Role and regulation of IL-8 and MCP-1 in LPS-induced uveitis in rabbits

Intravitreal injection of lipopolysaccharide (LPS) induces leukocyte infiltration and protein leakage into the aqueous humor. In the present study, we investigated the role of IL-8 and MCP-1 and regulation of these chemokines by TNFalpha and IL-1 in LPS-induced uveitis in rabbits. After intravitreal injection of LPS, generation of IL-8 in the aqueous humor showed a biphasic pattern with the first peak at 12 hr and the second one at 24 hr, while MCP-1 was produced in a monophasic pattern and peaked at 24 hr. Immunohistochemistry showed that ciliary epithelial cells and infiltrating leukocytes were the producing cells of IL-8 and MCP-1. Administration of anti-IL-8 IgG suppressed by 66% the peak levels of LPS-induced aqueous neutrophil counts at 24 hr but did not suppress aqueous mononuclear cell counts or protein levels. anti-MCP-1 IgG inhibited aqueous mononuclear cell counts by 41% and protein levels by 28%, but did not inhibit aqueous neutrophil counts. The levels of LPS-induced aqueous IL-8 and MCP-1 at 12 hr were inhibited by anti-TNFalpha mAb but not by an IL-1 receptor antagonist (IL-1Ra), while concentrations of the two chemokines at 24 hr were inhibited by both anti-TNFalpha mAb and IL-1Ra. A combination of anti-TNFalpha mAb and rrIL-1Ra had an additive effect on the 24 hr-chemokine levels and inhibited up to 90% chemokine production. Taken together, our results show that IL-8 mediates neutrophil infiltration, while MCP-1 mediates mononuclear cell infiltration and protein leakage in LPS-induced uveitis in rabbits. Levels of aqueous IL-8 and MCP-1 at 12 hr are regulated by TNFalpha, while levels at 24 hr are regulated by TNFalpha and IL-1.     

Effect of <Emphasis Type="Italic">N</Emphasis>-Acetylcysteine on Lipopolysaccharide-Induced Uveitis in Rats

Purpose In this study we investigated the in vivo effect of N-acetylcysteine (NAC) on lipopolysaccharide (LPS)-induced uveitis in rats. Methods To induce uveitis, LPS (100 μg) was injected into subcutaneous tissue of Wistar rats (170–190 g). NAC was injected intraperitoneally. Intracameral levels of protein, cells, nitrite, tumor necrosis factor (TNF)-α, and interleukin (IL)-6 were determined by spectrophotometry, hemocytometry, and enzyme-linked immunosorbent assay. Expression of TNF-α, IL-6, endothelial leukocyte adhesion molecule-1 (E-selectin), intercellular adhesion molecule-1 (ICAM-1), and inducible nitric oxide synthase (iNOS) mRNA was examined by real-time polymerase chain reaction. Results LPS injection elevated intracameral protein and cells, and the elevation was inhibited by NAC. LPS injection induced expression of TNF-α, IL-6, E-selectin, and ICAM-1 mRNA in the iris/ciliary body at 3 h, and iNOS mRNA at 6 h. The LPS-induced elevation of the mRNA levels was inhibited by NAC. NAC inhibited LPS-induced intracameral elevation of TNF-α, IL-6, and nitrite. Conclusion NAC decreased LPS-induced uveitis in vivo by reducing the expression of proinflammatory cytokines and adhesion molecules. Jpn J Ophthalmol 2007;51:14–20 ? Japanese Ophthalmological Society 2007     

Endotoxin-induced uveitis in the rat. The significance of intraocular interleukin-6.

