Human RPE-monocyte co-culture induces chemokine gene expression through activation of MAPK and NIK cascade

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Abstract

Cell–cell contact between human retinal pigment epithelium (hRPE) cells and monocytes occurs in many retinal diseases involving blood-retinal barrier breakdown. This study investigates chemokine secretion induced by co-culture of hRPE cells and monocytes and illustrates the roles of p38 kinase, ERK, JNK/SAPK and NF-κB-inducing kinase signaling pathways for hRPE IL-8 and MCP-1 secretion induced in hRPE by co-culture with monocytes. Co-culture of hRPE cells with monocytes increased steady-state IL-8 and MCP-1 mRNA and protein secretion. Stimulation of hRPE cells by monocytes resulted in prominent increases in p38, ERK1/2 and JNK/SAPK phosphorlation, IκBα degradation, and NF-κB nuclear translocation. The induced IL-8 and MCP-1 proteins were almost completely supporessed by U0126, a specific mitogen-activated protein kinase kinase (MEK) inhibitor, or by SB203580, a selective p38 inhibitor. Chemokine secretion was completely blocked by simultaneous administration of U0126 and SB203580. Induction of IL-8 and MCP-1 was abrogated by Ro318220, an inhibitor of PKC, as well as by genistein or herbimycin A, inhibitors of PTK. In addition, anti-inflammatory drugs dexamethasone (DEX) and cyclosporin A (CSA) both blocked activation of JNKS/SAPK and the cell–cell contact induced production of hRPE IL-8 and MCP-1, while activation of p38 and ERK was only inhibited by DEX, but not by CSA. These results suggest that activation of DEX-sensitive, CSA-resistant MEK/ERK and p38 pathways, and activation of NF-κB, PKC, and PTK are essential for IL-8 and MCP-1 expression by hRPE cells.

Introduction

The neuroectodermally derived retinal pigment epithelial (RPE) cells actively participate in the inflammatory process of infectious and non-infections retinal diseases, most of which are involved breakdown of the blood retinal barrier. For example, human RPE (hRPE) cells express several components that may be involved in inflammatory responses including cell adhesion molecules ICAM-1 and HLA-DR antigens both in vitro (Elner et al., 1992, Chan et al., 1986) and in uveitis (Whitcup et al., 1992). In previous studies, we have shown that hRPE cells are able to bind leukocytes on their cell surfaces and to respond to inflammatory stimuli and monocyte adhesion by secreting chemokines, principally IL-8 and MCP-1 (Elner et al., 1990, Elner et al., 1991, Elner et al., 1992, Elner et al., 1997, Yoshida et al., 2001). IL-8 and MCP-1 belong to the C–X–C and the C–C chemokine families, respectively. IL-8 is a chemoattractant, an activator of neutrophils and eosinophils, and a mediator of angiogenesis (Sehmi et al., 1992, Koch et al., 1992). MCP-1 is a chemoattractant and an activator for lymphocytes and monocytes, causing monocyte/macrophage infiltration into tissues (Taub et al., 1995, Rollins et al., 1991). IL-8 and MCP-1, as principal chemokines secreted by hRPE (Elner et al., 1997), are therefore thought to be the major mediators responsible for neutrophil and monocyte chemotactic activities in retinal diseases (Grossniklaus et al., 2002). Monocyte extravasation and cell–cell contact between monocytes/macrophages and RPE cells are sequential events during these infections and non-infectious pathogenesis of the retinal diseases, such as proliferative vitreoretinopathy (PVR) and age-related macular degeneration (ARMD) (Grossniklaus et al., 1992).

