Peroxynitrite and Ocular Inflammation

nitration damage occurs earlier, on day 5 p.i. before any histologic or immunohistochemical evidence of macrophage infiltration. Therefore, the retinal damage of EAU appears to be derived from an alternative mechanism; and this mechanism operates apart from the effects of macrophages, especially in the release of reactive nitrogen species and reactive oxygen species. These early events that set the destructive pathway in EAU have not been elucidated.

SUPEROXIDE AND NITRIC OXIDE IN EAU

At physiological pH, peroxynitrite formed in vivo can directly nitrate phenolic rings to form 3-nitrotyrosine from tyrosine residues. In recent years, although other metabolites of nitric oxide have also emerged as biological oxidants, it is generally agreed that peroxynitrite is generally considered the most plausible entity for causing biological nitration and oxidation.7 Peroxynitrite has been implicated in the pathogenesis of a series of diseases, including acute and chronic inflammatory processes, sepsis, ischemic-reperfusion and a variety of neurodegenerative and retinal disorders.8

The presence of nitric oxide synthase (NOS) has recently been shown in mammalian mitochondria.9,10 Thus, with an abundance of substrate,10 nitric oxide is continuously produced in the mitochondria. Mitochondria are also a copious source of superoxide, which is generated at the sites of complexes I and III of the electron transport chain.11 In tissues and in mitochondria, peroxynitrite forms from a facile reaction of superoxide and nitric oxide concomitantly generated in close proximity. These facts suggest that mitochondria are continuously challenged by peroxynitrite formed within the organelles themselves. In the past, nitration of Mn superoxide dismutase, mitochondrial aconitase, the voltage-dependent anion channel, mitochondrial ATPase and cytochrome c have been detected in animals undergoing inflammatory processes.12,13 Photoreceptor cells are known to have the highest rates of glycolysis and respiration among all retinal cells.14 For these reasons, inner segments of photoreceptor are densely packed with mitochondria.15

Our study was designed to determine the primary nitration target(s) of peroxynitrite and to detect the onset of this post-translational modification in EAU. The retina contains numerous proteins that complement its complex visual functions. Three of these proteins, all of which are essential for mitochondrial energetics and metabolism functions, were found to be selective prime targets of peroxynitrite nitration. Moreover, the protein nitration was found to commence early in the inflammatory process, far before the entry of inflammatory cells known to release superoxide and nitric oxide in the retina. In experimental uveitis, the insult that initiates the spiral of degenerative processes in the photoreceptors has not been defined in the past.

Nitration of Retinal Proteins in Uveitis

Experimental uveitis was induced by a hind foot-pad injection of 60 mg of bovine S-antigen in Freund’s complete adjuvant containing 4 mg/ml of heat killed Mycobacterium tuberculosis H37 RA (Difco, Detroit, MI). To investigate the

early phase of the disease, animals were sacrificed on days 5 and 10 p.i., with the normal peak of inflammation being at day 14 p.i. In ultraviolet/visible (UV/VIS) absorption and Western blot analyses, in vitro nitrated bovine serum albumin (BSA) was used as a model protein to establish the sensitivity and specificity of 3-nitrotyrosine absorption embedded in proteins.16 Bovine serum albumin containing 19 tyrosine residues/molecule was nitrated in good yield in vitro by the peroxynitrite donor 3-morpholinosydnonimine (SIN-1; Sigma, St Louis, MO) to give 354 nm absorption at pH 7 (see insert in Fig. 1).

The tyrosine residues were nitrated at a much higher level in BSA compared with the level of nitrotyrosine formed in SIN-1-reacted retina and in inflamed retina nitrated in inflammation. Since the molar absorption coefficient for nitrotyrosine is only 4400/M/cm,17 the maximal obtainable intensity for 354 nm, the pH 7 band for the SIN-1-reacted retinal proteins is small (Fig. 1A). In these spectra, the tyrosine-nitrated proteins (spectrum 4, Fig. 1A) revealed 360 nm nitrotyrosine chromophore after subtracting spectrum 3, sum of controls, spectra 1 and 2 (Fig. 1A).

