Expression of the chemokines MIP-1alpha, MCP-1, and RANTES in experimental autoimmune uveitis.

PURPOSE: To determine the location of the CC chemokines macrophage inflammatory protein (MIP)-1alpha, monocyte chemoattractant protein (MCP)-1, and regulated on activation of normal T-cell-expressed and secreted (RANTES) during disease progression in experimental autoimmune uveitis (EAU) and their relationship with the presence of the T helper cell (Th)1-type cytokine IFNgamma. METHODS: EAU was induced by immunization of Lewis rats with retinal extract. Consecutive cryostat sections were prepared from eyes at different stages of EAU, graded for severity of uveitis and stained by using antibodies to MCP-1, MIP-1alpha, and RANTES and to cell surface markers. Supernatants from superficial cervical lymph node cells were examined by ELISA for IFNgamma, IL-4, and IL-10. RESULTS: MIP-1alpha and IFNgamma were present most frequently and most extensively at peak disease but also were detectable in the choroid 8 days after immunization, before clinical disease onset. MCP-1 and RANTES were present at peak disease, but much less frequently. RANTES was occasionally found in the choroid before clinical disease. By days 19 to 21 after immunization, although infiltrating cells were present, there were only residual low levels of chemokine staining. MCP-1 and RANTES were detected on CD3-positive cells and on some ED1-positive cells, whereas MIP-1alpha was also associated with vessels and areas of exudate. Lymph node cells cultured from animals with peak disease had increased levels of IFNgamma and IL-10, but for IFNgamma this occurred only after stimulation in vitro with retinal extract. CONCLUSIONS: Although MCP-1 and RANTES were associated predominantly with cells infiltrating the retina, MIP-1alpha was also associated with resident cells. All three are likely to exacerbate EAU-MIP-1alpha, to the greatest degree.

Control of chemokine production at the blood-retina barrier.

Chemokine production at the blood-retina barrier probably plays a critical role in determining the influx of tissue-damaging cells from the circulation into the retina during inflammation. The blood-retina barrier comprises the retinal microvascular endothelium and the retinal pigment epithelium. Chemokine expression and production by human retinal microvascular endothelial cells (REC) have never been reported previously, so we examined the in vitro expression and production of monocyte chemoattractant protein-1 (MCP-1), regulated on activation of normal T-cell expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, interleukin (IL)-8, epithelial cell-derived neutrophil activating protein-78 (ENA-78) and growth related oncogene alpha (GROalpha) in these cells, both unstimulated and stimulated by cytokines likely to be present during the evolution of an inflammatory response. We compared this to expression and production of these chemokines in vitro in human retinal pigment epithelial cells (RPE). MCP-1 was expressed and produced constitutively by REC but all the chemokines were produced in greater amounts upon stimulation with the proinflammatory cytokines IL-1beta and tumour necrosis factor-alpha (TNF-alpha). MCP-1 and IL-8 were produced at much higher levels than the other chemokines tested. MIP-1alpha and MIP-1beta were present only at low levels, even after stimulation with IL-1beta and TNF-alpha. Cytokines with greater anti-inflammatory activity, such as IL-4, IL-10, IL-13, transforming growth factor-beta (TGF-beta) and IL-6, had little effect on chemokine production either by REC alone or after stimulation with IL-1beta and TNF-alpha. RPE, although a very different cell type, showed a similar pattern of expression and production of chemokines, indicating the site-specific nature of chemokine production. Chemokine production by REC and RPE is probably significant in selective leucocyte recruitment during the development of inflammation in the retina.

CXCR4 receptor expression on human retinal pigment epithelial cells from the blood-retina barrier leads to chemokine secretion and migration in response to stromal cell-derived factor 1 alpha.

Retinal pigment epithelial (RPE) cells form part of the blood-retina barrier and have recently been shown to produce various chemokines in response to proinflammatory cytokines. As the scope of chemokine action has been shown to extend beyond the regulation of leukocyte migration, we have investigated the expression of chemokine receptors on RPE cells to determine whether they could be a target for chemokine signaling. RT-PCR analysis indicated that the predominant receptor expressed on RPE cells was CXCR4. The level of CXCR4 mRNA expression, but not cell surface expression, increased on stimulation with IL-1beta or TNF-alpha. CXCR4 protein could be detected on the surface of 16% of the RPE cells using flow cytometry. Calcium mobilization in response to the CXCR4 ligand stromal cell-derived factor 1alpha (SDF-1alpha) indicated that the CXCR4 receptors were functional. Incubation with SDF-1alpha resulted in secretion of monocyte chemoattractant protein-1, IL-8, and growth-related oncogene alpha. RPE cells also migrated in response to SDF-1alpha. As SDF-1alpha expression by RPE cells was detected constitutively, we postulate that SDF-1-CXCR4 interactions may modulate the affects of chronic inflammation and subretinal neovascularization at the RPE site of the blood-retina barrier.

