Susan McKillop-Smith

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-1β or TNF-α. 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 1α (SDF-1α) indicated that the CXCR4 receptors were functional. Incubation with SDF-1α resulted in secretion of monocyte chemoattractant protein-1, IL-8, and growth-related oncogene α. RPE cells also migrated in response to SDF-1α. As SDF-1α 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.

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)‐1α, MIP‐1β, interleukin (IL)‐8, epithelial cell‐derived neutrophil activating protein‐78 (ENA‐78) and growth related oncogene α (GROα) 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‐1β and tumour necrosis factor‐α (TNF‐α). MCP‐1 and IL‐8 were produced at much higher levels than the other chemokines tested. MIP‐1α and MIP‐1β were present only at low levels, even after stimulation with IL‐1β and TNF‐α. Cytokines with greater anti‐inflammatory activity, such as IL‐4, IL‐10, IL‐13, transforming growth factor‐β (TGF‐β) and IL‐6, had little effect on chemokine production either by REC alone or after stimulation with IL‐1β and TNF‐α. 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.

Synthetic and plant-derived cannabinoid receptor antagonists show hypophagic properties in fasted and non-fasted mice

Background and purpose:  Obesity is a severe health problem in the modernized world and understanding the central nervous mechanisms underlying food‐seeking behaviour and reward are at the forefront of medical research. Cannabinoid receptors have proven an efficient target to suppress hunger and weight gain by their pharmacological inactivation.

The human histone gene expression regulator HBP/SLBP is required for histone and DNA synthesis, cell cycle progression and cell proliferation in mitotic cells

Histone proteins are essential for chromatin formation, and histone gene expression is coupled to DNA synthesis. In metazoans, the histone RNA binding protein HBP/SLBP is involved in post-transcriptional control of histone gene expression. In vitro assays have demonstrated that human HBP/SLBP is involved in histone mRNA 3′ end formation and translation. We have inhibited human HBP/SLBP expression by RNA interference to determine its function during the mitotic cell cycle. Inhibition of HBP/SLBP expression resulted in the inhibition of histone gene expression and DNA synthesis, the inhibition of cell cycle progression in S phase and the inhibition of cell proliferation. These findings indicate that human HBP/SLBP is essential for the coordinate synthesis of DNA and histone proteins and is required for progression through the cell division cycle.

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.

Effect of anti-macrophage inflammatory protein-1α on leukocyte trafficking and disease progression in experimental autoimmune uveoretinitis

This study has enabled us to identify the influence of the chemokine, macrophage inflammatory protein‐1α (MIP‐1α), on leukocyte behavior at the blood‐retina barrier in vivo and its link with the inflammatory process and disease pathogenesis. MIP‐1α has not previously been thought to be effective under conditions of physiological shear flow. However, short‐term anti‐MIP‐1α treatment inhibited leukocyte slowing and accumulation and subsequent extravasation of leukocytes at the blood‐retina barrier in animals with experimental autoimmune uveoretinitis. This was effective predominantly in the post‐capillary venules which have been shown to be the main site of passage of leukocytes across the blood‐retina barrier. Long‐term anti‐MIP‐1α treatment also prevented decreased leukocyte velocity and reduced disease severity as measured clinically, histologically and in terms of blood‐retina barrier breakdown.

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‐1β, tumour necrosis factor‐alpha (TNF‐α) and transforming growth factor‐beta (TGF‐β) 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‐γ) rapidly down‐regulated these responses. IFN‐γ did not appear to be acting directly on IL‐1β 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‐γ 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.