Similarly, in the inflamed retina (Fig. 2A), subtraction of spectrum 1, non-immunized control retina from spectrum 2, EAU day 5 retina revealed an absorption peak centered at 350 nm, indicative of nitrotyrosine chromophore and an absorption for cytochrome c at 407 nm (Fig. 2A).18 Although the absorption bands were not completely resolved, as was commonly seen in the in vivo samples, they clearly demonstrated the presence of nitrotyrosine chromophore and released cytochrome c. These observations are consistent with other reported tissue studies in which tyrosine-nitration can also be detected by immunohistochemistry,5 by electrochemically monitored HPLC,19 or by Western blotting20; but no one method alone will totally ascertain the formation of nitrotyrosine in vivo. In this study, to assay and confirm the protein tyrosine-nitration in both in vitro and in vivo samples, we used UV/VIS absorption for initial screening, Western blot in conjunction with mass spectrometry for confirmation, and immunohistochemical staining for subsequent localization in the retina.

Identification of Nitrated Retinal Protein

Exposure of naı¨ve retina to the peroxynitrite donor SIN-1 resulted in seven tyrosine-nitrated proteins, as revealed by Western blot analysis (Fig. 1C, lane 1). The molecular masses of these proteins are 68, 52, 50, 41, 39, 35, and 29 kDa, as estimated by the relative mobility (Rf value) of these proteins compared with that of the protein standards. The relative intensities of these nitrated bands in Western blot appeared to follow closely the intensities seen in the total retinal protein profile (Fig. 1B, lane 1). In this system, major proteins were all nitrated as compared with the controls (Fig. 1C, lane 2).

The EAU eyes were obtained from the animals on days 0, 5, 10, 12, and 14 p.i. Day 0 denotes non-immunized control animals. In the EAU retina, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 12% gel)

Figure 1 UV/VIS absorption and Western blot of retinal proteins nitrated by SIN-1. A. Analysis of UV/VIS spectra of nitrated retinal proteins at pH 7. Spectrum 1: retinal protein end absorption; spectrum 2: degradation products of SIN-1 following reaction; spectrum 3: sum of spectra 1 and 2; spectrum 4: nitrated retinal proteins. Subtraction of spectrum 3 from 4 resulted in an absorption centered near 360 nm, indicative of nitrotyrosine chromophore. The insert shows the absorption of nitrated BSA. B. Coomassie Blue staining of retinal proteins incubated with (lane 1) and without (lane 2) SIN-1. C. Western blot probed with anti-nitrotyrosine. Lane 1: reti- naþSIN-1; lane 2: retina-SIN-1. Note that all major proteins in the retina are equally nitrated.

Figure 2 Retinal protein nitration in the early phase of EAU. A. UV/VIS absorption spectra of control animal (D0) (spectrum 1) and early phase of EAU (spectrum 2). Subtraction of spectrum 1 from 2 reveals absorption of nitrated tyrosine at 350 nm and cytochrome c at 407 nm (arrow). B. Coomassie Blue staining of electrophoresed retinal proteins. Lane 1: D0; and lane 2: D5 p.i. C. Western blot of nitrated retinal proteins. Lanes 1 (D0), 2 (D5), 3 (D10), 5 (BSAþSIN-1), and 6 (BSA-SIN-1) were probed with antinitrotyrosine. Lane 4 (D5) was probed with preimmune serum. Band A: mitochondrial import stimulation factor; band B: phosphoglycerate mutase; and band C: cytochrome c.

revealed 10 major protein bands (Fig. 2B, lane 2); this profile was similar to that of the non-immunized control animals (Fig. 2B, lane 1). Western blots of EAU samples were then run in parallel with nitrated BSA (Fig. 2C, lane 5). The blots of EAU retinas indicated three relatively intense tyrosine-nitrated protein bands,

Figure 3 Western blot analyses of nitrated cytochrome c in the early phase of EAU. Lane 1 (D0) and lane 2 (D5) were blotted with anti-rat cytochrome c, and lane 3 (D5) and lane 4 (D10) were blotted with anti-nitrotyrosine antibody. Band A: cytochrome c trimer; band B, cytochrome c and band C: nitrated cytochrome c.

located at 32, 29, and 16 kDa (Fig. 2C, lanes 2 and 3). Moreover, these three bands appeared early in the inflammation, on days 5 and 10 p.i., long before the peak of inflammation at day 14 p.i.