The potential role of interleukin-6 (IL-6) was studied as an inflammatory mediator of endotoxin (or lipopolysaccharide [LPS])-induced uveitis (EIU) in the rat. In young Lewis rats, levels of intraocular IL-6, but not serum IL-6, correlated with the severity of uveitis and with aqueous humor protein levels in response to foot pad injections of LPS (P less than 0.001). Adult Lewis rats did not develop uveitis and had no intraocular IL-6, although IL-6 was released systemically. Resistance to EIU and absence of IL-6 levels in the aqueous humor, despite the ability to release serum IL-6, also were observed in brown Norway rats, irrespective of age and weight. Intravitreal injection of as little as 1 ng of human recombinant IL-6 induced uveitis in young Lewis rats. In adult Lewis rats, and in young animals made tolerant to LPS, intravitreal IL-6 still caused substantial leakage of plasma proteins into the anterior chamber but no influx of inflammatory cells. As early as 2 hr after intravitreal injection of IL-6, immunohistochemical analysis showed invasion of the iris, corneal stroma, and anterior chamber by polymorphonuclear leukocytes (PMN) and expression of major histocompatibility complex (MHC) class II antigen in the retina by large cells that were macrophage-marker ED2 negative. This was followed by massive PMN infiltration of the retinal layers and vitreous. The MHC class II antigen expression of ciliary and iris epithelium occurred at a later stage (greater than 8 hr).(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition by triptolide of chemokine, proinflammatory cytokine, and adhesion molecule expression induced by lipopolysaccharide in corneal fibroblasts

PURPOSE: The production of proinflammatory cytokines and chemokines as well as the surface expression of intercellular adhesion molecule (ICAM)-1 by corneal fibroblasts contribute to corneal inflammation. The effects of triptolide on the expression of these proteins induced by lipopolysaccharide (LPS) in human corneal fibroblasts were examined in comparison with those of dexamethasone. METHODS: The release of interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, IL-6, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), monocyte chemotactic protein (MCP)-1, macrophage inflammatory protein (MIP)-1beta, and IL-8 from cultured corneal fibroblasts was measured with assay kits. Surface expression of ICAM-1 on the cultured cells was measured with a whole-cell enzyme-linked immunosorbent assay. RESULTS: Lipopolysaccharide (LPS) induced the release of the proinflammatory cytokine IL-6 and that of the chemokines G-CSF, MCP-1, MIP-1beta, and IL-8 as well as surface expression of ICAM-1 by corneal fibroblasts, whereas IL-1beta, TNF-alpha, and GM-CSF were not detected in the culture supernatants of cells incubated with or without LPS. Triptolide and dexamethasone each inhibited in a concentration-dependent manner the LPS-induced release of IL-6, G-CSF, MCP-1, and IL-8 by corneal fibroblasts. Whereas the inhibitory effect of dexamethasone on LPS-induced IL-6 release was greater than that of triptolide, the inhibitory effect of triptolide on LPS-induced G-CSF release was more pronounced than was that of dexamethasone. Dexamethasone also inhibited LPS-induced MIP-1beta release, whereas triptolide did not. Both compounds inhibited the LPS-induced surface expression of ICAM-1. CONCLUSIONS: Triptolide inhibits the LPS-induced expression of IL-6, chemokines (G-CSF, MCP-1, IL-8), and ICAM-1 in cultured human corneal fibroblasts. This compound might thus be expected to limit the infiltration of immune cells into the cornea.     

Effects of rolipram, a selective inhibitor of type 4 phosphodiesterase, on lipopolysaccharide-induced uveitis in rats

PURPOSE: To investigate effects of rolipram, an inhibitor of type 4 phosphodiesterase, on lipopolysaccharide (LPS)-induced uveitis in Wistar rats. METHODS: A total of 100 microg LPS was injected into the rat footpad. Rolipram (Wako Pure Chemical, Osaka, Japan) was injected intraperitoneally 30 minutes before administration of LPS. Levels of intracameral protein, cells, E-selectin, tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and nitrite were determined. E-selectin, TNF-alpha, IL-6, and inducible nitric oxide synthase (iNOS) mRNAs and immunohistochemical reactivity of nuclear factor (NF)-kappa B and TNF-alpha were also examined in the iris-ciliary body. RESULTS: After LPS injection, intracameral protein and cells increased from 18 to 30 hours later. Rolipram, however, inhibited elevation of protein and cells. After LPS injection, mRNA levels of E-selectin, TNF-alpha, and IL-6 in the iris-ciliary body increased 3 hours later, and iNOS mRNA increased 6 hours later. Elevation of mRNA levels for E-selectin, TNF-alpha, and IL-6 was inhibited by rolipram. After LPS injection, intracameral TNF-alpha and IL-6 levels increased 4 to 6 hours later, and nitrite levels increased 14 to 20 hours later. Elevation of TNF-alpha and IL-6 levels was decreased by rolipram. Rolipram did not affect iNOS mRNA and nitrite levels. Immunoreactivity of NF-kappa B was strong 1 hour after LPS injection, and was decreased by rolipram. Immunoreactivity of TNF-alpha was strong 4 hours after LPS injection and was decreased by rolipram. CONCLUSIONS: NF-kappa B translocation and expression of E-selectin, TNF-alpha, and IL-6 are involved in the pathogenesis of LPS-induced uveitis and are inhibited by rolipram. The inhibitory effect of rolipram in uveitis may be independent of iNOS synthesis.     

