Lipopolysaccharide-primed heterotolerant dendritic cells suppress experimental autoimmune uveoretinitis by multiple mechanisms.

Exposure of bone-marrow-derived dendritic cells (BMDC) to high-dose ultrapure lipopolysaccharide for 24 hr (LPS-primed BMDC) enhances their potency in preventing inter-photoreceptor retinoid binding protein: complete Freund's adjuvant-induced experimental autoimmune uveoretinitis (EAU). LPS-primed BMDC are refractory to further exposure to LPS (= endotoxin tolerance), evidenced here by decreased phosphorylation of TANK-binding kinase 1, interferon regulatory factor 3 (IRF3), c-Jun N-terminal kinase and p38 mitogen-activated protein kinase as well as impaired nuclear translocation of nuclear factor κB (NF-κB) and IRF3, resulting in reduced tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-12 and interferon-β secretion. LPS-primed BMDC also show reduced surface expression of Toll-like receptor-4 and up-regulation of CD14, followed by increased apoptosis, mediated via nuclear factor of activated T cells (NFATc)-2 signalling.

A key role for PTP1B in dendritic cell maturation, migration, and T cell activation.

Dendritic cells (DC) are the major antigen-presenting cells bridging innate and adaptive immunity, a function they perform by converting quiescent DC to active, mature DC with the capacity to activate naïve T cells. They do this by migrating from the tissues to the T cell area of the secondary lymphoid tissues. Here, we demonstrate that myeloid cell-specific genetic deletion of PTP1B (LysM PTP1B) leads to defects in lipopolysaccharide-driven bone marrow-derived DC (BMDC) activation associated with increased levels of phosphorylated Stat3.

Culture and characterization of microglia from the adult murine retina.

Purpose: To develop a protocol for isolating and culturing murine adult retinal microglia and to characterize the phenotype and function of the cultured cells.

Method: Retinal single-cell suspensions were prepared from adult MF1 mice. Culture conditions including culture medium, growth factors, seeding cell density, and purification of microglia from the mixed cultures were optimised.

Inhibition of the alternative pathway of complement activation reduces inflammation in experimental autoimmune uveoretinitis.

We have shown previously that complement factor H (CFH) and complement factor B (CFB) are constitutively expressed by retinal pigment epithelial cells and their production is regulated by inflammatory cytokines, suggesting that the alternative pathway (AP) of complement activation might play a role in retinal inflammation. In this study, we further investigated the role of the AP in retinal inflammation using experimental autoimmune uveoretinitis (EAU) as a model. Mice with EAU show increased levels of C3d deposition and CFB expression in the retina.

Immune activation in retinal aging: a gene expression study.

Purpose: To investigate changes in gene expression during aging of the retina in the mouse.

Methods: Total RNA was extracted from the neuroretina of young (3-month-old) and old (20-month-old) mice and processed for microarray analysis. Age-related, differentially expressed genes were assessed by the empiric Bayes shrinkage-moderated t-statistics

Method: Statistical significance was based on dual criteria of a ratio of change in gene expression >2 and a P < 0.

Up-regulation of complement factor B in retinal pigment epithelial cells is accompanied by complement activation in the aged retina.

Complement activation is involved in the pathogenesis of age-related macular degeneration. How complement is activated in the retina is not known. Previously we have shown that complement factor H (CFH) is constitutively expressed by retinal pigment epithelial (RPE) cells and the production of CFH is negatively regulated by inflammatory cytokines and oxidative insults.

Characterization of a spontaneous mouse retinal pigment epithelial cell line B6-RPE07.

Purpose: A spontaneously arising retinal pigment epithelial (RPE) cell line (B6-RPE07) was cloned from a primary culture of mouse RPE cells and maintained in culture for more than 18 months. Morphologic and functional properties of this cell line have been characterized.

Methods: The morphology of the B6-RPE07 cells was examined by phase-contrast light microscopy, electron microscopy, and confocal microscopy.

Immunological responses in mice to full-thickness corneal grafts engineered from porcine collagen.

Tissue-engineered (TE) corneas were fabricated from porcine collagen cross-linked with 1-ethyl-3-(3-dimethyl aminoproplyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS), and were transplanted into BALB/c mice orthotopically using a full-thickness penetrating keratoplasty (PKP) procedure. The biocompatibility was evaluated by assessing both local and systemic immune responses. Myeloid cells including granulocytes and macrophages were the main infiltrating cells in recipient cornea and in retro-TE corneal membrane which developed 7-10 days post surgery.

Interferon-alpha: a key factor in autoimmune disease?

Purpose: Interferon (IFN)-alpha is an effective drug for treatment of uveitis in Behçet's disease. This study was undertaken to investigate the mechanism of action of IFN-alpha in the treatment of various types of noninfectious sight-threatening uveitis.

Methods: Eleven patients with refractory uveitis, and 13 healthy individuals were enrolled.

CD4+CD25+ T regulatory cells induced by LPS-activated bone marrow dendritic cells suppress experimental autoimmune uveoretinitis in vivo.

Background: Tolerance-inducing DC are considered to be less mature than immunogenic DC, but the conditions promoting a less mature DC phenotype are not clear. We have previously shown that lipopolysaccharide (LPS) can have differential effects on DC function depending on the timing of DC exposure to LPS. Here, we show that early LPS-activated bone marrow derived DC (early DC, eDC), when administered subcutaneously to mice in vivo, promote tolerance to EAU induced via immunisation with interphotoreceptor retinol binding protein (IRBP) peptide 161-180.

FTY720 in corneal concordant xenotransplantation.

Background: : Currently, there are no effective treatments for the control of corneal xenograft rejection. We evaluated the efficacy and mode of action of a novel immunosuppressant, FTY720, in a model of corneal xenograft transplantation.

Methods: : Rat-to-mouse corneal xenografts were performed and the effects of treatment with daily intraperitoneal injections of FTY720 (0.

Antigen-specific inhibition of experimental autoimmune uveoretinitis by bone marrow-derived immature dendritic cells.

Purpose: To investigate the effect of maturation status of bone marrow-derived dendritic cells (BMDCs) on the in vivo immune response to interphotoreceptor retinoid-binding protein (IRBP) 161-180 peptide in experimental autoimmune uveoretinitis (EAU).

Methods: Immature and mature BMDCs were generated without or with the stimulation by lipopolysaccharide (LPS), and their mRNA cytokine profile and phenotype were analyzed by RNase protection assay and flow cytometry. The effect of immature and mature DCs in inducing antigen-specific T-cell proliferation and cytokine profile was further investigated in an IRBP peptide-induced model of EAU.

Secretion of interleukin-10 or interleukin-12 by LPS-activated dendritic cells is critically dependent on time of stimulus relative to initiation of purified DC culture.

Dendritic cells (DC) are key regulators of adaptive immunity with the potential to induce T cell activation/immunity or T cell suppression/tolerance. DC are themselves induced by "maturation" signals such as bacterial lipopolysaccharide (LPS). We demonstrate here that LPS can stimulate DC to display similar maturation phenotypes but to differentiate toward an interleukin (IL)-10(high)- or IL-12(high)-secretor profile depending on the timing of maturation signal induction.