The effects of intraocular injection of interleukin-13 on endotoxin-induced uveitis in rats

PURPOSE. Interleukin (IL)-13 is a strong immunomodulatory cytokine that inhibits macrophages from secreting proinflammatory mediators. This study was conducted to investigate the effect of intraocular injection of IL-13 on the development of endotoxin-induced uveitis (EIU) in the Lewis rat. METHODS. One injection into the anterior chamber of recombinant human IL-13 (6 ng in 10 l saline) was performed either simultaneously with a single injection of lipopolysaccharide (LPS) from Salmonella typhimurium into the footpad or 6 hours before the IL-13 injection. EIU was evaluated by slit lamp examination at 6, 16, and 24 hours after LPS injection. Counts of inflammatory cells were performed on cryostat sections after specific immunostaining. Anterior chamber paracentesis was performed, and kinetic analysis of the IL-13 injected in the anterior chamber was performed by ELISA. Cytokine and chemokine gene expression in the iris-ciliary body and the retina was evaluated by reverse transcription-polymerase chain reaction. RESULTS. A significant inhibition of ocular inflammation was observed in IL-13-treated rats at 16 and 24 hours after LPS injection. Unilateral injection of IL-13 inhibited EIU only in the injected eye. High levels of IL-13 were detected in the aqueous humor at 2 hours after local IL-13 injection to remain high up to 18 hours. In contrast, IL-13 was not detected in the corresponding sera. Quantitative analysis of inflammatory cells in ocular tissues showed a significant decrease in OX-42 ϩ cells (microglia, activated macrophages, dendritic cells, and polymorphonuclear leukocytes) and ED1 ϩ cells (monocytes-macrophages and dendritic cells) in treated rats. A decreased expression of TNF-␣, IL-1␤, IL-6, monocyte chemoattractant protein (MCP)-1, and macrophage inflammatory protein (MIP)-2 mRNAs was observed in the iris-ciliary body and the retina from IL-13-treated rats, whereas IFN-␥ was upregulated in the iris-ciliary body. CONCLUSIONS. Injection of IL-13 into the anterior chamber may inhibit the ocular inflammation induced by LPS injection by reducing intraocular cytokine and chemokine mRNA expression in ocular tissues. (Invest Ophthalmol Vis Sci. 2001;42: 2022-2030 E ndotoxin-induced uveitis (EIU) is an animal model of acute ocular inflammation induced in the rat by systemic or local injection of lipopolysaccharide (LPS) from Gram-negative bacterial cell walls. 3,7-9 The injection of endotoxin induces systemic and local ocular inflammatory manifestations that have been partly attributed to the synthesis of proinflammatory cytokines and chemokines. Indeed, systemic injection of LPS stimulates the intraocular production of different cytokines, mainly IL-1, IL-6, IFN-␥, and TNF-␣, 7,10 -14 and chemokines, such as monocyte chemoattractant protein (MCP)-1, a prototype of CC chemokine; IL-8, a prototype of CXC chemokine; macrophage inflammatory protein (MIP)-2, a rat functional IL-8 equivalent; and cytokineinduced neutrophil chemoattractant (CINC), a peptide of the CXC family. IL-13 is an anti-inflammatory cytokine produced by T helper (Th)2 lymphocytes, 20 which inhibits the synthesis of proinflammatory cytokines and chemokines (IL-1, IL-6, TNF-␣, IL-8, and MIP-1␣, a CC chemokine) by LPS-activated monocytes. 21-24 IL-13 has been shown to induce an upregulation of IL-1-receptor antagonist (IL-1ra) 26 The inhibitory effect of IL-13 has been demonstrated in Th1 autoimmune diseases

Protein kinase Czeta (PKCzeta) regulates ocular inflammation and apoptosis in endotoxin-induced uveitis (EIU): signaling molecules involved in EIU resolution by PKCzeta inhibitor and interleukin-13.: Protein kinase C? in endotoxin-induced-uveitis

We show that inhibitory effect of interleukin-13 on endotoxin-induced uveitis in the Lewis rat is dependent on signaling activity of protein kinase Czeta (PKCzeta). To understand the effect of interleukin-13 or PKCzeta inhibitor treatment, the activation status of rat bone marrow-derived macrophages was studied in vitro. At 6 hours, lipopolysaccharide-stimulated macrophages produced tumor necrosis factor-alpha (TNF-alpha) with nuclear factor kappaB (NF-kappaB)/p65 expression. Treatment led to absence of NF-kappaB/p65 expression and low levels of TNF-alpha, suggesting accelerated inactivation of macrophages. At 24 hours after lipopolysaccharide stimulation, nuclear NF-kappaB/p65 decreased and nuclear NF-kappaB/p50 increased, associated with nuclear BCL-3 and a low level of TNF-alpha, indicating onset of spontaneous resolution. Treatment limited PKCzeta cleavage, with expression of nuclear NF-kappaB/p50 and BCL-3 and low nuclear NF-kappaB/p65 promoting macrophage survival, as evidenced by Bcl-2 expression. At 24 hours, intraocular treatment decreased membranous expression of PKCzeta by ocular cells, reduced vascular leakage with low nitric-oxide synthase-2 expression in vascular endothelial cells, and limited inflammatory cell infiltration with decreased intraocular TNF-alpha, interleukin-6, and nitric-oxide synthase-2 mRNA. Importantly, treatment decreased nuclear NF-kappaB/p65, increased transforming growth factor-beta2, and reduced caspase 3 expression in infiltrating macrophages, implying a change of their phenotype within ocular microenvironment. Treatment accelerated endotoxin-induced uveitis resolution through premature apoptosis of neutrophils related to high expression of toll-like receptor 4 and caspase 3

Oligonucleotide-polyethylenimine complexes targeting retinal cells: structural analysis and application to anti-TGFbeta-2 therapy.

PURPOSE: The aim of this study was to characterize oligonucleotide-polyethylenimine (ODN/PEI) complex preparation for potential transfection of retinal cells in vitro and in vivo. METHODS: The effect of medium preparation [HEPES-buffered saline (HBS), water] on particle size and morphology was evaluated. Cultured Lewis rat retinal Müller glial (RMG) cells were transfected using fluorescein isothiocyanate (FITC)-ODN/PEI complexes specifically directed at transforming growth factor beta (TGFbeta)-2. Efficacy of transfection was evaluated using confocal microscopy, and regulation of gene expression was assayed using quantitative real-time RT-PCR and ELISA assay. One, 24, and 72 h after injection of FITC-ODN/PEI complexes into the vitreous of rat eyes, their distribution was analyzed on eye sections. RESULTS: Complexes prepared in HBS were smaller than complexes prepared in pure water and presented a core-shell structure. These particles showed a high cellular internalization efficacy, along with a significant and specific down-regulation of TGFbeta-2 expression and production in RMG cells, correlating with specific inhibition of cell growth at 72 h. In vivo, complexes efficiently transfect retinal cells and follow a transretinal migration at 24 h. After 72 h, ODN seems to preferentially target RMG cells without inducing any detectable toxicity. CONCLUSIONS: Specific down-regulation of TGFbeta-2 expression using ODN/PEI complexes may have potential interest for the treatment of retinal diseases associated with glial proliferation