Effects of cloricromene,a coumarin derivative,on endotoxin-induced uveitis in Lewis rats

PURPOSE: To investigate the effects of cloricromene, a coumarin derivative, in rats subjected to endotoxin-induced uveitis (EIU). METHODS: Endotoxin uveitis was induced in male Lewis rats by a single footpad injection of 200 microg lipopolysaccharide (LPS). Cloricromene was topically applied to the rat eye twice at 1 hour before and 7 hours after injection of LPS. A separate group of animals was treated with vehicle. Rats were killed 16 hours after injection and the eyes enucleated for histologic examination and immunohistochemical analysis. The effect of treatment was also evaluated by slit lamp examination, by the number of intraocular inflammatory cells on histologic sections, and by measuring the protein and TNFalpha levels in the aqueous humor. Nitrite and nitrate production was also measured in the aqueous humor. RESULTS: The histopathology of the iris-ciliary body included inflammatory cell infiltration and nuclear modification of vessel endothelial cells. Cloricromene treatment reduced the inflammatory cell infiltration and improved histologic status of the ocular tissue. Immunohistochemical analysis for P-selectin, intracellular adhesion molecule (ICAM)-1, nitrotyrosine, and poly(ADP-ribose) synthetase (PARS) revealed a positive staining in inflammatory cell infiltration from LPS-treated rats. The degree of staining for P-selectin, ICAM-1, nitrotyrosine, and PARS was markedly reduced in tissue sections obtained from LPS-recipient rats that had received cloricromene. Cloricromene strongly inhibited cell infiltration, protein exudation, TNFalpha production, and nitrite-nitrate formation. CONCLUSIONS: This study provides the first evidence that cloricromene, a coumarin derivative, attenuates the degree of inflammation and tissue damage associated with EIU in rats.     

Effects of scutellariae radix extract and its components (baicalein, baicalin, and wogonin) on the experimental elevation of aqueous flare in pigmented rabbits

PURPOSE: To evaluate the possible inhibitory effects of hot water extract of Scutellariae radix and its major components (baicalein, baicalin, and wogonin) on experimental elevation of aqueous flare in pigmented rabbits. METHODS: To produce aqueous flare elevation in rabbits, prostaglandin E(2) (PGE(2)), 25 microg/mL, was applied to the cornea with the use of a glass cylinder, or lipopolysaccharides (LPS), 0.5 microg/kg, were injected into an ear vein. Animals were pretreated by the oral administration of 150 g/day of food containing 0.02%, 0.07%, or 0.2% (w/w) extract of Scutellariae radix for 5 days, or by intravenous injection of baicalein, baicalin, or wogonin, 60 microg/kg or 600 microg/kg, 30 minutes before experimental uveitis was induced. Aqueous flare was measured with a laser flare-cell meter. Aqueous flare intensity was expressed as the area under the curve (AUC) in arbitrary units. RESULTS: The AUC of PGE(2)- and LPS-induced aqueous flare elevation was 1,343 and 5,066 arbitrary units, respectively. Pretreatment by oral administration of 0.07% or 0.2% extract of Scutellariae radix did not inhibit PGE(2)-induced aqueous flare elevation (AUC: 1,252 and 1,210, respectively), but it did inhibit LPS-induced aqueous flare elevation (AUC: 2,248 and 1,973, respectively). Pretreatment by intravenous injection of 600 microg/kg of baicalein, baicalin, or wogonin inhibited LPS-induced aqueous flare elevation (AUC: 2,289, 2,163, and 1,509, respectively). Pretreatment with 60 microg/kg of wogonin also inhibited LPS-induced aqueous flare elevation (AUC: 1,980). CONCLUSION: Hot water extract of Scutellariae radix may have an inhibitory effect on experimental anterior uveitis induced by LPS in pigmented rabbits.     

Intraocular production of a cytokine (CINC) responsible for neutrophil infiltration in endotoxin induced uveitis.

