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Interferon-gamma induced protein of 10 kDa (IP-10) is a C-X-C chemokine that attracts T lymphocytes and inhibits angiogenesis. In this study, we investigated the expression of IP-10 by human retinal pigment epithelial cells (HRPE) and compared IP-10 expression to that of interleukin-8 (IL-8), which is a leukocytic chemoattractant and pro-angiogenic factor. Cultured HRPE cells were incubated with either IL-1 beta (0.2-20 ng/ml) or tumor necrosis factor (TNF)-alpha (0.2-20 ng/ml) alone or in combination with interferon-gamma (IFN-gamma) (1000 U/ml). HRPE cells were also incubated with: (1) media conditioned by activated human T lymphocytes (CM), or (2) the same CM treated with neutralizing antibodies to IL-1, TNF, and/or IFN-gamma. IL-8 and IP-10 protein levels were measured by ELISA and mRNA levels by Northern blot analysis of HRPE cells. HRPE cells produced very high levels of IP-10 in response to either IL-1 beta/IFN-gamma, TNF-alpha/IFN-gamma or CD3-activated T-lymphocyte CM. The levels of IP-10 were at least tenfold higher (p<.001) than IL-8 measured in the same samples. Neutralizing antibodies to TNF and IFN-gamma, but not to IL-1, abrogated the ability of the CD3-activated T lymphocytes CM to induce HRPE IP-10 (p<.001). HRPE cells produce differential levels of IP-10 and IL-8 in response to various combinations of recombinant and T-lymphocyte-secreted pro-inflammatory cytokines. This may be important in evolving inflammatory and angiogenic ocular responses.

Purpose: It was previously demonstrated that toll-like receptor 4 (TLR4) is involved in species-specific human retinal pigment epithelial (HRPE) photoreceptor outer segment recognition and oxidant production. This study was performed to demonstrate the classical role of TLR4 in HRPE endotoxin (lipopolysaccharide; LPS) binding leading to HRPE proinflammatory cytokine secretion., Methods: Cultured HRPE cells were examined for TLR4 expression by immunofluorescence, Western blot analysis, and RT-PCR. HRPE cells labeled with fluorescent monoclonal antibodies (mAbs) to TLR4 and its associated adhesion molecule, CD14, were analyzed by real-time microscopy and resonance energy transfer (RET), determining associations of fluorescent LPS, TLR4, and CD14. LPS-induced HRPE secretion of interleukin (IL)-8 was measured with and without specific blocking mAb to TLR4 and CD14. HRPE TLR4 expression was measured after LPS exposure in the presence and absence of blocking antibodies to TLR4 and CD14., Results: All three HRPE cell lines demonstrated constitutive TLR4 expression by immunofluorescence, Western blot analysis, and RT-PCR. Examination of HRPE cells labeled with fluorescent mAb to TLR4, CD14, and LPS demonstrated RET among the three molecules, indicating that LPS-CD14 binding preceded LPS-TLR4 binding and the close association of CD14 and TLR4. LPS-induced IL-8 was significantly reduced (P < 0.05) in the presence of blocking mAb to TLR4 or CD14. Upregulation of HRPE TLR4 gene and protein expression occurred in response to LPS exposure and was inhibited by mAb to TLR4 or CD14., Conclusions: HRPE TLR4 is a multifunctional molecule that participates in photoreceptor outer segment membrane recognition, oxidant production, LPS recognition, and cytokine production. These roles indicate potential involvement in retinal degenerative and inflammatory processes.

Purpose: To investigate the role of the phosphatidylinositol 3-kinase (PI3K) pathway and the signal mediator AP-1 in monocyte chemotactic protein (MCP)-1 and interleukin (IL)-8 gene expression in human retinal pigment epithelial (hRPE) cells., Methods: hRPE cells were stimulated with IL-1beta and TNF-alpha and by coculturing with monocytes in the presence or absence of a series of kinase inhibitors. The induction of MCP-1 and IL-8 protein and mRNA was determined by ELISA and RT-PCR, respectively. Western blot analysis, kinase assays, and electrophoretic mobility shift assays were used to detect the activation of signaling mediators and transcription factors., Results: Concomitant with the induction of chemokine expression by the stimuli, there was phosphorylation of PI3K and its downstream targets-namely, Akt, GSK, and FKHR. Ly294002, a specific inhibitor of PI3K, resulted in time- and dose-dependent blockade of MCP-1 mRNA expression and protein production. The IC(50) for inhibition of MCP-1 secretion induced by IL-1beta, TNF-alpha, and hRPE-monocyte binding was 16, 12, and less than 3 micro M, respectively. In contrast, Ly294002 did not inhibit the IL-8 expression induced by any of the stimuli. Ly294002 as well as U0126, SB202190, and SP600125, the selective inhibitors of MEK, p38, and JNK, respectively, strongly inhibited induced c-fos expression, whereas Ly294002 did not inhibit induction of MEK, p38, or JNK. Blockade of PI3K/Akt abolished IL-1beta-induced nuclear translocation of AP-1, whereas the induction of IkappaB degradation was unchanged., Conclusions: The Ly294002-sensitive PI3K/Akt pathway regulates MCP-1, but not IL-8 expression in hRPE cells independent of MAPK and IkappaB. PI3K-dependent induction of hRPE c-fos and AP-1 nuclear translocation may be a target for therapies aimed at modulating MCP-1 in retinal diseases.