The 32 kDa (upper) and 29 kDa (lower) bands were excised separately from an electrophoresed gel and subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS). The Sequest database search revealed that the upper band is mitochondrial import stimulation factor and the lower band, rat phosphoglycerate mutase. In both mitochondrial import stimulation factor and phosphoglycerate mutase, six peptides each were identified to match the known sequences. Using Western blot (15% gel) gel, the 14 kDa band from EAU days 5 and 10 p.i. (Fig. 2C, band C) was identified as cytochrome c. This sample was also blotted in parallel with both rat cytochrome c antibody (lanes 1 and 2, Fig 3) and nitrotyrosine antibody (lanes 3 and 4, Fig. 3). The identity of the cytochrome c band was also confirmed by LC-MS/MS, using cytochrome c from both whole retina and isolated mitochondria. Sequential studies covering days 0, 5, 10, 12, and 14 p.i. revealed that three Tyr-nitrated proteins, including mitochondrial import stimulation protein, phosphoglycerate mutase and cytochrome c, were at near maximal intensities on days 5 and 10 p.i., then leveled off gradually from day 10 to the peak of inflammation on day 14 (Fig. 4). During the period from days 0 to 10, the retinal morphology was well preserved. Day 12 signified the onset of disease, and the entrance of inflammatory cells was visible.

Figure 4 (See color insert.) Retinal morphology and protein nitration during the course of EAU. Nitrated retinal proteins from days 0 (D0), 5 (D5), 10 (D10), 12 (D12), and 14 (D14) p.i. were immunoblotted with anti-nitrotyrosine (B), the relative intensities of nitrated proteins were quantified (C) and correlated with morphologic changes in EAU (A). Maximal intensities of tyrosine-nitration were seen in days 5 and 10, with well preserved retinal structures. Day 12 marked the onset of inflammation with arrival of inflammatory cells.

Localization of Nitrated Retinal Protein in the Early Phase of EAU

To assess the cellular source of peroxynitrite in the early phase of EAU, tyrosinenitrated proteins in the retina were localized. Using immunohistochemical methods, positive nitrotyrosine staining was localized exclusively at the photoreceptor inner segments in day 5 p.i. retina (Fig. 5B). No nitrotyrosine staining

Figure 5 (See color insert.) Localization of tyrosine-nitrated proteins in the retina. Polyclonal nitrotyrosine antibody and anti-rabbit IgG conjugated with biotin were used for the detection. A: non-immunized control animals and B: EAU day 5 p.i. Note the intense localization of nitrated proteins seen only in the photoreceptor inner segments (B).

was seen in the non-immunized controls (Fig. 5A). The specificity of primary antibody was established by (1) replacing the primary antibody with phosphatebuffered saline and (2) reacting the primary antibody with authentic nitrotyrosine before staining. Both procedures abolished the nitrotyrosine staining in the inflamed retinas.

Displacement of Cytochrome C from Electron Transport Assembly

The release of cytochrome c in EAU animals on days 5 and 10 p.i. was examined by isolating intact mitochondria.21 The retinal cytosol and intact mitochondria were separated initially. In cytosolic fractions, the presence of cytochrome c was not detected on days 5 and 10 p.i., indicating that cytochrome c was not released into the cytosol at the early phase of disease. When the isolated mitochondria were sonicated briefly to rupture the outer membranes,22 a substantial release of cytochrome c was observed on both days 5 and 10 (Fig. 6, band A in lanes 2 and 3). A functional cytochrome c binds to both mitochondrial respiratory complexes III and IV and is, therefore, stable to sonication but sensitive to detergents.23 No detergent was used in these isolation procedures. It appears that in this early phase of inflammation, although cytochrome c (more likely nitrated cytochrome c) was already displaced from its normal binding site in the electron transport chain, the mere separation of cytosol from intact mitochondria did not result in the significant release of cytochrome c. However, upon mechanical rupture of

