Serum anti-carbonic anhydrase antibodies and oxidant-antioxidant balance in patients with acute anterior uveitis.

PURPOSE: To study the existence of anti-carbonic anhydrase antibodies (anti-CA-I&II) in acute anterior uveitis (AAU) patients and to analyze the relationship between the levels of these antibodies and the total antioxidant capacity (TAC), total oxidant capacity (TOC), oxidative stress index (OSI), and malondialdehyde (MDA) level. METHODS: Forty-five AAU cases and 43 healthy controls were enrolled in this prospective study. RESULTS: The average anti-CA I and II antibody levels were 0.433 ± 0.306 and 0.358 ± 0.261 IU/mL, respectively, in the AAU group and 0.275 ± 0.147 and 0.268 ± 0.108 IU/mL, respectively, in the control group (p = 0.004 and p = 0.036, respectively). In addition, it was found that the TOC, OSI, and MDA levels in the AAU subjects were statistically significantly higher than those of the control subjects. CONCLUSIONS: These results suggest that autoimmune responses against CA I and CA II and an altered serum oxidant-antioxidant balance may be involved in the pathogenesis of AAU.

Lack of association between the protein tyrosine phosphatase non-receptor type 22 R263Q and R620W functional genetic variants and endogenous non-anterior uveitis.

OBJECTIVE: Endogenous uveitis is a major cause of visual loss mediated by the immune system. The protein tyrosine phosphatase non-receptor type 22 (PTPN22) gene encodes a lymphoid-specific phosphatase that plays a key role in T-cell receptor (TCR) signaling. Two independent functional missense single nucleotide polymorphisms (SNPs) located within the PTPN22 gene (R263Q and R620W) have been associated with different autoimmune disorders. We aimed to analyze for the first time the influence of these PTPN22 genetic variants on endogenous non-anterior uveitis susceptibility. METHODS: We performed a case-control study of 217 patients with endogenous non-anterior uveitis and 718 healthy controls from a Spanish population. The PTPN22 polymorphisms (rs33996649 and rs2476601) were genotyped using TaqMan allelic discrimination assays. The allele, genotype, carriers, and allelic combination frequencies were compared between cases and controls with χ(2) analysis or Fisher's exact test. RESULTS: Our results showed no influence of the studied SNPs in the global susceptibility analysis (rs33996649: allelic P- value=0.92, odds ratio=0.97, 95% confidence interval=0.54-1.75; rs2476601: allelic P- value=0.86, odds ratio=1.04, 95% confidence interval=0.68-1.59). Similarly, the allelic combination analysis did not provide additional information. CONCLUSIONS: Our results suggest that the studied polymorphisms of the PTPN22 gene do not play an important role in the pathophysiology of endogenous non-anterior uveitis.

Anti-inflammatory effects of specific cyclooxygenase 2,5-lipoxygenase, and inducible nitric oxide synthase inhibitors on experimental autoimmune anterior uveitis (EAAU).

PURPOSE: Inflammation, in general, causes the release of a variety of inflammatory mediators that in turn induce cyclooxygenase (COX) 2, nitric oxide synthase (iNOS) and 5-lipoxygense (LP) synthesis, producing large amounts of inflammatory prostaglandins (PG), nitric oxide (NO), and leukotriene (LT) B4. Therefore, inhibition of these enzymes may abrogate intraocular inflammation in experimental autoimmune anterior uveitis (EAAU). METHODS: Lewis rats were immunized with melanin-associated antigen (MAA) isolated from bovine iris and ciliary body. These animals were divided into three groups. The first group of rats received subcutaneous injection of COX 2 inhibitor CS 236 at different time points. The second and third groups of animals received subcutaneous aminoguanidine (AG), an iNOS inhibitor, and nordihydroguaiaretic acid (NDGA), a 5-LP inhibitor, respectively. Control animals received vehicle. Rat eyes were examined daily by slit-lamp biomicroscopy from Day 7 to 30 post injection for uveitis. Animals were also sacrificed at various time points for histologic analysis. RESULTS: Control animals developed severe EAAU in both eyes. The disease started in these animals on Day 12 post immunization and lasted for ten days. Interestingly, CS 236, a potent COX 2 inhibitor, completely abrogated EAAU when the animals were treated daily from the Day 0 to 14 or Day 0 to 20 after MAA injection. Furthermore, daily CS 236 treatment after the onset of EAAU (Day 14-20) significantly reduced the severity (both clinical and histologic) of EAAU and shortened the duration of disease. iNOS inhibitor (AG) and 5-LP inhibitor (NDGA) partially attenuated EAAU. CONCLUSIONS: Our results show that EAAU was partially attenuated by AG and NDGA. Interestingly, CS 236, a potent COX 2 inhibitor, completely inhibited EAAU in male Lewis rats most likely by inhibiting the initial phase and onset of the disease.

