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Amirah Mohd Zaki, Grazyna Galatowicz, Virginia L Calder, ; Enhanced expression of conjunctival IL-9 co-localized with mast cells in seasonal allergic conjunctivitis and its effect on mast cell cytokine secretion via IL-9R.. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2484.
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Mast cells (MC) are well known as the primary responders in allergy, secreting pro-inflammatory mediators including cytokines into the extracellular environment. IL-9 is a pro-inflammatory cytokine that is associated with the immunopathogenesis of allergic diseases including asthma. However, its role in allergic conjunctivitis is still unknown. The aim of this study was to investigate conjunctival expression of IL-9, and its role in cytokine secretion by mast cells.
Human anonymised conjunctival tissue biopsies (3μm) were obtained from SAC donors (n=8, from all males; age: 18-65 years) at 8 hours’ post allergen challenge, and normal, non-inflamed, conjunctival tissues from anonymised donors (n=8 from 3 males; age 31-57 years). Donor tissues were collected after obtaining informed consent and Local Ethics approval in accordance with the Declaration of Helsinki. Sequential tissue sections were stained for anti-human IL-9 (Abcam), anti-human MC tryptase (AA1;DAKO) and primary antibody was omitted as a negative control. Two independent, masked observers enumerated positively stained cells per biopsy area (at least three fields). To study the function of IL-9 in vitro, bone marrow derived murine mouse mast cells (BMMCs) were exposed to PMA/ionomycin, ionomycin alone or anti-IgE in the presence or without anti-IL-9 or anti-IL-9R antibodies (R&D). Cytokine secretion at 24, 48, 72, 96, and 120 hours was assayed using multiplex bead arrays (Luminex).
IL-9 expressing cells were detected mainly within the subepithelial and stromal areas. There was a significant increase in numbers of IL-9+ cells in SAC tissues (mean=10.25 ± 1.26) compared to controls (mean= 25.00 ± 3.72; P<0.01). MC numbers were increased in SAC tissues and co-localised with IL-9. IL-9 was detected 24 hours’ post stimulation and reached its maximum at 48 hr in response to PMA/ionomycin and anti-IgE and 96 hr in response to ionomycin. Following neutralization of IL-9, secretion of IL-4, IL-5 and IL-13 was upregulated whereas these cytokine levels decreased upon blocking IL-9R (P<0.05).
IL-9 expression within conjunctival tissues was upregulated during challenge and is secreted by mast cells. In vitro studies revealed that IL-9 affects IL-4, IL-5 and IL-13 secretion from BMMC.
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