Figure 6 Nitration and release of cytochrome c in the early phase of EAU. The release of cytochrome c was not seen in the cytosolic fraction separated from the intact mitochondria. However, after mild sonication of mitochondria fraction to disrupt the outer membranes, substantial release of cytochrome c/nitrated cytochrome c was detected by Western blot probed with anti-rat cytochrome c. Lane 1: non-immunized control (D0); lane 2: D5 and lane 3: D10.

mitochondrial outer membranes, cytochrome c was released into the supernatant. No cytochrome c release was detected in the controls, even with sonication to disrupt the mitochondrial outer membranes (Fig. 6).

ROLE OF PEROXYNITRITE IN EAU

From total proteins in EAU retina, we have detected three mitochondria-related proteins that were specifically nitrated in the early phase of EAU, prior to any macrophage or other phagocytic infiltration (Figs. 2, 4). Using LC-MS/MS, we identified two nitrated proteins near 30 kDa as mitochondrial import stimulation factor and phosphoglycerate mutase. The third protein (14 kDa) was identified as cytochrome c (Fig. 3). Levels of tyrosine-nitration were also correlated with the extent of cellular infiltration and photoreceptor degeneration in the course of EAU (Fig. 4). In the early phase of EAU, the tyrosine-nitrated retinal proteins were localized exclusively in the photoreceptor inner segments, which are densely populated with mitochondria (Fig. 5). Further, in vivo nitrated cytochrome c was found to be displaced from its original binding site at the electron transport chain assembly. The in vitro nitration of naı¨ve retina was also carried out by peroxynitrite donor SIN-1 to result in nitration of all seven major proteins with similar intensities (Fig. 1). Therefore, in vitro nitration in the solution phase lacks the selectivity displayed by the in vivo nitration.

Peroxynitrite has been implicated in the pathogenesis of a series of diseases. In these systems, peroxynitrite generation requires nitric oxide and superoxide.8 The concomitant generation of these two agents at a localized site results in the formation of peroxynitrite by a combination reaction, threefold faster than the rate of superoxide dismutation by superoxide dismutase.24 We and others have reported in the past that both superoxide and nitric oxide are among

the most important primary oxidant species generated by macrophages in inflammatory diseases such as uveitis.3,4,25 However, our study showed that

nitration of mitochondrial proteins occurred prior to the infiltration of macrophages (Fig. 4), indicating that the generation of reactive species and formation of peroxynitrite occurred within the retinal cells and was not from macrophages or other infiltrating inflammatory cells.

Photoreceptor cells, which are responsible for all visual processes, have the highest rate of glycolysis and respiration, as revealed by the metabolic mapping of mammalian retina using H3-2-deoxyglucose autoradiography.14 Because of this high metabolic requirement, the inner segments in the photoreceptor cells are packed with mitochondria, with a density unseen in any other cells.15 Mitochondria are also an important cellular source of superoxide. It is estimated that 1–2% of the oxygen consumed undergoes partial reduction, generating superoxide.11 In recent years, mitochondrial production of nitric oxide by mitochondrial NOS was recognized.9,10 Nitric oxide produced by mitochondrial NOS and L-arginine is readily diffusible through cell membranes, whereas superoxide is not; therefore, it is conceivable that a charged combination product, such as peroxynitrite will be principally formed in the same compartment as superoxide, probably near the inner membranes.26 In the absence of macrophages, the actively respiring mitochondria in the inner segments of photoreceptor cells would be the early source of peroxynitrite, causing the nitration of cellular proteins at the proximity.