Anti-inflammatory effects of aronia extract on rat endotoxin-induced uveitis.

PURPOSE: Aronia crude extract (ACE) with high levels of polyphenol compounds has been reported to have antioxidative effects in vitro and in vivo. In this study, attention was focused on the antioxidant effect of ACE. The purpose of the present study was to investigate the effect of ACE on endotoxin-induced uveitis (EIU) in rats. In addition, the endotoxin-induced expression of the inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 proteins was investigated in a mouse macrophage cell line (RAW 264.7) treated with ACE in vitro, to clarify the anti-inflammatory effect. METHODS: EIU was induced in male Lewis rats by a footpad injection of lipopolysaccharide (LPS). Immediately after the LPS inoculation, 1, 10, or 100 mg ACE or 10 mg prednisolone was injected intravenously. After 24 hours, the aqueous humor was collected from both eyes, and the number of infiltrating cells, protein concentration, nitric oxide (NO), prostaglandin (PG)-E2, and TNF-alpha levels in the aqueous humor were determined. RAW 264.7 cells treated with various concentrations of ACE were incubated with 10 mug/mL LPS for 24 hours. Levels of NO, PGE2, and TNF-alpha were determined by an enzyme-linked immunosorbent assay. The expression of iNOS and COX-2 proteins was analyzed by Western blot analysis. RESULTS: The number of inflammatory cells, the protein concentrations, and the levels of NO, PGE2, and TNF-alpha in the aqueous humor in the groups treated with ACE were significantly decreased in a dose-dependent manner. In addition, the anti-inflammatory effect of 100 mg ACE was as strong as that of 10 mg prednisolone. The anti-inflammatory action of ACE was stronger than that of either quercetin or anthocyanin administered alone. ACE also suppressed LPS-induced iNOS and COX-2 protein expressions in RAW 264.7 cells in vitro in a dose-dependent manner. CONCLUSIONS: The results suggest that ACE has a dose-dependent anti-ocular inflammatory effect that is due to the direct blocking of the expression of the iNOS and COX-2 enzymes and leads to the suppression of the production of NO, PGE2, and TNF-alpha.

Endotoxin-induced uveitis in cyclooxygenase-2-deficient mice.