AIMS/BACKGROUND: The subcutaneous injection of bacterial endotoxin in Lewis rats produces an acute intraocular inflammation evolving over a 24 hour period. This endotoxin induced uveitis (EIU) is characterised by a biphasic protein exudation and a cellular infiltrate composed of macrophages and polymorphonuclear neutrophils (PMNs). This model was used to study the mechanism of cellular infiltration in ocular inflammation. METHODS: EIU was induced by a subcutaneous injection of lipopolysaccharide (LPS) (S typhimurium) at 350 micrograms/kg. The levels of cytokine induced neutrophil chemoattractant (CINC) were measured every 2 hours in the serum and in the aqueous humour by ELISA. The intraocular inflammation was quantified by protein measurement and leucocyte counting. RESULTS: The kinetics of CINC production in the systemic circulation showed a rapid rise, peaking 2 hours after LPS injection, followed by a progressive decline over the next 8 hours. In the eye, the CINC levels increased above the serum levels 10 hours after EIU induction corresponding to the time of cellular infiltration. When leucocyte entry in the eye was inhibited by 56% and 64% with an antiadhesion molecule antibody, there was only a slight reduction in the aqueous humour CINC levels of 9% and 16%, respectively, indicating that CINC was produced by ocular tissue cells. The specific effect of CINC in the eye was confirmed when a direct intraocular injection of 250 ng of purified CINC was followed by significant PMN infiltration, in the absence of protein exudation. CONCLUSION: The data indicate that the production of the CINC chemotactic factor by ocular tissue participates in the inflammatory reaction in EIU.     

A cholinergic agonist attenuates endotoxin-induced uveitis in rats

PURPOSE: Investigation of physiological anti-inflammatory mechanisms can contribute to the treatment of inflammatory disorders. The purpose of the present study was to investigate the effect of nicotine, a selective cholinergic agonist, on endotoxin-induced uveitis (EIU) in rats and the underlying molecular mechanism. METHODS: Lipopolysaccharide (LPS; endotoxin) and nicotine were injected intraperitoneally. Clinical scores were evaluated by slit lamp. Intracameral protein content and the number of cells were determined. Immunohistochemical reactivity of alpha7 nicotine acetylcholine receptor (alpha7nAChR) was examined in the iris and ciliary body (ICB). mRNA and protein levels of cytokines and chemokines were measured by real-time PCR and enzyme-linked immunosorbent assay. RESULTS: After LPS injection, clinical scores, as well as protein content and number of cells in the aqueous humor increased during 18 to 36 hours. Nicotine inhibited the endotoxin-induced elevation of these levels. mRNA and protein of alpha7nAChR expression levels were significantly increased by LPS and/or nicotine injection. Nicotine showed no effects on endotoxin-induced elevation of mRNA levels in ICB. However, nicotine decreased the endotoxin-induced elevation of interleukin (IL)-6, IL-1beta, tumor necrosis factor (TNF)-alpha, cytokine-induced neutrophil chemoattractant (CINC)-1, and monocyte chemotactic protein (MCP)-1, but did not affect IL-10 in the serum and aqueous humor. CONCLUSIONS: Nicotine attenuated endotoxin-induced uveitis through directly decreasing the levels of multiple cytokines and chemokines in the aqueous humor, but did not affect the mRNA levels of these factors. The findings suggest that the nicotinic anti-inflammatory pathway may be involved in the pathogenesis of EIU.     

Multiplex bead immunoassay analysis of aqueous humor reveals distinct cytokine profiles in uveitis

