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E.V. Salazar, G. Bernal, L. Baute, B.E. Brito; Endotoxin–Induced Uveitis (EIU) Inflammatory Process is Inhibit by Anti–TLR–4 Monoclonal–Antibody . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4524.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate if anti–Toll like Receptor–4 (TLR–4) inhibits EIU development in a mouse model.
C3H/HeN mice, 8 weeks old, were injected intravitreally (i.v.) with 2 µl of anti–TLR4 (20 µg/ml, MTS 510, donated by Dr. K. Miyake). The controls were injected with apyrogenic saline or irrelevant antibody (IAb). Six hours later, EIU was induced by intraperitoneal (i.p.) injection of 200 µl (2 mg/ml) LPS and controls mice were injected with saline. For RT–PCR analysis, mice were sacrificed 3 hours after i.p. LPS challenge and iris/ciliary body were dissected and snap frozen in liquid nitrogen. RNA was amplified using specific primers for TLR4 and IP–10. Another group of mice were sacrificed after 24 hours and both eyes were enucleated and fixed in 10% buffered formalin for histology. Paraffin sections of 5 µm were stained with hematoxylin–eosin (H–E) and ocular infiltrating cells were counted. Aqueous humor was collected by anterior chamber puncture; both eyes of each animal were pooled. Results were expressed as mean±SEM. Statistical differences were studied by Mann–Whitney U–test.
Constitutive TLR4 mRNA expression was detected in the iris/ciliary body of mouse. IP–10 mRNA expression was only observed in those mice that did not received an intravitreal injection of anti–TLR–4 before the LPS i.p. challenge, and was absent in those mice that were treated with the antibody. Twenty four hours after EIU induction, there was significantly reduction in the number of ocular infiltrating cells observed in H–E histological section of eyes (p<0.0006, 88%), as well as in aqueous humor (p<0.002, 94%) in those mice treated iv with anti–TLR4 (anti–TLR–4 i.v./LPS i.p.; 2.42±0.78 cells, n=7; 0.18x106±0.10 cells/ml, n=16, respectively), in comparison to their controls (saline i.v./LPS i.p.; 16.50±3.08 cells, n=6; 3.23x106±0.25 cells/ml, n=17 and IAb i.v./LPS i.p.; 16.40±1.93 cells, n=5; 3.30x106±0.26 cells/ml, n=13, respectively).
Anti–TLR–4 monoclonal antibody, significantly reduce the number of ocular infiltrating cells and inhibit the chemokine IP–10 mRNA expression by the iris/ ciliary body. This suggests that intraocular TLR–4 expression might have a very important role during the development of Endotoxin–Induced Uveitis.
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