Retinal Mueller Glial Cells Trigger the Hallmark Inflammatory Process

We studied changes in the retinal proteome from horses with equine recurrent autoimmune uveitis compared to controls. We found markers for breakdown o...
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Retinal Mueller Glial Cells Trigger the Hallmark Inflammatory Process in Autoimmune Uveitis Stefanie M. Hauck,*,† Stephanie Schoeffmann,† Barbara Amann,‡ Manfred Stangassinger,‡ Hartmut Gerhards,§ Marius Ueffing,†,| and Cornelia A. Deeg‡ Institute of Human Genetics, GSF-National Research Center for Environment and Health, Ingolstaedter Landstr. 1, D-85764 Neuherberg, Germany, Institute of Animal Physiology, LMU Munich, Veterina¨rstr. 13, D-80539 Munich, Germany, Department of Equine Surgery and Medicine, LMU Munich, Veterina¨rstr. 13, D-80539 Munich, Germany, and Institute of Human Genetics, Technical University of Munich, Trogerstr. 32, D-81675 Munich, Germany Received December 13, 2006

Spontaneous equine recurrent uveitis (ERU) is an incurable autoimmune disease affecting the eye. Although retinal-autoantigen specific T-helper 1 cells have been demonstrated to trigger disease progression and relapses, the molecular processes leading to retinal degeneration and consequent blindness remain unknown. To elucidate such processes, we studied changes in the total retinal proteome of ERU-diseased horses compared to healthy controls. Severe changes in the retinal proteome were found for several markers for blood-retinal barrier breakdown and whose emergence depended upon disease severity. Additionally, uveitic changes in the retina were accompanied by upregulation of aldose 1-epimerase, selenium-binding protein 1, alpha crystallin A chain, phosphatase 2A inhibitor (SET), and glial fibrillary acidic protein (GFAP), the latter indicating an involvement of retinal Mueller glial cells (RMG) in disease process. To confirm this, we screened for additional RMG-specific markers and could demonstrate that, in uveitic retinas, RMG concomitantly upregulate vimentin and GFAP and downregulate glutamine synthetase. These expression patterns suggest for an activated state of RMG, which further downregulate the expression of pigment epithelium-derived factor (PEDF) and begin expressing interferon-gamma, a pro-inflammatory cytokine typical for T-helper 1 cells. We thus propose that RMG may play a fatal role in uveitic disease progression by directly triggering inflammatory processes through the expression and secretion of interferon-gamma. Keywords: inflammation • blood-retinal barrier • Mueller glia • autoimmune disease • clinical proteomics • uveitis

Introduction Recurrent uveitis is a major sight threatening disease among humans and horses.1,2 Equine recurrent uveitis (ERU) is a spontaneous disease affecting approximately 10% of the equine population.3 Due to its high prevalence and resulting blindness, ERU is also considered a major problem for the affected animals themselves,3,4 as well as being the only spontaneous model for human autoimmune uveitis.2 Recurrent uveitis is characterized by repeated attacks of CD4+ T-cells targeting intraocular structures, particulary the retina.5 As the retina forms part of the central nervous system, affected areas with destroyed architecture are unable to reorganize and remain therefore functionally compromised. Autoimmune reactions are directed toward retina-expressed proteins in both horses2,6 and * To whom correspondence should be addressed. Institute of Human Genetics, GSF National-Research Center for Environment and Health, Ingolstaedter Landstr. 1, D-85764 Neuherberg, Germany; E-mail, [email protected]; Telephone, 00498931873565; Fax, 00498931874426. † GSF-National Research Center for Environment and Health. ‡ Institute of Animal Physiology, LMU Munich. § Department of Equine Surgery and Medicine, LMU Munich. | Technical University of Munich. 10.1021/pr060668y CCC: $37.00

 2007 American Chemical Society

humans.7,8 Identified autoantigens are S-antigen (S-Ag), interphotoreceptor retinoid binding protein (IRBP)7 and cellular retinaldehyde-binding protein (cRALBP),6 all expressed exclusively in the retina and pineal gland. Although some features of the self-aggressive immune reaction have been identified and characterized, little is known about the role of the major target tissue itself in the inflammatory process. Proteome analysis is a comprehensive approach to disease pathogenesis and etiology, whereby protein expression patterns reflect the complexity of molecular processes and are potentially able to provide the molecular basis for specific diagnosis and understanding of disease pathogenesis.9 Because disease mechanisms are highly complex and involve many different proteins, a high resolution approach is therefore useful for detecting all molecules participating in the inflammatory process.10 The goal of our study was to analyze the retinal proteome under normal and pathogenic uveitic conditions as a positive step toward elucidation of the system dynamics of this target tissue, as well as identification of regulatory circuits implicated in this pathology. Journal of Proteome Research 2007, 6, 2121-2131