PURPOSE: Endotoxin-induced uveitis (EIU) is a model that mimics human acute anterior uveitis. Cyclooxygenase (COX)-2 is an enzyme that initiates the conversion of arachidonic acid (AA) into prostaglandins (PGs), whereas 5-lipoxygenase (5-LO) generates leukotrienes (LT). The purpose of this study was to delineate the role of COX-2 in acute ocular inflammation. METHODS: EIU was induced in wild-type (WT), heterozygotic (COX-2(+/-)) and COX-2 null (COX-2(-/-)) mice by injection of lipopolysaccharide (LPS). Other mice were coinjected with LPS and IFN gamma. Ocular histology, serum cytokines, and AA products determined by ELISA, and relevant ocular messengers determined by RT-PCR were compared among the different groups. RESULTS: Histology showed that the EIU score was significantly enhanced in COX-2(-/-) mice in comparison to WT and COX-2(+/-). PGE(2) was increased in WT and COX-2(+/-) EIU but not in COX-2(-/-) EIU. LTB(4) in serum and ocular 5-LO transcripts were increased in COX-2(-/-) EIU mice in comparison with WT and COX-2(+/-) EIU mice. IL-6 increased, whereas IFN gamma decreased both in serum and ocular transcripts in COX-2(-/-) EIU mice in comparison with WT and COX-2(+/-). Furthermore, EIU was suppressed in mice treated with recombinant IFN gamma, as shown by the decreased EIU scores, the presence of serum LTB(4) and IL-6 and ocular 5-LO and IL-6 mRNA, and the increases in serum IFN gamma and ocular IFN gamma, particularly in COX-2(-/-) mice. CONCLUSIONS: These data suggest that disturbance of the AA pathway exacerbates EIU in COX-2-deficient mice. IFN gamma moderately reverses this exacerbation and protects against EIU.

Effects of intravenous administration of FR122047 (a selective cyclooxygenase 1 inhibitor) and FR188582 (a selective cyclooxygenase 2 inhibitor) on prostaglandin-E2-induced aqueous flare elevation in pigmented rabbits.

PURPOSE: Two isoforms of cyclooxygenase (COX-1 and COX-2) exist. To determine in vivo effects of the intravenous administration of FR122047 (a selective COX-1 inhibitor), FR188582 (a selective COX-2 inhibitor), diclofenac sodium or dexamethasone phosphate disodium on prostaglandin-E2 (PGE2)-induced aqueous flare elevation and mRNA levels for COX-1 and COX-2 in pigmented rabbits. METHODS: To produce aqueous flare elevation in rabbits, PGE2, 25 microg/ml, was applied to the cornea with the use of a glass cylinder. FR122047, FR188582, diclofenac sodium or dexamethasone phosphate disodium was intravenously injected before PGE2 application. Aqueous flare was measured with a laser flare-cell meter. The mRNA levels for COX-1 and COX-2 in the iris-ciliary body were determined by real-time polymerase chain reaction. RESULTS: FR122047, FR188582 and diclofenac sodium (15 micromol/kg each) injected intravenously 30 min before PGE2 application inhibited 29 +/- 5, 40 +/- 12 and 50 +/- 9% of aqueous flare elevation, respectively. Simultaneous injection of FR122047 (15 micromol/kg) and FR188582 (15 micromol/kg) 30 min before PGE2 application inhibited 61 +/- 8% of flare elevation. Dexamethasone phosphate disodium (15 micromol/kg) injected intravenously 300 min before PGE2 application inhibited 68 +/- 8% of aqueous flare elevation. Less than 3-fold changes in mRNA levels for COX-1 and COX-2 in the iris-ciliary body were noted after PGE2, FR122047, FR188582, diclofenac sodium or dexamethasone phosphate disodium treatment. CONCLUSION: It is possible that enzyme activities of both COX-1 and COX-2 may be involved in the mechanism of PGE2-induced aqueous flare elevation in pigmented rabbits.

Malondialdehyde and antioxidant enzyme levels in the aqueous humor of rabbits in endotoxin-induced uveitis.

PURPOSE: To investigate the role of oxidative stress in endotoxin-induced uveitis. METHODS: Lipopolysaccharide was injected intravitreally into the right eyes of rabbits. Sterile saline was injected intravitreally into the left eyes as a control. Inflammation was assessed according to clinical score, aqueous humor cell count, and protein levels. Malondialdehyde, superoxide dismutase, glutathione peroxidase, catalase, and nitrite levels were measured in the aqueous humor. RESULTS: The clinical grade (p < 0.01), inflammatory cell count (p < 0.001), and protein content (p < 0.001) were significantly higher in the aqueous humor of eyes with uveitis than in that of controls. Malondialdehyde (p < 0.01) and nitrite (p < 0.001) levels in the aqueous humor of eyes with uveitis were significantly higher than in the control group. Superoxide dismutase (p < 0.001), glutathione peroxidase (p < 0.001), and catalase (p < 0.001) levels were significantly lower in the aqueous humor of eyes with uveitis than in that of the controls. CONCLUSIONS: Oxygen free radicals may be implicated as a mediator of inflammation in endotoxin-induced uveitis. The increase in free radicals in the aqueous humor may play a role in the pathogenesis of endotoxin-induced uveitis.