Mitochondrial DNA contains 37 genes coding for two rRNAs, 22 tRNAs and 13 polypeptides. The mitochondrial DNA-encoded polypeptides are all subunits of enzyme complexes of the oxidative phosphorylation system.27 Therefore, most of the proteins required for the mitochondrial functions are encoded by nuclear genes, synthesized by cytoplasmic ribosomes and imported to mitochondria post-translationally.28 Therefore, there are cytosolic protein factors that chaperone and target cytoplasmic precursor proteins to mitochondrial membrane receptors. Mitochondrial import stimulation factor serves these functions.29 Although mitochondrial import stimulation factor originates as a cytosolic factor, during the chaperone process, it sits on the mitochondrial membrane receptors to transfer preproteins; therefore, mitochondrial import stimulation factor is exposed to the peroxynitrite generated within mitochondria.29

Phosphoglycerate mutase catalyzes the interconversion of 2- and 3- phosphoglycerate in the glycolytic/gluconeogenic pathways. These reactions are essential components in the metabolism of glucose and/or 2, 3-bisphosphogly- cerate in all cells.30 Although this protein does not reside intramitochondrially, it

However, when mild sonication was applied to disrupt outer mitochondrial membranes, substantially more cytochrome c was detected. In the respiratory chain assembly, cytochrome c is bound to complex III and cytochrome oxidase by electrostatic interaction and is therefore stable to sonication but sensitive to most detergents.23 In the present study, no detergent was used in processing retina and mitochondria. In the previous reports, when cytochrome c was released from apoptotic or permeabilized mitochondria, it was often found that cytochrome c was already dissociated from the electron transport chain before pathologic membrane rupture.22 Therefore, it appears that the release of cytochrome c requires two simultaneous impairments: 1) rupture or permeabilization of mitochondrial outer membranes; and 2) detachment of cytochrome c from the respiratory chain complex. In this study, the integrity of mitochondrial outer membranes was still mostly intact on day 5; but cytochrome c was already displaced from its normal binding site in the respiratory chain due to tyrosinenitration in the molecule.

The initial signal leading to upregulation of mitochondrial NOS in S-antigen induced EAU has not been dealt in the past. In an organ-specific autoimmune disease such as EAU, the CD4-positive T-cells are present in the retina early in the inflammation. For example, after adaptive transfer of S-antigen specific T-cells, these T-cells were seen in the retina within 24 hours, although loss of retinal stratification was not observed until after 120 hours.32 The local antigen presentation to these S-antigen autoreactive T-cells can result in the generation of tumor necrosis factor-a (TNF-a) by the antigen-presenting cells. Tumor necrosis factor-a, an inflammatory agonist, is known to upregulate NOS, and subsequently to produce reactive oxygen species.33 Tumor necrosis factor-a can also increase mitochondrial Ca2þ, a known stimulator of mitochondrial reactive oxygen species.34 In this process, TNF-a initially mobilizes Ca2þ from its endoplasmic storage to the mitochondria; Ca2þ then triggers mitochondrial NOS activity.35

CONCLUSION

In the early phase of EAU, prior to leukocyte infiltration, we found three major nitrated retinal proteins and these were mitochondrial import stimulation factor, phosphoglycerate mutase and cytochrome c, all of which are mitochondriarelated proteins. Immunohistochemical staining revealed that these nitrated proteins are exclusively localized in the inner segments of photoreceptor cells, a layer known to be densely populated with mitochondria. These findings provide evidence for a rather selective tyrosine-nitration process that modifies specific proteins in vivo. In this early stage of inflammation, mitochondria are the major source of peroxynitrite and mitochondrial proteins the prime target for damage by the mitochondrial oxidative stress. Hence, for the first time, these findings implicate the photoreceptor damage at the molecular level by peroxynitrite generated in the mitochondria. Such oxidative damage may lead to microglial

activation, recruitment of blood-born monocytes/macrophages and neutrophils in the amplification of retinal damage and clinical and histologic findings of amplified uveitis.

ACKNOWLEDGMENT

This study was supported in part by grants EY015714 and EY03040 from National Institutes of Health.

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