3-nitrotyrosine and Uveitis

The aim of this study was to investigate the effects of palmitoylethanolamide (PEA), an endogenous fatty acid amide belonging to the family of the N-acylethanolamines (NAEs), in rats subjected to endotoxin-induced uveitis (EIU). EIU was induced in male rats by a single footpad injection of 200μg lipopolysaccharide (LPS). PEA was administered intraperitoneally at 1h before and 7h after injection of LPS. Another group of animals was treated with vehicle. Dexamethasone (DEX) was administered as a positive control. Rats were sacrificed 16h after injection and the eyes tissues were collected for histology, immunohistochemical and western blot analyses. The histological evaluation of the iris-ciliary body showed an increase of neutrophilic infiltration and nuclear modification of vessel of endothelial cells. PEA treatment decreased the inflammatory cell infiltration and improved histological damage of eye tissues. In addition, PEA treatment reduced pro-inflammatory tumor necrosis factor (TNF-α) levels, protein extravasion and lipid peroxidation. Immunohistochemical analysis for intracellular adhesion molecule (ICAM)-1 and nitrotyrosine showed a positive staining from LPS-injected rats. The degree of staining for ICAM-1 and nitrotyrosine was significantly reduced in eye sections from LPS-injected rats treated with PEA. In addition, an increase of inducible nitric oxide synthase (iNOS) and nuclear factor (NF-κB) was also evaluated in inflammed ocular tissues by western blot. PEA strongly inhibited iNOS expression and nuclear NF-κB translocation. Thus, in this study we demonstrated that PEA reduces the degree of ocular inflammation in a rat model of EIU.

Topics: Active Transport, Cell Nucleus; Amides; Animals; Anti-Inflammatory Agents; Dexamethasone; Disease Models, Animal; Endothelial Cells; Ethanolamines; Inflammation Mediators; Intercellular Adhesion Molecule-1; Lipid Peroxidation; Lipopolysaccharides; Male; Malondialdehyde; Neutrophil Infiltration; Nitric Oxide Synthase Type II; Palmitic Acids; Rats, Inbred Lew; Transcription Factor RelA; Tumor Necrosis Factor-alpha; Tyrosine; Uvea; Uveitis

The study investigated the effects of the aldose reductase (AR) inhibitor benzofuroxane derivative 5(6)-(benzo[d]thiazol-2-ylmethoxy) benzofuroxane (herein referred to as BF-5m) on the biochemical and tissue alterations induced by endotoxic uveitis in rats. BF-5m has been administered directly into the vitreous, in order to assess the expression and levels of (i) inflammatory markers such as the ocular ubiquitin-proteasome system, NF-κB, TNF-α, and MCP-1; (ii) prooxidant and antioxidant markers such as nitrotyrosine, manganese superoxide dismutase (MnSOD), and glutathione peroxidase (GPX); (iii) apoptotic/antiapoptotic factors caspases and Bcl-xl; (iv) markers of endothelial progenitor cells (EPCs) recruitment such as CD34 and CD117. 5 μL of BF-5m (0.01; 0.05; and 0.1 μM) into the right eye decreased in a dose-dependent manner the LPS-induced inflammation of the eye, reporting a clinical score 1. It reduced the ocular levels of ubiquitin, 20S and 26S proteasome subunits, NF-κB subunits, TNF-α, MCP-1, and nitrotyrosine. BF-5m ameliorated LPS-induced decrease in levels of MnSOD and GPX. Antiapoptotic effects were seen from BF-5m by monitoring the expression of Bcl-xl, an antiapoptotic protein. Similarly, BF-5m increased recruitment of the EPCs within the eye, as evidenced by CD34 and CD117 antibodies.

Topics: Aldehyde Reductase; Animals; Antioxidants; Apoptosis; Benzofurans; Disease Models, Animal; Enzyme Inhibitors; Eye; Inflammation Mediators; Lipopolysaccharides; Male; Oxidative Stress; Proteasome Endopeptidase Complex; Rats; Rats, Sprague-Dawley; Tyrosine; Ubiquitin; Uveitis

Macrophages infiltrating an inflamed or injured tissue undergo development of coordinated sets of properties that contribute to tissue damage, repair, and remodeling. The purpose of this study was to determine whether macrophages isolated from normal or inflamed retina are programmed to a distinct set of properties and to examine whether the development of experimental autoimmune uveoretinitis (EAU) affects macrophage function.. EAU was induced in Lewis rats, and a retina-derived macrophage-enriched population was generated by density centrifugation during the prepeak, peak, and resolution phases of the disease. Cell surface phenotype was assessed by two- and three-color flow cytometry, and function was determined in vitro by nitric oxide (NO) production, with or without further cytokine stimulation or by immunohistochemistry to determine expression of beta-glucuronidase, nitric oxide synthase (NOS)-2, and nitrotyrosine.. Myeloid-derived cells from normal retina were programmed similar to TGF-beta-stimulated uncommitted bone-marrow-derived macrophages (BMDMs). Contrary to BMDM behavior, retina-isolated macrophages displayed distinct properties and phenotype at different phases of the disease course and remained resistant throughout, to further cytokine challenge in vitro. During peak disease, retina-isolated macrophages had characteristics of IFN-gamma/TNF-alpha primed cells (nitrotyrosine positive and NO producing). Despite equivalent numbers of macrophages during resolution, their function reverted to characteristics of TGF-beta primed cells (beta-glucuronidase positive).. Resident retinal myeloid-derived cells are primed and are resistant to further cytokine stimulation, and, similar to macrophages derived during EAU recovery, behave operationally as though TGF-beta primed. During peak inflammation, infiltrating macrophages adapt to concurrent hierarchical Th1 T-cell response (IFN-gamma/TNF-alpha), generating NO. The results provide evidence of in vivo programming of macrophages within the retina.

Topics: Animals; Autoimmune Diseases; Cytokines; Female; Flow Cytometry; Fluorescent Antibody Technique, Indirect; Glucuronidase; Immunoenzyme Techniques; Immunophenotyping; Macrophage Activation; Macrophages; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Rats; Rats, Inbred Lew; Retina; Retinitis; Tyrosine; Uveitis