Corneal endothelial integrity in mice lacking extracellular superoxide dismutase.

PURPOSE: To evaluate corneal endothelial morphology in mice without secreted extracellular superoxide dismutase (SOD) in normal ageing and in a lipopolysaccharide (LPS)-induced inflammation model and to measure the contents of SOD isoenzymes in the mouse cornea and the superoxide radical concentrations in corneas with and without extracellular SOD. METHODS: The central corneal endothelium of wild-type and extracellular SOD-null mice were studied in micrographs at eight different ages and after a unilateral intravitreal injection of LPS, with the contralateral eye serving as the control. The activities of the SOD isoenzymes in the mouse cornea were determined with a direct assay, the superoxide radical concentration was assessed by lucigenin-induced chemiluminescence, and the extracellular SOD distribution was mapped with immunohistochemistry. RESULTS: The activities of the cytosolic Cu- and Zn-containing SOD, the mitochondrial Mn-containing SOD and extracellular SOD were 4300, 15, and 340 U/g wet weight, respectively. Extracellular SOD was found in the epithelium, stroma, and endothelium. The concentration of extracellular superoxide radicals was doubled in extracellular SOD-null corneas, and the endothelial cell density decreased more with age in extracellular SOD-null than in wild-type control corneas. In the LPS-induced inflammation model, the cell density decreased more, and the cells became more irregular in extracellular SOD-null than in wild-type corneas. CONCLUSIONS: In the mouse cornea, absence of extracellular SOD leads to a higher concentration of extracellular superoxide radicals, an enhancement in the spontaneous age-related loss of endothelial cells, and an increased susceptibility to acute inflammatory endothelial damage. Extracellular SOD is likely to have a protective role in the corneal endothelium.

Apoptosis is a prominent feature of acute anterior uveitis in the Fischer 344 rat.

AIMS: To examine the hypothesis that apoptosis of infiltrating cells contributes to spontaneous resolution of uveitis in clinically relevant rodent models. METHODS: Experimental melanin induced uveitis (EMIU) was induced in Fischer 344 rats by immunisation with 250 microg bovine ocular melanin. Endotoxin induced uveitis (EIU) was induced by injection of 200 microg Escherichia coli lipopolysaccharide. Formalin fixed, paraffin embedded ocular cross sections were stained by terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate biotin nick end labelling (TUNEL) to identify apoptotic cells. Indirect immunoperoxidase staining of paraformaldehyde lysine periodate fixed tissue cross sections was used to demonstrate expression of inducible nitric oxide synthase (iNOS). RESULTS: TUNEL positive mononuclear cells were observed in the anterior uvea during both EMIU and EIU at all selected time points. However, whereas the majority of mononuclear cells appeared apoptotic from the outset of disease, neutrophils were notably TUNEL negative at all time points examined. Many infiltrating neutrophils expressed iNOS. CONCLUSION: Apoptosis occurs early in the course of rat EMIU and EIU, and may contribute to resolution of these diseases. In general, infiltrating mononuclear cells die rapidly, while neutrophils survive, producing inducible nitric oxide synthase which may contribute to disease pathogenesis.

Increased matrix metalloproteinases in the aqueous humor of patients and experimental animals with uveitis.

