Control of chemokine production at the blood-retina barrier - PubMed (original) (raw)

Control of chemokine production at the blood-retina barrier

I J Crane et al. Immunology. 2000 Nov.

Abstract

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.

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Figures

Figure 1

Figure 1

Time-course of chemokine production in response to interleukin-1β (IL-1β) (0·25 ng/ml, 50 IU/ml) over 48 hr for human retinal microvascular endothelial cells (REC) and human retinal pigment epithelial cells (RPE). The chemokine concentration (pg/ml) in culture supernatants was detected using enzyme-linked immunosorbent assay (ELISA). Monocyte chemoattractant protein-1 (MCP-1), closed squares; regulated on activation of normal T-cell expressed and secreted (RANTES), open squares; interleukin-8 (IL-8), closed circles; growth related oncogene α (GROα), open circles; and epithelial cell-derived neutrophil activating protein-78 (ENA-78), closed triangles. Results shown are from one representative experiment.

Figure 2

Figure 2

Chemokine production by human retinal microvascular endothelial cells (REC) (open bars) and human retinal pigment epithelial cells (RPE) (hatched bars) in response to interleukin-1β (IL-1β), tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ). Chemokine was detected in supernatants after 24 hr of culture using enzyme-linked immunosorbent assay (ELISA). IL-1β was used at 0·25 ng/ml (50 IU/ml), TNF-α at 10 ng/ml (1100 IU/ml) and IFN-γ at 20 ng/ml (136 IU/ml). Results shown are combined from at least three experiments using REC from different donors. Error bars indicate the standard error of the mean (SEM).

Figure 3

Figure 3

Chemokine production by human retinal microvascular endothelial cells (REC) (left column) and human retinal pigment epithelial cells (RPE) (right column) in response to interleukin (IL)-4, IL-6, IL-10, IL-13 and transforming growth factor-β (TGF-β) alone (open bars) or in combination with interleukin-1β (IL-1β) (hatched bars) or tumour necrosis factor-α (TNF-α) (cross-hatched bars). Chemokine was detected by enzyme-linked immunosorbent assay (ELISA) in RPE supernatants after 24 hr of culture. IL-1β was used at 0·25 ng/ml, and IL-4, IL-6, IL-10, IL-13, TGF-β and TNF-α all at 10 ng/ml. Results shown are combined from at least three experiments using REC and RPE from different donors. Error bars indicate the standard error of the mean (SEM).

Figure 4

Figure 4

Chemokine mRNA expression as detected by reverse transcription–polymerase chain reaction (RT–PCR). RNA was extracted from cells 6 hr after stimulation of human retinal microvascular endothelial cells (REC), human retinal pigment epithelial cells (RPE) and human umbilical vein endothelial cells (HUVEC) (HU) cultures (C, no cDNA). Lane 0, (control) no cDNA; lane 1, unstimulated cultures; lane 2, 0·25 ng/ml of interleukin-1β (IL-1β); lane 3, 10 ng/ml of tumour necrosis factor-α (TNF-α); lane 4, IL-1β (0·25 ng/ml) + TNF-α (10 ng/ml). Results shown for macrophage inflammatory protein-1α (MIP-1α), MIP-1β, regulated on activation of normal T-cell expressed and secreted (RANTES) and epithelial cell-derived neutrophil activating protein-78 (ENA-78) had 5 µl of cDNA in the PCR reaction whereas those for β-actin, monocyte chemoattractant protein-1 (MCP-1), interleukin-8 (IL-8) and growth related oncogene α (GROα) had 1 µl of cDNA.

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