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V. L. Calder, G. Galatowicz, S. W. Y. Chan, F. C. L. Cheung, R. Martinelli, M. E. Stern; Human Mast Cell Chemokine Production and Signalling Pathways. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1502.
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© ARVO (1962-2015); The Authors (2016-present)
Conjunctival mast cells (MC) are important effector cells in ocular allergy and secrete a range of mediators. Human cord blood-derived mast cells (CBMC) are a useful in vitro model as they are phenotypically and functionally similar to conjunctival MC in their response to IgE stimulation. The aim of this study was to characterise CBMC chemokine profiles and signalling pathways.
Human cord blood CD34+stem cells (105) were cultured in 200ul StemspanTM medium containing SCF [100ng/ml], IL-6 [50ng/ml] and IL-3 [1ng/ml], adding 10% fetal calf serum on week 9. On week 11, differentiated CBMC were confirmed by coexpression of CD117 (c-kit) and FcεR1. For cross-linking, CBMC were incubated with 4µg/ml IgE for 16h before 25µg/ml anti-IgE Ab was added and culture supernatants harvested at 24h. Multiplex chemokine assays were performed to detect RANTES, IP-10, MCP-1, MIP-1α, MIP-1ß, eotaxin-1, Mig and IL-8 by flow cytometry. Phosphorylation of signalling molecules (Erk-1,-2, p38 MAPK) was assayed by flow cytometry and confirmed by Western blotting. NFATc1, c-Jun and NF-ΚBp65 nuclear translocation was quantitated in cell cytospins.
CBMC were immunophenotyped as 100% c-kit+ and 90% FcεR1+. Following FcεR1 stimulation, secretion of IL-8 (CXCL8; 5.5 ± 0.2), CCL3 (MIP-1α; 0.2 ± 0.01), CCL4 (MIP-1ß; 7.6 ± 0.5), CCL2 (MCP-1; 9.5 ± 0.02) ng/ml were detected in comparison with unstimulated cells [p<0.001], whereas CCL5 (RANTES), CCL11 (eotaxin-1), CXCL9 (Mig) and CXCL10 (IP-10) were undetectable. FcεR1 stimulation induced phosphorylation of erk1,2 and p38. Erk, but not p38, phosphorylation was upregulated in the presence of SCF. Phosphorylation of erk 1,2 was p38- and JNK-independent but partially reduced by PI3 kinase inhibitors. P38 activation was JNK and PI3 kinase independent. By 30 min FcεR1 stimulation, CBMC were NF-AT+ (75%), NF-ΚB and c-Jun (both 35%). The effect of inhibitors on chemokine production is being investigated.
The CBMC demonstrated a differential chemokine response following IgE cross-linking, involving specific signalling pathways.
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