It has been shown that the cell-to-cell contact of RPE with monocytes stimulates proliferation of RPE cells (Osusky and Ryan, 1996, Kirchhof et al., 1989) and accelerates maturation and differentiation of monocytes into macrophages (Osusky et al., 1997a, Osusky et al., 1997b). These mutual effects involve production of unidentified bioactive factors (Kirchhof et al., 1989). Our latest data show that hRPE IL-8 and MCP-1 secretion can be induced by direct contact with monocyte in a time-dependent manner, but not by the monocytes in the same medium that are physically separated from hRPE cells, suggesting that the monocyte-RPE cell contact induces chemokine secretion by hRPE cells (Elner et al., 2001, Yoshida et al., 2001). Blocking by anti-CD11b, -CD14, -CD54, and -CD102 monoclonal antibodies inhibits cell contact-induced RPE IL-8 and MCP-1 production (Elner et al., 2001). Immunohistochemistry study further reveals that the vast majority of IL-8 and MCP-1 are produced by RPE cells, not by the previously contacted monocytes (Elner et al., 2001). These findings corroborate the previous study that demonstrates vascular endothelial IL-8 and MCP-1 production by monocyte-endothelial contact (Lukacs et al., 1995). Subsequently, resident tissue cells such as bronchial epithelial, synovial, glioblastoma and glomerular mesangial cells have also been shown to produce IL-8 or MCP-1 when triggered by monocyte contact (Drumm et al., 2000, Thiele et al., 2000, Kasahara et al., 1998, Hisada et al., 2000, Kuroiwa et al., 1999, Rimbach et al., 2000). The importance of leukocyte-resident cell interaction at the inflammatory sites is underscored by increasing evidence linking such interactions to the production of potent inflammatory mediators. For example, the gingival-lymphoid cell contact has been shown to induce IL-1α, IL-1β, and IL-6 expression by gingival fibroblasts (Murakami et al., 1999). Co-culture of rat Kupffer cells with syngeneic hepatoma cells leads to enhanced nitric oxide production (Kurose et al., 1997).

The signal pathways involved in hRPE IL-8 and MCP-1 gene expression and protein production by several proinflammatory factors have been reported by this laboratory (Bian et al., 2001), the intracellular signals that are triggered by monocyte contact remain unknown. Two previous studies have suggested that the activation of transcription factor NF-κB is required for MCP-1 gene expression induced by macrophage contact with endothelial and mesangial cells (Rimbach et al., 2000, Hisada et al., 2000). In this study, we explored the signaling cascades induced by monocytes that lead to increased secretion of hRPE IL-8 and MCP-1 by hRPE cells. This study demonstrates that activation of p38 kinase, extracellular signal-regulated kinase 1 and 2 (ERK1/2 or p42/44 mitogen activated protein kinases, MAPR), c-Jun N-terminal protein kinase (JNK, also called stress-activated protein kinase (SAPK)) and NF-κB-inducing kinase (NIK) are required for monocyte contact-induced IL-8 and MCP-1 gene expression and protein production by hRPE.

Section snippets

Materials

U0126 was obtained from Promega (Madison, WI, USA). SB202190, SB203580, genistein, herbimycin, and Ro318220 were obtained from Calbiochem (San Diego, CA, USA). Bay 11-7085, CAPE (caffeic acid phenethyl ester) and parthenolide were obtained from BIOMOL (Plymouth Meeting, PA, USA). Unless otherwise specified, the concentrations used in this study for U0126, SB202190, SB203580, herbimycin, Ro318220, and BAY 11-7085 were 20, 30, 10, 10, 10 and 40 μm, respectively; for genistein and CAPE were 25 

Activation of MAP kinases during hRPE-monocyte co-culture

To determine whether MAP kinases are involved in hRPE chemokine production following monocyte: hRPE co-culture, monocytes (106 or 105 cells per well) were overlaid onto hRPE cells at 37°C for 15, 30 and 60 min (Western blotting or kinase assay), 24 hr (ELISA), and 6 hr (mRNA) in the presence or absence of U0126 and SB203580 or SB202190. U0126 is a potent inhibitor of dual specificity kinase MEK, the kinase immediately upstream from ERE1/2 (Garcia et al., 1993). SB203580 and SB202190 are selective

Discussion

Activation of leukocytes and their infiltration into the choroid, retina and vitreous play a critical roles in many ocular diseases, including PVR (Charteris et al., 1992), ARMD (Lopez et al., 1991, Grossniklaus et al., 2002), and patently inflammatory ocular diseases such as uveitis. IL-8 and MCP-1 are likely to be the two major chemokines coordinating the recruitment and activation of leukocytes at the sites of retinal disease since they are responsible for the majority of hRPE-derived

Acknowledgements

Grant support: NIH Grants EY09441, EY007003, and Research to Prevent Blindness-Olga Keith Weiss Award (V.M.E.).

References (70)

  • K. Gewert et al.

    Effects of dexamethasone on mitogen-activated protein kinases in mouse macrophages: implications for the regulation of 85 kDa cytosolic phospholipase A(2)

    Biochem. Pharmacol.

    (2000)
  • H.E. Grossniklaus et al.

    Immunohistochemical properties of surgically excised subretinal neovascular membranes in age-ralated macular degeneration

    Am. J. Ophthalmol.

    (1992)
  • Y. Hisada et al.

    Cell to cell interaction between mesangial cells and macrophages induces the expression of monocyte chemoattractant protein-1 through nuclear factor-kappaB activation

    Biochem. Biophys. Res. Commun.