PURPOSE: To extensively characterize the complex network of cytokines present in uveitis aqueous humor (AqH), and the relationships between cytokines and the cellular infiltrate. METHODS: AqH from noninflammatory control subjects and patients with idiopathic, Fuchs' heterochromic cyclitis (FHC), and herpes-viral or Beh?et's uveitis were analyzed for IL-1beta, -2, -4, -5, -7, -8, -10, -12, -13, -15, TNFalpha, IFNgamma, CCL2 (MCP-1), CCL5 (RANTES), CCL11 (Eotaxin), TGFbeta2, and CXCL12 (SDF-1), using multiplex bead immunoassays. The cellular infiltrate was also determined for each sample. RESULTS: Idiopathic uveitis AqH, compared with noninflammatory controls, was characterized by high levels of IL-6, IL-8, CCL2 and IFNgamma, the levels of which correlated with each other. For IL-6 and IL-8 these levels were proportional to the number of neutrophils present. By contrast, the levels of both TGFbeta2 and CXCL12 decreased in idiopathic uveitis AqH with increasing inflammation. Cluster analysis showed a degree of segregation between noninflammatory and idiopathic uveitis AqH. Further examination using random forest analysis yielded a complete distinction between these two groups. The minimum cytokines required for this classification were IL-6, IL-8, CCL2, IL-13, TNFalpha, and IL-2. CONCLUSIONS: Application of multiplex bead immunoassays has allowed us to identify distinct patterns of cytokines that relate to both clinical disease and the cellular infiltrates present. Bioinformatics analysis allowed identification of cytokines that differentiate idiopathic uveitis from noninflammatory control AqH and are likely to be important for the pathogenesis of uveitis.     

Synergistic uveitic effects of tumor necrosis factor-alpha and interleukin-1 beta.

Tumor necrosis factor (TNF) and interleukin-1 (IL-1), cytokines with multiple, overlapping biologic activities, have been shown to interact synergistically in nonocular tissues. To test the hypothesis that coinjection of TNF and IL-1 interact synergistically in the eye, low, marginally inflammatory doses of human recombinant TNF-alpha (4000 U), IL-1 beta (40 U), and TNF-alpha+IL-1 beta (TNF-alpha/IL-1 beta) were injected into the vitreal chamber of the rabbit eye, and inflammation was assessed at 6, 24, 48, and 168 hr post-cytokine injection. TNF-alpha/IL-1 beta induced an anterior uveitis that was barely detectable at 6 hr, increased at 24 hr, peaked at 48 hr, and largely resolved by 168 hr. Synergy was observed for infiltration of inflammatory leukocytes into aqueous humor at 24 and 48 hr and for protein and prostaglandin E levels in aqueous humor at 48 hr. Based upon protein levels in vitreous humor, TNF-alpha/IL-1 beta also induced a posterior uveitis. This posterior uveitis was not apparent until 48 hr and then increased significantly at 168 hr. At 48 and 168 hr, the effects of TNF-alpha/IL-1 beta on protein levels in vitreous humor were consistent with a synergistic interaction. Results of separate experiments using higher dose combinations of TNF-alpha/IL-1 beta and a longer time course suggested that the effects of TNF-alpha/IL-1 beta on the blood vitreous barrier persisted beyond 168 hr. The results of this study support the hypothesis that TNF-alpha and IL-1 beta interact synergistically when injected into the rabbit eye.(ABSTRACT TRUNCATED AT 250 WORDS)     

Murine endotoxin-induced uveitis, but not immune complex-induced uveitis, is dependent on the IL-8 receptor homolog.

PURPOSE: To determine the roles of the murine interleukin-8 receptor homolog (mIL-8Rh, neutrophil chemokine CXC receptor 2) and macrophage inflammatory protein-1alpha (MIP-1alpha, a CC chemokine) in two eye inflammation models: endotoxin-induced uveitis (EIU) and immune complex-induced uveitis (reverse passive Arthus reaction (RPAR) uveitis). METHODS: For the EIU model, 250 ng E.coli endotoxin was injected into the vitreous of mIL-8Rh-/- mice or heterozygous littermate mIL-8Rh+/- controls and into MIP-1alpha-/- mice or congenic MIP-1alpha+/+ controls. Eyes were harvested after 24 h for histologic characterization of infiltrating cells and IL-6 bioassays. For the RPAR model, mouse antiserum against human serum albumin (HSA) was injected into the vitreous of mIL-8Rh-/-, mIL-8Rh+/-, MIP-1alpha-/-, and MIP-1alpha+/+ mice. Twenty-four hours later, animals were challenged with intravenous HSA. Eyes were harvested after 4 h for analysis. RESULTS: RPAR resulted in the deposition of immune complexes at the ciliary area and iris with the subsequent development of uveitis. Genetic deficiency of mIL-8Rh reduced the median number of infiltrating cells in EIU by 63% (p < 0.01) but had no effect on RPAR-induced inflammation. In the EIU model, macrophages comprised a much higher percentage (45%) of infiltrating cells in mice lacking mIL-8Rh than in controls (17%). Loss of the MIP-1alpha gene had no apparent effect on RPAR uveitis and a 39% reduction of infiltrating cells in EIU that was not statistically significant. IL-6 activity in aqueous humor was much less in mice with RPAR uveitis than in those with EIU. Neither gene deletion had a significant impact on IL-6 levels in either disease model. CONCLUSIONS: Chemokines acting via mIL-8Rh have a significant role in the induction of neutrophil infiltration during EIU but not during RPAR uveitis. MIP-1alpha is not critical for either EIU or RPAR-induced uveitis. The differential dependence on IL-8-like chemokines is in accord with the two forms ofuveitis having different etiologies and, therefore, potentially different optimal therapies.     

