Objective: To investigate the dynamics and distribution of toll-like receptor 4 (TLR4) in uvea-resident tissue macrophages during endotoxin-induced uveitis (EIU) in Wistar rats.
Methods: Fifty Wistar rats were randomly divided into five groups (n = 10 per group) based on the following time points: before LPS injection (0 h, control group) and 6, 12, 24, and 48 h after LPS injection. All the rats (except the control group) received a footpad injection of 200 microg of vibrio cholera lipopolysaccharide (LPS). The intensity of anterior segment inflammation was evaluated after the LPS injection. Ten rats each were killed before LPS injection and 6, 12, 24, 48 h after injection. The iris-ciliary body complex and choroid from each eye were removed and cut into segments. Immunohistochemical localization of TLR4 and a resident tissue macrophage marker, cluster of differentiation 163 (CD163), was performed on whole mount isolated iris-ciliary body complexes and choroids. TLR4+ and CD163+ cells in the iris were counted, and the cell density (cells/mm(2)) was calculated. For CD163+ cells, the percent of round pleiomorphic cells in positive staining cells was calculated. The distribution patterns and the phenotypes of cells expressing these two proteins were further characterized by double-labeled immunofluorescence studies. Positive cell density and the percent of round-pleiomorphic CD163+ cells were analyzed by one-way ANOVA followed by least significant difference procedure (LSD) tests for multiple comparisons.
Results: The iris-ciliary body complex did not express TLR4 in normal rats. Six h after the LPS injection, a small number of TLR4+ cells were detected in the irides of two rats. The density of TLR4+ cells in the iris was (506.1 +/- 39.5) cells/mm(2) (12 h), (492.3 +/- 54.5) cells/mm(2) (24 h) and (663.8 +/- 150.2) cells/mm(2) (48 h), respectively. The number of TLR4+ cells significantly increased 12, 24 and 48 h after the injection (F = 167.2, P < 0.001, ANOVA). No changes of morphology of TLR4+ cells were detected 12-48 h after the injection. CD163 was expressed in the uvea in all rats. CD163+ round tissue macrophages were present at all time periods (0-48 h). The proportion of these cells was 13% at 0 h and increased to approximately 80% at 12-48 h. These changes occurred mainly in the macrophages located in the stroma bordering the iris endothelial layer. Double-labeling immunofluorescence demonstrated the co-expression of TLR4 and CD163 in round stroma cells with TLR4 protein located at the cell membrane and CD163 protein in the cytoplasm. TLR4+ cells could not be detected in choroid in any of the rats.
Conclusions: Iris tissue macrophages expressed TLR4 and TLR4+ cells increased in the iris during EIU. It indicates that TLR4 may play an important role in the pathogenesis of acute anterior uveitis.