    (2000)
  • P.F. Lopez et al.

    Pathologic features of surgically excised subretinal neovascular membranes in age-related macular degeneration

    Am. J. Ophthalmol.

    (1991)
  • N.W. Lukacs et al.

    Production of chemokines, interleukin-8 and monocyte chemoattractant protein-1, during monocyte: endothelial cell interactions

    Blood

    (1995)
  • S. Matsuda et al.

    Mechanisms of action of cyclosporine

    Immunopharmacology

    (2000)
  • Y. Miyazaki et al.

    Dexamethasone inhibition of TGF beta-induced cell growth and type II collagen mRNA expression through ERK-integrated AP-1 activity in cultured rat articular chondrocytes

    Osteoarthritis Cartilage

    (2000)
  • T. Ohtsuka et al.

    Glucocorticoid-mediated gene suppression of rat cytokine-induced neutrophil chemoattractant CINC/gro, a member of the interleukin-8 family, through impairment of NF-kappa B activation

    J. Biol. Chem.

    (1996)
  • S. Pece et al.

    Signaling from E-cadherins to the MAPK pathway by the recruitment and activation of epidermal growth factor receptors upon cell–cell contact formation

    J. Biol. Chem.

    (2000)
  • J.W. Pierce et al.

    Novel inhibitors of cytokine-induced IkappaBalpha phosphorylation and endothelial cell adhesion molecule expression show anti-inflammatory effects in vivo

    J. Biol. Chem.

    (1997)
  • G. Rimbach et al.

    Macrophages stimulated with IFN-gamma activate NF-kappa B and induce MCP-1 gene expression in primary human endothelial cells

    Mol. Cell. Biol. Res. Commun.

    (2000)
  • B.J. Rollins et al.

    Recombinant human MCP-1/JE induces chemotaxis, calcium flux, and the respiratory burst in human monocytes

    Blood

    (1991)
  • T. Shalom-Barak et al.

    Interleukin-17-induced gene expression in articular chondrocytes is associated with activation of mitogen-activated protein kinases and NF-kB

    J. Biol. Chem.

    (1998)
  • M. Bryckaert et al.

    Both FGF1 and bcl-x synthesis are necessary for the reduction of apoptosis in retinal pigmented epithelial cells by FGF2: role of the extracellular signal-regulated kinase 2

    Oncogene

    (1999)
  • C.C. Chan et al.

    HLA-DR antigens on retinal pigment epithelial cells rfrom patients with uveitis

    Arch. Ophthalmol.

    (1986)
  • M.A. Collart et al.

    Regulation of tumor necrosis factor alpha transcription in macrophages: involvement of four kappa B-like motifs and of constitutive and inducible forms of NF-kappa B

    Mol. Cell. Biol.

    (1990)
  • K. Drumm et al.

    Soot-exposed mononuclear cells increase inflammatory cytokine mRNA expression and protein secretion in cocultured bronchial epithelial cells

    Respiration

    (2000)
  • S.G. Elner et al.

    Acetoacetylated lipoprotein uptake by retinal pigment epithelium (RPE) cells (abstr)

    Invest. Ophthalmol. Vis. Sci.

    (1984)
  • S.G. Elner et al.

    RPE cell-monocyte binding induced chemokine production is mediated by CD14

    Invest. Ophthalmol. Vis. Sci.

    (2001)
  • S.G. Elner et al.

    Modulation and function of intercellular adhesion molecule-1 (CD54) on human retinal pigment epithelial cells

    Lab. Invest.

    (1992)
  • V.M. Elner et al.

    Neutrophil chemotactic factor (IL-8) gene expression by cytokine-treated retinal pigment epithelial cells

    Am. J. Pathol.

    (1990)
  • S.G. Elner et al.

    Monocyte chemotactic protein gene expression by cytokine-treated human retinal pigment epithelial cells

    Lab. Invest.

    (1991)
  • H.L. Evanoff et al.

    A sensitive ELISA for the detection of human monocyte chemoattractant protein-1 (MCP-1)

    Immunol. Invest.

    (1992)
  • E. Fiorini et al.

    Adhesion of immature and mature T cells induces in human thymic epithelial cells (TEC) activation of IL-6 gene transcription factors (NF-kappaB and NF-IL-6)– and IL-6 gene expression: role of alpha3 betal 1 and alpha6 betal 4 integrins

    Dev.Immunol

    (2000)
  • R. Fukunaga et al.

    MNK1, a new MAP kinase-activated protein kinase, isolated by a novel expression screening method for identifying protein kinase substrates

    EMBO J.

    (1997)
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