingly, neutralization of IL-113 and bFGF inhibited the IL-6 response by 60% and 15-20%, respectively. Antibody neutralization results were supported by the induction of fibroblast IL-6 secretion by recombinant IL-II3 (68-fold), TGF-I]I (2.5-tbld), and bFGF (l.5-fold). Neutralization of other potential eosinophil-derived mediators including GM-CSF, PDGF-BB, NGE and IL-4, had an insignificant effect. CONCLUSIONS: Our results suggest a major role for eosinophilderived IL-113, TGF-13 and bFGF in fibroblast activation and eosinophilassociated fibrotic responses, including subepithelial fibrosis in asthma. Funding: NIH, EMS Foundation