PURPOSE: Matrix metalloproteinases (MMPs) play a major role in connective tissue remodelling, wound healing and embryogenesis. They have also been implicated in pathological tissue degradation in diseases such as rheumatoid arthritis (RA), osteoarthritis (OA), and tumor invasion. The aim of this study was to define the potential role of MMPs in the inflammatory process of uveitis by identifying these proteases in the aqueous humor (AH) of patients with uveitis and in rabbits with endotoxin-induced uveitis (EIU). METHODS: Aqueous humor samples from 6 patients with uveitis and 5 control patients who had undergone elective cataract surgery were examined. The profile of MMPs in the AH of experimentally-induced acute anterior uveitis in rabbits was also assessed. Western blot analysis and SDS-PAGE substrate zymography were used to detect metalloenzymes and their natural inhibitor, tissue inhibitor of metalloproteinase (TIMP-1) in aqueous samples. RESULTS: Aqueous humor from all patients contained interstitial collagenase (MMP-1), stromelysin (MMP-3), gelatinase B (MMP-9) and TIMP-1. Although the amount of MMPs varied considerably, TIMP-1 levels remained unchanged in the aqueous of uveitis patients. Using substrate gel zymography, we were able to reveal several gelatinolytic bands, including one major band at approximately 92-kDa whose activity differed between uveitis and cataract AH. The gelatinase activity found in human AH samples was shown to be inhibited by 10 mM EDTA and activated in vitro by APMA, indicating that these enzymes were indeed of the metalloproteinase class. Aqueous humor samples from the rabbit EIU model revealed a 100-kDa molecular weight species likely to correspond to gelatinase B. This gelatinolytic activity was maximal at 6 hours after the lipopolysaccharide (LPS) injection, declined at 12 and 24 hours post LPS, and was absent at later time points. The induction of gelatinase activity in rabbit AH preceded the increase in cell number during the inflammatory process in the anterior chamber. CONCLUSIONS: Metalloproteinases found in normal human AH may participate in physiological turnover of extracellular matrix in the eye. Elevated levels of MMPs were found in the AH of patients with uveal inflammation and animals with LPS-induced uveitis, where they are likely to be critical to tissue destructive and repair processes. It is likely that pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 alpha (IL-1 alpha), which are known modulators of MMPs, induce their secretion in acute anterior uveitis.

Role of nitric oxide synthase isozymes in endotoxin-induced uveitis.

PURPOSE: The authors previously reported that in vitro treatment with N(G)-nitro L-arginine (L-NNA), an inhibitor of nitric oxide synthase (NOS), reduces aqueous humor (AH) protein and cellular infiltration in endotoxin-induced uveitis in the rat eye. The objective of the current study was to determine the role(s) of respective major forms (constitutive and inducible) of NOS by comparing the effects of relatively selective inhibitors of these NOS isozymes. METHODS: N(G)-nitro L-arginine (L-NNA), a relatively selective inhibitor for constitutive NOS (c-NOS), and N-iminoethyl L-ornithine (L-NIO), a more selective inhibitor for inducible NOS (i-NOS), were administered in vivo. Male Lewis rats were footpad injected with bacterial lipopolysaccharide (LPS, 200 microgram) and were injected intraperitoneally at 0 hours, 6 hours, or both, after LPS injection with 10 mg of NIO, NNA, or saline as a control. Nitric oxide synthase activity in the ocular tissue and AH protein and cell content were determined at various times after treatment with LPS. RESULTS: After in vivo treatment, L-NIO was found to be a more potent inhibitor than L-NNA for ocular i-NOS (87% versus 43% inhibition), and L-NNA was more potent than L-NIO for ocular c-NOS (81% versus 39%). Two injections of L-NNA, one at time 0 and one 6 hours after LPS injection, inhibited the AH protein increase by 71%, but L-NIO did so by only 30%. L-NNA inhibited cellular infiltration by 86%, whereas L-NIO had no significant effect on cellular infiltration. A significant inhibition of cellular infiltration and AH protein increase also was observed with a single injection of 10 mg of L-NNA but not of L-NIO when the inhibitors were given simultaneously with LPS. Thus, reduction of uveitis symptoms correlates with the inhibition of c-NOS. CONCLUSIONS: The constitutive form of NOS in ocular tissue, presumably in vascular endothelial cells, appears to play a critical role at the onset of the development of endotoxin-induced uveitis.