Systemic CD4(+) T cell phenotype and activation status in intermediate uveitis

AIM: To investigate peripheral blood lymphocyte phenotype in patients with intermediate uveitis using CD69, chemokine receptor, and cytokine expression. METHODS: Peripheral blood lymphocytes of 18 patients with idiopathic intermediate uveitis and 6 patients with presumed sarcoid intermediate uveitis were evaluated for CD4(+) T cell expression of CD69, CCR4, CCR5, CXCR3 and the intracellular cytokines IFNgamma, TNFalpha, and interleukin (IL)-10 by flow cytometry, and for IL-2, IL-4, IL-5, IL-10, IFNgamma, and TNFalpha production following unstimulated and activated culture using cytokine bead array and compared with healthy control subjects. RESULTS: The expression of CD69 and TNFalpha by peripheral blood CD4(+) lymphocytes of patients with idiopathic intermediate uveitis and presumed sarcoid intermediate uveitis was significantly higher than control subjects (p = 0.002 and p<0.05, respectively). The ratios of the concentrations of IL-2:IL-5 and IFNgamma:IL-5 in supernatants of activated peripheral blood lymphocyte cultures were significantly higher in patients with presumed sarcoid intermediate uveitis than control subjects. CONCLUSIONS: This study implicates TNFalpha in the pathogenesis of intermediate uveitis, highlighting the potential role of anti-TNF treatments for this disease. Studies of Th1:Th2 cytokine ratios suggested polarisation of the immune response towards Th1 in presumed sarcoid intermediate uveitis despite clinically quiescent systemic disease.     

Comparison of uveitis induced by interleukin-8 (IL-8) and endotoxin in rabbits

IL-8 is a potent chemoattractant which has been postulated to play a role in the cytokine cascade associated with uveitis. The authors studied the effect of intravitreal IL-8 on the induction of uveitis in the rabbit. IL-8 at varying concentrations (1 ng, 10 ng or 100 ng) or endotoxin (100 ng) was injected intravitreally within the rabbit eye. At 6, 24 and 48 hours following injection the induction of uveitis was evaluated by clinical scoring, anterior chamber (AC) leukocyte count, AC protein concentration and histopathology in 15 rabbits. Only the 100 ng concentration of IL-8 induced uveitis at 6 and 24 hours by clinical scoring and AC leukocyte count; the AC protein concentration remained normal. In contrast, endotoxin caused a severe uveitis with a significant increase in all the parameters evaluated. The authors conclude that intravitreal IL-8, in the concentrations studied, induces a limited uveitis which is detectable at six hours and resolves within 48 hours. It is characterized by leukocyte infiltration without an increased AC protein concentration. Thus, IL-8 may play a role in the cytokine cascade involved in the induction of uveitis.     

Comparison of uveitis induced by interleukin-8 (IL-8) and endotoxin in rabbits

IL-8 is a potent chemoattractant which has been postulated to play a role in the cytokine cascade associated with uveitis. The authors studied the effect of intravitreal IL-8 on the induction of uveitis in the rabbit. IL-8 at varying concentrations (1 ng, 10 ng or 100 ng) or endotoxin (100 ng) was injected intravitreally within the rabbit eye. At 6, 24 and 48 hours following injection the induction of uveitis was evaluated by clinical scoring, anterior chamber (AC) leukocyte count, AC protein concentration and histopathology in 15 rabbits. Only the 100 ng concentration of IL-8 induced uveitis at 6 and 24 hours by clinical scoring and AC leukocyte count; the AC protein concentration remained normal. In contrast, endotoxin caused a severe uveitis with a significant increase in all the parameters evaluated. The authors conclude that intravitreal IL-8, in the concentrations studied, induces a limited uveitis which is detectable at six hours and resolves within 48 hours. It is characterized by leukocyte infiltration without an increased AC protein concentration. Thus, IL-8 may play a role in the cytokine cascade involved in the induction of uveitis     