Cytokine regulation of granulocyte-macrophage colony-stimulating factor (GM-CSF) production by human retinal pigment epithelial cells.

GM-CSF is an important regulator of macrophage, granulocyte and dendritic cell behaviour and function. These cell types have been implicated in the retinal damage characteristic of endogenous posterior uveitis. Dendritic cells in the choroid have access to retinal antigens processed by the retinal pigment epithelial (RPE) cells of the blood-retinal barrier and are thought to be candidates for the presentation of antigen in uveoretinitis. We therefore investigated the production of GM-CSF and its regulation in human RPE cells. IL-1beta, tumour necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta) all stimulated GM-CSF production by RPE cells and a combination of these cytokines increased GM-CSF production over five-fold compared with that with the individual cytokines alone. Interferon-gamma (IFN-gamma) rapidly down-regulated these responses. IFN-gamma did not appear to be acting directly on IL-1beta or via the synthesis of another protein. GM-CSF mRNA expression showed the same pattern of response to these cytokines, indicating transcriptional or pre-transcriptional regulation, and there was no evidence that IFN-gamma was acting by destabilizing GM-CSF mRNA. These results are generally important in understanding the ways in which cytokine regulation differs between different cell types and also more specifically for determining ways in which a cytokine with a significant role in the development of autoimmune uveoretinitis may be manipulated.

Cytokine regulation of RANTES production by human retinal pigment epithelial cells.

The mechanism whereby inflammatory cells gain access to the retina in posterior intraocular inflammatory disease remains unclear. The chemokine RANTES has the potential to influence the migration of memory T cells and monocytes across the blood-retinal barrier during inflammatory eye disease. We have therefore examined the production of RANTES by cultured human retinal pigment epithelial cells (RPE), which form a part of the blood-retinal barrier, in response to cytokines likely to be present in the microenvironment. IL-1 beta and TNF alpha stimulated RANTES production by these cells. IFN-gamma acted synergistically with TNF alpha to increase RANTES production. In contrast, IL-4 downregulated RANTES production stimulated by TNF alpha. RT-PCR studies showed that RANTES mRNA from RPE followed the same pattern of expression in response to cytokines as did RANTES production indicating that RANTES production was controlled at, or prior to, transcription. RANTES is produced in vitro by RPE in response to the proinflammatory cytokines IL-1 beta, TNF alpha, and IFN-gamma and is therefore likely to play a role in the development of the inflammatory eye disease endogenous posterior uveitis.

TGF-beta and IL-1 beta act in synergy to enhance IL-6 and IL-8 mRNA levels and IL-6 production by human retinal pigment epithelial cells.

Cytokines produced by human retinal pigment epithelial (RPE) cells may function as important regulators of intraocular inflammation. We investigated the effect of transforming growth factor-beta (TGF-beta) on interleukin-1 beta (IL-1 beta) induction of IL-6 and IL-8 mRNA levels and protein production by human RPE cells. Both TGF-beta and IL-1 beta alone induce IL-6 mRNA and IL-6 production in human RPE cells and synergize to enhance IL-6 mRNA levels and IL-6 production over a range of TGF-beta (0.1-10 ng/ml) and IL-1 beta concentrations (5-500 U/ml). TGF-beta was also found to enhance IL-1 beta induction of IL-8 mRNA levels at lower IL-1 beta concentrations (50 U/ml) but had no effect at higher IL-1 beta concentrations (500 U/ml). However, TGF-beta had no synergistic effect on IL-1 beta induction of IL-8 secretion. These results suggest that expression of IL-6 and IL-8 in human RPE cells is regulated by different transcriptional and translational mechanisms, and that RPE cells are important regulators of cytokine production within the ocular microenvironment.

Adhesion molecule expression in acute and fibrotic sympathetic ophthalmia.

Samples of iris ciliary body, choroid and retina from normal eyes and from 2 cases of sympathetic ophthalmitis (one acute and one late stage fibrosis) were examined for the expression of the VLA integrins beta 1 and alpha 1-6, and the integrin beta 3, in addition to ICAM-1, VCAM-1, ELAM-1 and CD44 using an APAAP staining technique. The expression of VLA-4, VLA-5, VCAM-1, ICAM-1, and CD44 was significantly increased and ELAM-1 was slightly increased in acute sympathetic ophthalmitis in comparison to fibrotic and normal eyes. VLA-6 was moderately increased in acute and fibrotic cases and VLA-2 VLA-3 and beta 3 were moderately expressed on all tissues examined. The differential expression of molecules known to be involved in lymphocyte activation and adhesion in acute sympathetic ophthalmitis suggests that certain adhesion molecules play a role in the pathogenesis of intraocular inflammation and may be suitable targets for immunotherapy.