CD14 is the primary receptor for lipopolysaccharide (LPS)that plays important roles in host defense and subserves other host-related biological functions. We previously identified CD14 on cultured human retinal pigment epithelial (HRPE) cells using immunocytochemical techniques. In this study, we investigated immunoreactive HRPE CD14 expression by immunohistochemically staining HRPE cells and HRPE cells in sections of human eyes with anti-CD14 monoclonal antibodies (mAb). Constitutive HRPE gene and protein expression were confirmed by semiquantitative PCR and western blotting. ELISA for cell-associated and secreted (s) HRPE CD14 revealed that specific digestion by phosphoinositol-specific phospholipase C (PI-PLC) significantly reduced (P<0·01) cell-associated HRPE CD14 which was not modulated by LPS or γ-IFN. ELISA of the conditioned media (CM) of HRPE cells treated with PI-PLC contained significantly more (P<0·001) sCD14, but sCD14 was not modulated by LPS or γ-IFN. FACS analysis confirmed HRPE cell surface CD14. To show functional CD14, fluorescently-labelled LPS and CD14 were demonstrated to show significant co-localization on live, cultured HRPE cells in close proximity (<7 A˚) as demonstrated by resonance energy transfer of the fluorescent ligands (P<0·0001). Significant inhibition (P<0·001) of LPS-induced IL-8 secretion, as measured by ELISA, occurred in the presence of function blocking anti-CD14 mAb. Significant inhibition of LPS-induced HRPE IL-8 secretion by PKC, PTK, PI3 kinase, and p38 kinase inhibitors indicated cell mediators responsible for LPS-induced HRPE chemokine secretion. This study demonstrates that HRPE cells express functional CD14 in vitro and in situ along at the outer blood-retina barrier. [Copyright &y& Elsevier]

Purpose. Human retinal pigment epithelial (HRPE) cells are involved in ocular inflammation by secretion of chemokines such as IL-8 and MCP-1. It has been shown in this and other laboratories that interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) are potent inducers of HRPE IL-8 and MCP-1 secretion. The induced IL-8 and MCP-1 expression is often modulated by other proinflammatory factors in a synergistic manner. Modulation of IL-8 and MCP-1 production by interleukin-4 (IL-4), a important mediator in Th2-mediated immunity, and granulocyte/macrophage-colony-stimulating factor (GM-CSF), one of the cytokines secreted by HRPE has been reported in non-ocular cells. The aim of the present investigation was to study effects of these two cytokines alone or in combination with IL-1β or TNF-α on HRPE IL-8 and MCP-1 generation. Methods. The primary culture of HRPE cells was stimulated with various doses of IL-4, GM-CSF, IL1-β and TNF-α alone or in combination for 8 or 24 hr. The supernatants were subjected to enzyme-linked immunosorbent assay (ELISA) for IL-8 and MCP-1. The mRNAs were isolated from the corresponding cells for Northern blot analysis. Results. IL-1β and TNF-α induced dose-dependent increases in HRPE IL-8 and MCP-1 secretion with maximal stimulation observed at 2–5 ng/ml. IL-4 alone (100 ng/ml) resulted in a slight increase of MCP-1 and IL-8 secretion. When IL-4 was co-administrated with IL-1β or TNF-α, two to three-fold increases in IL-8 and MCP-1 were observed over the maximal levels induced by IL-1β or TNF-α alone. Northern blot analyses revealed that IL-4 did not alter the steady-state MCP-1 mRNA stimulated by IL-1β and TNF-α, or alter the IL-8 mRNA stimulated by TNF-α, although the IL-1β-induced IL-8 mRNA was slightly enhanced by higher concentrations of IL-4 (100 ng/ml). Conclusion. The synergistic action by IL-4 occurs predominately at the post-transcriptional level. In contrast to IL-4, GM-CSF alone or in combination with IL-1β or TNF-α did not generate additional secretion of HRPE IL-8 and MCP-1. HRPE IL-8 and MCP-1 gene expression and protein production are stimulated by IL-1β or TNF-α through pathways differentially modulated by IL-4 and GM-CSF. [ABSTRACT FROM AUTHOR]

Objective: To determine vitreous levels of interleukin 8 (IL-8) and interferon-induced protein 10 (IP-10), which are members of the C-X-C chemokine family that promote and inhibit neovascularization, respectively. Methods: We measured the levels of IL-8 and IP-10 by specific enzyme-linked immunosorbent assays in the vitreous from 30 patients with proliferative diabetic retinopathy (PDR) and 10 control patients undergoing vitrectomy for idiopathic macular holes or idiopathic macular puckers. Results: Detectable levels of IL-8 were found in 23 of 24 patients with active PDR, 4 of 6 patients with inactive PDR, and 6 of 10 controls. Levels of IL-8 were significantly increased in vitreous samples from the patients with active PDR (P=.02) when compared with vitreous samples from the controls. The IL-8 levels detected in vitreous samples from patients with inactive PDR were not significantly elevated over those found in the control samples. Interferoninduced protein 10 was detected in the vitreous samples from 23 of 24 patients with active PDR, all patients with inactive PDR, and 9 of 10 controls. Significant elevations of IP-10 were measured in samples from patients with active PDR (P=.004) and in those with inactive PDR (P=.00) over those from controls. In addition, levels of IP-10 were significantly elevated in vitreous samples from patients with inactive PDR compared with vitreous samples from patients with active PDR (P=.02). Conclusion: Both IL-8 and IP-10 participate in the pathogenesis of PDR. [ABSTRACT FROM AUTHOR]