Effects of alpha(2)-adrenergic agonists on lipopolysaccharide-induced aqueous flare elevation in pigmented rabbits

PURPOSE: To evaluate the effects of the alpha(2)-adrenergic agonists (clonidine, apraclonidine, and guanfacine) on lipopolysaccharide (LPS)-induced aqueous flare elevation in pigmented rabbits. METHODS: Anterior uveitis was induced with an intravenous injection of LPS (0.5 microg/kg) in an ear vein. The reproducibility of experimental uveitis induced by LPS (0.5 microg/kg) was also determined. Clonidine (0.01, 0.05, 0.25, or 1%), apraclonidine (1%), or guanfacine (1%) was topically instilled in the right eye 30 and 5 minutes before and 30 minutes after LPS application (N = 6 animals, respectively). Clonidine (0.25%) was topically administered three times at 30-minute intervals from 240 or 120 minutes before, or 120 or 240 minutes after LPS application (N = 6 animals, respectively). Then 1 mg/kg of yohimbine was injected into an ear vein 30 minutes before each topical three-time instillation of clonidine 1%, apraclonidine 1% or guanfacine 1% (N = 6 animals, respectively). Aqueous flare was measured with a laser flare-cell meter. Aqueous flare elevation was expressed as the area under the curve (AUC) in arbitrary units. Rabbits received the first LPS intravenous injection, and the control values of the AUC were obtained. Three months later, the alpha(2)-agonist and the second LPS administration were given to the same animals. RESULTS: The AUCs (5,184 +/- 1,255 units) after the first application of LPS were similar to those (5,033 +/- 1,290) after the second application 3 months after the first administration. Topical instillation of clonidine inhibited LPS-induced aqueous flare elevation in a dose-dependent manner (0.01-0.25%). Topical instillation of clonidine 1%, apraclonidine 1% or guanfacine 1% inhibited LPS-induced aqueous flare elevation by 98 +/- 2.0% (mean +/- SD), 86 +/- 14% and 94 +/- 5.7%, respectively. Pretreatment with intravenous yohimbine prevented the inhibitory effect on flare elevation induced by each agent. CONCLUSION: The present findings suggested that topical instillation of some alpha(2)-agonists may have an inhibitory effect on ocular inflammation, which is mediated in part by alpha(2)-receptors.     

Characterization of T lymphocyte subtypes in endotoxin-induced uveitis and effect of pentoxifylline treatment

PURPOSE: The aims of the study were twofold: 1) to investigate the role of T lymphocyte subtypes in the pathogenesis of endotoxin-induced uveitis (EIU) and 2) to study the possible beneficial effect of pentoxifylline, an inhibitor of neutrophil motility, and Tumor Necrosis Factor-alpha on this disease. METHODS: Forty-two inbred male Lewis rats were divided into seven equal groups. 200 microg of Escherichia coli 055: B55 lipopolysaccharide (LPS) was injected in one hind footpad of the Group 2, 3, 4, 5, 6, and 7 rats. Group 5, 6, and 7 rats also received concomitant intraperitoneal pentoxifylline (PTX) during food pad injection of LPS. Group 1 rats were used as controls with intra-peritoneal normal saline injection. Eight, 24, and 48 hours after treatment, the rats were euthanized. Neutrophil leukocyte, mononuclear cells, and CD4+, CD8+, and CD45RA+ cell infiltration in the anterior uveal tissue were determined either by hematoxylin-eosin or monoclonal antibody staining. Tumor Necrosis Factor-alpha (TNF-alpha) levels were also measured in the aqueous and blood samples. We compared the numbers of infiltrating cells in the different groups. RESULTS: We found that peak infiltration of lymphocyte, neutrophils, and CD4+ cells occurred at 24 hours. However, CD8+ and CD45RA+ cell number reached their highest levels at 48 hours. There was no inflammatory cell infiltration in the control rats. Concomitant pentoxifylline treatment did not affect any of these parameters, although it effectively reduced TNF-alpha concentrations in the anterior chamber and the serum. CONCLUSION: We conclude that, 1) T lymphocytes might be involved in the pathogenesis of endotoxin-induced uveitis. 2) The potential role of pentoxifylline in the treatment of human uveitis is questionable. However, these are initial findings and need confirmation by additional studies.     

