Cellular localisation and dynamics of nitric oxide synthase expression in the rat anterior segment during endotoxin-induced uveitis

Paul G. McMenamin, Julie M. Crewe

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Abstract

The present study examined the temporal pattern and cellular localisation of nitric oxide synthase in Endotoxin-Induced Uveitis (EIU). Lewis rats (n = 40) received a single footpad injection of 200 μg of bacterial lipopolysaccharide. Animals were killed at 0, 2, 4, 6, 12, 24, 48 and 72 hr after injection and ocular tissues prepared as iris-]ciliary body wholemounts or frozen sections of the anterior segment. The expression of constitutive nitric oxide synthase (cNOS) and inducible nitric oxide synthase (iNOS) was investigated at all time points by immunohistochemistry. A further group of animals (n = 6) were killed at the peak of the disease (12 hr) and the cellular co-localisation of iNOS on resident and infiltrating immune cells was investigated by double immunohistochemistry utilising the biotinylated monoclonal antibodies ED1, ED2 and Ox6. Expression of cNOS on iris vessels did not alter during the course of EIU. Quantitative analysis of iris-ciliary body wholemounts revealed the first evidence of iNOS+ at 2 hr which increased dramatically at 4 and 6 hr with a peak at 12 hr. The expression of iNOS in the early phase of the disease (2-6 hr) was associated with small round marginating and newly extravasated cells that on morphological criteria were most likely neutrophils and monocytes. At 12 hr cells of more mixed morphologies began to express iNOS and double labelling revealed 70% of these cells were also ED1+ (a lysosomal antigen present in monocytes/macrophages and dendritic cells), 52% were Ox6+ (MHC class II) (dendritic cells, activated macrophages and some T-cells) and 19% were ED2+ (pan-specific resident tissue macrophages). Expressed in an alternative manner, 10% of the total ED1+ cell population, 11% of the ED2+ cells and 44 % of Ox6+ cells co-expressed iNOS. Expression of iNOS decreased significantly by 24 hr to near baseline levels and was absent by 48 and 72 hr. Within the ciliary processes iNOS+ dendriform cells were noted at 6 hr and accumulations of many small round iNOS+ cells were present at 12 hr. The ciliary epithelium did not at any time express iNOS at the protein level detectable by immunohistochemistry. The results of this study suggest that iNOS expression early in EIU is associated with infiltrating or newly recruited neutrophils and monocytes/macrophages in the iris whereas later in the disease resident tissue macrophages and MHC class II+ cells (activated macrophages and putative dendritic cells) in the iris and ciliary body may synthesise nitric oxide. The role of this late phase of nitric oxide synthesis may include lymphocytostasis and immunosuppression as proposed in other tissue sites. The outcome of the present study may help in planning therapeutic strategies using NOS inhibitors.

Original languageEnglish
Pages (from-to)157-164
Number of pages8
JournalExperimental Eye Research
Volume65
Issue number2
DOIs
Publication statusPublished - 1 Jan 1997
Externally publishedYes

Keywords

  • Dendritic cells
  • Endotoxin
  • Inflammation
  • Iris
  • Macrophages
  • Synthase
  • Uveitis

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