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V.L. Calder, G. Galatowicz; Human Cord Blood Mast Cell IgE Cross–Linking: An in vitro Model of Conjunctival Mast Cell Responses? . Invest. Ophthalmol. Vis. Sci. 2005;46(13):934.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Conjunctival mast cells are important effector cells in ocular allergy and secrete a range of mediators, but can only be isolated in low cell numbers. The aim of this study was to characterise human cord blood–derived mast cells (HCBMC) phenotypically and functionally in response to IgE stimulation.The effects of two anti–allergy drugs – epinastine & olopatadine – were also investigated. Methods: Human cord blood CD34+stem cells (105) were cultured in 200ul StemspanTM serum–free medium containing SCF [100ng/ml] and IL–6 [50ng/ml], with IL–3 [1ng/ml] added during the first 14 days. On week 9–11, 10% fetal calf serum was added. At week 11, cells were characterised by flow cytometry for CD117 (c–kit) and FcεR1, and cytospins prepared for tryptase (AA1) and chymase (CCI) staining. For cross–linking, cells were incubated with IgE [4µg/ml; 16 hr] before anti–IgE Ab[25µg/ml] was added and histamine release determined by ELISA. Intracellular cytokine expression for IL–4 and IL–13 was investigated at 4, 24 & 48hr. Multiplex cytokine assays were performed to detect IL–2, IL–4, IL–5, IL–6, IL–10, TGFα & ß by flow cytometry. Results: At week 10, HCBMC were immunophenotyped as 100% c–kit+ and 90% FcεR1+, >95% AA1+ and >58% CCI+. Following FcεR1 stimulation, significant histamine secretion was detected at 1hr [35.9±5.9 ng/ml; p<0.01], which was inhibited by 51.4% with epinastine [0.1µg/ml] (p<0.05) and by 35.1% with olopatadine [10µg/ml] (p<0.05). Intracellular expression of IL–4 was 90.6±1.6% in unstimulated cells, which decreased to 47.3±0.3% at 48hr post stimulation (p<0.0001). Epinastine [10µg/ml] reduced IL–4 expression (39.1±2.9%; p<0.05). In contrast, basal expression of IL–13 was 8.2±0.3%, which increased to 14.0±1.5% at 48hr post stimulation. Production of IL–1ß was induced by 1hr and peaked at 4hr [95.4±14.6 ρg/ml]. IL–8 was significantly increased at 1hr and peaked at 24hr (7.5±0.08 ng/ml; p<0.003). IL–5 was detected even in unstimulated cells [55.8±2.7 ρg/ml]. The effect of the anti–allergic drugs on production of these cytokines is being investigated. Conclusions: The HCBMC were phenotypically comparable to conjunctival mast cells and demonstrated a differential cytokine response following IgE cross–linking. The anti–allergic drugs effectively reduced the release of histamine and had some effect on IL–4 expression. The data suggests these cells provide an in vitro system with which to investigate mast cell cytokine responses and we are currently investigating human conjunctival mast cells (in collaboration with Prof. J. Dart, Moorfields Eye Hospital).
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