Suppressive effects of histamine H1 receptor antagonist diphenhydramine on the leukocyte infiltration during endotoxin-induced uveitis.

Histamine has been shown to play an important role in the step of leukocyte rolling, the initial step to leukocyte infiltration into an inflamed region. We investigated the roles of histamine in the leukocyte recruitment during endotoxin-induced uveitis (EIU) in vivo using acridine orange digital fluorography. An injection of histamine into the vitreous cavity of a Lewis rat induced leukocyte rolling along the major retinal veins. In other experiments, EIU was induced in Lewis rats by footpad injection of lipopolysaccharide (LPS). Leukocyte rolling was also observed in the retinal veins of EIU rats. To block the histamine H1 receptor, diphenhydramine (DPH) was administered intraperitoneally 15 min before the LPS injection. DPH significantly inhibited leukocyte rolling along the major retinal veins of EIU rats, suppressing leukocyte infiltration into the vitreous cavity. The vasodilation in EIU was also significantly suppressed with DPH. Moreover, leukocyte infiltration into aqueous humor was significantly suppressed in DPH-treated rats. Although the inhibitory effects of DPH was less obvious at later time points, addition of DPH every 12 hr showed prolonged anti-inflammatory effects up to 48 hr after LPS injection. In contrast, protein leakage into the aqueous humor was not suppressed as much as leukocyte infiltration with DPH. These results suggest that histamine would play a pivotal role in leukocyte recruitment during EIU in rats. Blocking the histamine H1 receptor might help to prevent or minimize leukocyte infiltration in uveitis.     

Effects of L-NAME and timolol on aqueous IL-1beta,IL-6, IL-8, TNF-alpha and NO levels after Nd:YAG laser iridotomy in rabbits

PURPOSE: Pro-inflammatory cytokines are produced by tissues and play a vital role in the host inflammatory response and uveitis. Nitric oxide (NO) can be produced in large amounts as a response to experimentally-induced uveitis or cytokines. In this study, we measured the levels of pro-inflammatory cytokines such as interleukin (IL)-1beta, IL-6, IL-8, tumor necrosis factor (TNF)-alpha and free-radical in aqueous humor after Nd:YAG laser iridotomy in rabbits, and investigated whether timolol maleate an anti-glaucoma drug, or a NO synthase inhibitor, NG-nitro-L-arginine methyl esther (L-NAME) had an inhibitory effect on these molecules, since L-NAME is a known anti-inflammatory agent in rabbits. METHODS: Bilateral experimental Nd:YAG laser iridotomy (power 7.5 mJ, mode single burst, aiming beam 4) was performed on 18 rabbits under general plus topical anesthesia. Aqueous humor samples were taken by clear corneal paracentesis preoperatively, and 1 and 24 h postoperatively. Six rabbits (12 eyes) were given bilateral topical timolol maleate 0.5% (Timoptic) drop b.i.d. (group 1), six rabbits (12 eyes) received bilateral 0.1 ml subconjuntival injections of L-NAME (150 mg/kg) (group 2), and six rabbits (12 eyes) were treated with topical balanced salt solution (BSS) b.i.d. (control). RESULTS. Preoperative cytokine and NO levels were comparable in the three groups, with no significant differences. In addition, there was no significant difference in baseline cytokine levels between the right and left eyes. In all groups, pre- and postoperative mean IL-1beta levels were below the detection limit of the assay (<5.0 pg/ml). In the control group, postoperative mean IL-6, IL-8 and NO levels were significantly higher after Nd:YAG laser iridotomy than before (for each, p < 0.01). Timolol and L-NAME both inhibited the rise in IL-8 and TNF-alpha levels. Timolol also inhibited the rise in IL-6 but not NO. L-NAME had an inhibitory effect against NO, but not IL-6. CONCLUSIONS: L-NAME has an inhibitory effect on IL-8, TNF-alpha and NO, but not on IL-6. Timolol had inhibitory effects on IL-6, IL-8 and TNF-alpha, but not on NO. These preliminary experimental results might help in assssing the effect of Nd:YAG laser iridotomy in aqueous humor, and to understand the inhibitory effects of timolol and L-NAME against these molecules.