The role of nitric oxide synthase in endotoxin-induced uveitis: effects of NG-nitro L-arginine.

PURPOSE: To evaluate the role of nitric oxide synthase (NOS) in endotoxin-induced uveitis (EIU). METHODS: EIU was caused in Lewis rats by injecting lipopolysaccharide (LPS) into the foot pad, and the effects of an NOS inhibitor, NG-nitro L-arginine (NA), was comparatively studied by the simultaneous administration of NA and LPS. Total NOS and inducible NOS (iNOS) activities were differentially assayed in the anterior segment of the eye in EIU with and without NA treatment. The effects of NA on EIU were also evaluated by clinical manifestation, histology, and protein concentration in the aqueous humor. RESULTS: In untreated rats, there was no significant iNOS activity. With the EIU model, iNOS activity showed a marked increase in the anterior segment of the eye, reaching a maximum 9 hours after LPS injection (10,850 +/- 1,650 cpm/mg protein, mean +/- SEM). NA reduced the iNOS activity 9 hours after injection to 2,400 +/- 90 cpm/mg protein (P < 0.001). Aqueous humor protein concentration in the EIU model was 10.6 +/- 0.75 mg/ml, and the cell number was 216 +/- 12 cells/microliters. NA significantly reduced these factors to 4.25 +/- 0.48 mg/microliters for the protein concentration (P < 0.0005) and 25 +/- 6 cells/ml for the cell number (P < 0.0005). Histologic studies showed less prominent infiltration of polymorphonuclear cells in the anterior uvea than for conventional EIU. CONCLUSIONS: Induction of iNOS may play a key role in the pathogenesis of EIU. Because inhibition of iNOS activity reduced the inflammatory response, suppression of NO formation may inhibit the development of EIU.

Effects of antiflammins on endotoxin-induced uveitis in rats.

Antiflammins are phospholipase A2-inhibitory, anti-inflammatory, synthetic oligopeptides derived from the region of the highest amino-acid sequence similarity between uteroglobin and lipocortin I. Endotoxin-induced uveitis is a model for anterior uveitis of the eye, which has been suggested to be induced through phospholipase A2 activation. In a preliminary report we demonstrated that topical administration of antiflammins could inhibit endotoxin-induced uveitis in rats. In this study, the anti-inflammatory effects of antiflammins were compared with those of corticosteroids on endotoxin-induced uveitis as measured by phospholipase A2 enzyme activity, inflammatory cell counts in the aqueous humor, and histopathologic features. Antiflammins are as effective as corticosteroids in their ability to suppress endotoxin-induced uveitis.

Course and dynamics of immunologic iridocyclitis in the rabbit.

Dynamics of immunologic inflammation of the anterior part of the ocular bulb in rabbits were studied biomicroscopically with the slit lamp and on the basis of.biochemical parameters registered in aqueous humor. Inflammation appeared on the first day after injection of homologous antigen and lasted not longer than 5-6 days. Elevated levels of protein, seromucoid and sialic acid in the aqueous humor were correlated with intensity of biomicroscopic inflammatory lesions, whereas proteolytic activity deviated. The findings were discussed on the assumption that the relatively rapid rise in hydrolase activity, unaccompanied as yet by presence of leukocytes in the anterior chamberm may be due to early destructive changes in the cells of the ocular structures. The changes in the biochemical indices had local character and were not associated with similar changes in the blood serum. The described model, which is based on biochemical and biomicroscopic studies, can be useful in studies on the kinetics of specific inflammation and for testing anti-inflammatory and immunosuppressive drugs used in ophthalmology.