British Medical Journal

Aims—Previous studies have implied that interferon gamma (IFN-ã) is involved in the pathogenesis of endotoxin induced uveitis (EIU) in the rat. This study investigated the source of IFN-ã in the iris during EIU. Methods—Whole mounts of iris were isolated from Lewis rats before and at different times (from 4 hours to 14 days) after foot pad injection of 200 μg Salmonella typhimurium lipopolysaccharide (LPS). Immunohistological analysis was performed using monoclonal antibodies (mAbs) specific to rat IFN-ã (DB12 and DB13). mAbs specific to monocytes, macrophages, and dendritic cells and MHC class II were used to asses the inflammatory response in the eye (ED-1, ED-2, and OX-6). An antibody specific to neurofilaments (2H3) was used to stain nerve fibres in the normal iris. Results—LPS administration induced acute intraocular inflammation, characterised by a massive infiltration of monocytes/macrophages and increased numbers of MHC class II positive cells in the iris. IFN-ã immunoreactive cells were not detected in iris whole mounts of control rats. Strikingly, IFN-ã immunoreactivity was found in fibres from 4 hours until 10 days after LPS injection, with the most intense staining at 48–72 hours. Other DB12 or DB13 positive cells were not detected in the iris. The pattern of DB12 and DB13 staining in the inflamed iris was similar to the 2H3 staining of neurons in the iris of control rats. Conclusion—These results show that systemic LPS administration induces IFN-ã immunoreactivity in iris fibres and suggest that iris nerve fibres may be a source of IFN-ã during EIU. The IFN-ã immunoreactive material in the iris nerve fibres may be identical to neuronal IFN-ã. (Br J Ophthalmol 1998;82:695–699) Interferon gamma (IFN-ã) plays an important role in the regulation of immune and inflammatory responses and in the host defence against viral infections. It has multiple biological eVects, including activation of monocytes/ macrophages and polymorphonuclear cells, proliferation of Th1-type lymphocytes, induction of cytokines, such as interleukin 1 (IL-1) and tumour necrosis factor-ã (TNF), induction of nitric oxide (NO) production, induction of MHC antigen expression on a wide variety of cell types, including lymphocytes and macrophages, and antiviral activity (reviewed by Young and Hardy and Sen). The production of IFN-ã is mainly restricted to T lymphocytes and natural killer (NK) cells. T lymphocytes secrete IFN-ã in response to appropriate antigens or mitogens. Both T cells and NK cells are readily stimulated to produce IFN-ã by the cytokines IL-2 and IL-12, whereas corticosteroids, transforming growth factor (TGF-â), and IL-10 markedly suppress its synthesis. Using monoclonal antibodies against rat IFN-ã it was recently shown that neurons in peripheral ganglia contain IFN-ã immunoreactive molecules. 4 This material has now been further characterised and has been denoted as neuronal IFN-ã. It diVers from “immune” IFN-ã in that it has a much higher molecular weight (between 54 and 66 kD) but is similar in bioactivity concerning antiviral eVects and MHC inducing capacities. Several studies have implied that IFN-ã is involved in the pathogenesis of endotoxin induced uveitis (EIU), although its exact contribution remains unclear. IFN-ã mRNA was detected in the uvea 7 and protein was found in aqueous humour during EIU. Injection of IFN-ã into the eyes of rats induced uveitis that resembled the response to lipopolysaccharide (LPS) (reviewed by De Vos et al) and transgenic mice expressing IFN-ã in the eye developed profound intraocular inflammation, 11 indicating that this cytokine may have uveitogenic properties. Strikingly, systemic administration of anti-IFN-ã antibodies resulted in an exacerbation of EIU. Moreover, Whitcup et al showed recently that the beneficial eVect of intraocular IL-12 treatment on EIU was associated with increased levels of aqueous IFN-ã. It has been hypothesised that the “paradoxical” eVects of IFN-ã in EIU may be concentration dependent. The present study investigated the source of IFN-ã in the iris during EIU in the rat. Earlier attempts, using immunohistochemical analysis of tissue sections, did not reveal IFN-ã positive cells in the eye during EIU. Previously, we have used immunohistochemical analysis of tissue whole mounts to assess cellular changes in the retina and choroid during EIU 15 and noted the higher resolution of this technique compared with the former. In the study presented here, we show IFN-ã immunoreactivity is expressed in iris nerve fibres during endotoxin induced uveitis. Materials and methods EXPERIMENTAL PROTOCOL Inbred male Lewis rats (6–8 weeks of age), weighing 150–200 g, were purchased from Br J Ophthalmol 1998;82:695–699 695 Netherlands Ophthalmic Research Institute, Amsterdam, Netherlands