Cellular localisation of inducible nitric oxide in the rat iris after LPS

Purpose: To localise the cellular source of inducible nitric oxide (iNOS) in the rat iris in endotoxin-induced uveitis (EIU). Methods: Five groups of 8 female Lewis rats, aged 7-8 weeks, were sacrificed at 0, 2, 4, 6, 12, 24, 48 and 72 hrs after footpad administration of LPS (100μl, 2mg/ml, Salmonella typhimurium). Immunolocalisation of iNOS was performed on whole, flat mounted iris preparations at the above time points. Double immunolabeling was performed at the peak of the inflammation (12 hrs) to determine the phenotype of iNOS-positive cells. The mAb combinations NOS/ED1, iNOS/ED2 and iNOS/Ox6 were utilised. Total cell density and morphology were assessed and expressed as cells/mm². Results: No iNOS staining was present in normal (0 hrs) or in negative control irides. There was a rapid influx of small round iNOS⁺ cells infiltrating the iris at 2-4 hrs after LPS. This was followed by a gradual increase in iNOS expression reaching a maximum density at 12 hrs (835±143 cells/mm²). Thereafter the expression declined sharply and was not detectable at 48 and 72 hrs after LPS. Double labeling showed 70% of iNOS⁺ cells were small round/pleiomorphic ED1⁺ monocytes, 19% were ED2⁺ resident macrophages and approximately 52% were Ox6⁺ (MHC class II). Conclusions. These results suggest that newly extravasated monocytes are the major source of iNOS in the early phase of EIU and iNOS expression on both resident macrophages and dendritic cells is a secondary phenomenon later in the inflammation.