TLR-4/MD2 Has a Critical Role on Endotoxin-Induced Uveitis (EIU)

Abstract

Purpose: : To study if cell tranmigration and TNFα syntesis in endotoxin-induced uveitis is mediated through TLR-4/MD2 activation.

Methods: : C3H/HeN mice, 8 weeks old, were injected intravitreally (i.v.) with 2 µl of anti-TLR-4/MD2 (20 µg/ml, MTS 510, donated by Dr. K. Miyake). 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 control mice were injected with saline. Animals were study by intravitalmicroscopy or sacrificed after 24 hours for aqueous humor (AH) analysis from both eyes obtained by anterior chamber puncture and tested for TNF secretion by the WEHI 134 clone 13 bioassay. 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. mRNA was amplified using specific primers for TNF-alpha. Another group of mice were sacrificed and explant cultures of iris/cilliary body were challenged with LPS. Twenty four hours later, culture supernatant was tested for TNF secretion. Results were expressed as mean±SEM. Statistical differences were studied by Mann-Whitney U-test.

Results: : Twenty four hours after EIU induction, a statistical reduction in cell adhesion and infiltration was observed by intravitalmicroscopy. TNF levels were increase in AH of mice treated with saline i.v./LPS i.p. (4961±628 pg/ml) and IAb i.v./LPS i.p. (4943±494 pg/ml); while a significant reduction (89%; p<0.0001) was observed after anti-TLR-4 i.v./ LPS i.p. (537.2±68.3 pg/ml). TNF-alpha mRNA expression was detected in the iris/ciliary body of mice that did not received an intravitreal injection of anti-TLR-4/MD2 before the LPS i.p. challenge, while it was absent in mice treated with the antibody. Also, a 71% reduction of TNF levels was observed in iris/cilliary body explant cultures when pre-incubated with anti-TLR-4/MD2 (74±6.17 pg/ml), compared to explant cultures challenged with LPS (255±45.2 pg/ml).

Keywords: uveitis-clinical/animal model • inflammation • immunomodulation/immunoregulation