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Osamu Sakai, Yoshiyuki Tamada, Mitsuyoshi Azuma; Involvement of NFB on Cytokine Production From Mast Cells and Conjunctival Fibroblasts Cultured Under Allergic Conditions. Invest. Ophthalmol. Vis. Sci. 2011;52(14):6425.
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Mast cells play a central role in the pathogenesis of allergic conjunctivitis. In sensitized patients, challenge by a specific ocular allergen causes release of mediators from activated mast cells. Histamine is a well-known and extensively studied mediator. Several cytokines such as IL-4 and TNF-α, are also produced and released, and these cytokines cause the activation of conjunctival fibroblasts. Activated conjunctival fibroblasts produce and release several chemokines, which cause attraction and accumulation of inflammatory cells, and lead to aggravated allergic conjunctivitis. NFΚB is known to regulate expression of many genes related to inflammation. The purposes of present studies were to 1) develop an in vitro model of allergic conjunctivitis, and 2) determine the involvement of NFΚB in production of cytokines in mast cells and conjunctival fibroblasts.
The in vitro model: After sensitizing with anti-dinitrophenol (DNP) IgE, rat mast cells (RBL-2H3) were challenged with DNP-BSA for 24 hr. The conditioned medium above the cultured mast cells was then cultured with conjunctival fibroblasts from rats. The activation of NFΚB was evaluated by immunoblotting for decreased levels of endogenous inhibitor IΚB-α. In some experiments, IΚB kinase inhibitor BMS-345541 was used to inhibit NFΚB activation. Cytokines released into the culture medium from mast cells and conjunctival fibroblasts were measured by ELISA.
Mast cells cultured under allergic conditions produced and released IL-4 and TNF-α. This was associated with degradation of IΚB-α, suggesting activation of NFΚB. Conjunctival fibroblasts cultured in the conditioned medium from the activated mast cells showed decrease in IΚB-α; eotaxin-1 and CINC-1 were also produced and released. BMS-345541 significantly inhibited decrease of IΚB-α and production of cytokines in both types of cells.
Cytokines, which probably play a role in immunoregulation of the ocular surface in allergic patients, were produced and released in our cultured cells, and NFΚB was a crucial factor in production of cytokines. These results suggest new opportunities for developing and evaluating anti-allergic drugs that inhibit the NFΚB pathway.
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