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research articles Materials and Methods Retinal Samples. Uveitic retinas from horses presenting with at least three clinical signs of spontaneous ERU were included in the proteomic analysis studies. Clinical signs of intraocular inflammation included aqueous flare, synechiae, vitritis, pigment deposition on the anterior lens surface, retinal detachment, and cataract. For each clinical case, a history was provided by both owners and veterinarians treating the horses. We graded the clinical signs on a scale between 0 and 3 (no disease and severe disease, respectively).2 The retinas of six ageand sex-matched, eye-healthy horses served as controls. All animals were euthanized due to reasons unrelated to this study during quiescent stages of ERU. Following enucleation, the eyes were opened and the retinas removed completely. A small area (approximate diameter ) 0.3 cm) of retinal tissue remained adjacent to the optic nerve; this area was then excised for histopathology, fixed in Bouin’s solution (Sigma), and embedded in paraffin (Microm). Retinal sections from 10 ERU cases (4 in active disease stage and 6 in quiescent stage) and 10 healthy controls were used for validation of candidate protein regulation. Two-Dimensional Gel Electrophoresis (2DE). Fresh equine retinas were immediately stabilized with protease inhibitors (Roche), homogenized, lyophilized, and stored at -80 °C. For 2DE analysis, protein pellets were solubilized in 2DE lysis buffer (9 M urea, 2 M thiourea, 1% DTE, 4% CHAPS. Immobiline dry strips pH 3-11 NL, 24 cm (GE Healthcare) were immersed overnight in lysis buffer containing 150 µg normalized protein sample (Bradford assay), additional 1% Pharmalyte pH 3-10 (GE-Healthcare) and 0.5% Bromphenol blue. Isoelectric focusing was done on a Multiphor (GE-Healthcare) for 60 kVh at 20 °C, followed by separation on gradient SDS-PAGE gels (9-15%) at constant 3 W per gel (Ettan, GE Healthcare). Gels (g12 per retina) were silver-stained for subsequent image analysis and mass spectrometry. Mass Spectrometry for Protein Identification. Selected spots were excised from 2DE, destained, processed by proteolysis with trypsin as described,11,12 and analyzed by MALDITOF peptide mass fingerprinting and MS/MS on a MALDITOF/TOF tandem mass spectrometer (ABI 4700 Proteomics Analyzer, Applied Biosystems). For positive-ion reflector mode spectra, 2500 laser shots were averaged and processed with external calibration; PMF spectra were neither smoothed nor the background subtracted. Monoisotopic peak masses were determined automatically (4000 series explorer software, version 3.6) within a mass range of 800-4000 kDa, a signal-tonoise ratio minimum of 5, and local noise window width m/z 200. Up to seven of the most intense ion signals (signal-tonoise ratios above 30) were selected as precursors for MS/MS acquisition, excluding common trypsin autolysis peaks and matrix ion signals. In MS/MS positive ion mode, 4000 spectra were averaged with 1 kV of collision energy; collision gas air at a pressure of 1.6 × 10-6 Torr and default calibration. Monoisotopic peak masses were automatically determined with a signalto-noise ratio minimum of 10 and local noise window width m/z 200. Combined PMF and MS/MS queries were done with MASCOT Database search engine v1.913 (Matrix Science Ltd.) embedded into GPS-Explorer Software (version 3.6, Applied Biosystems) on the Swiss-Prot database (version 20060725; 230133 sequences; 84471903 residues) or MSDB metadatabase (version 20060831; 3 239 079 sequences; 1 079 594 700 residues) with the following parameter settings: entries restricted to mammalian (44 514 and 339 488 sequences, respectively); 65 2122

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ppm mass accuracy; 0.3 Da fragment mass tolerance; trypsin cleavage; one missed cleavage allowed; carbamidomethylation as fixed modification; and oxidation of methionines allowed as variable modification. Protein identification was considered positive (Tables 1 and 2 ) if (i) the probability-based MOWSE score14 obtained from both PMF analysis and MS/MS analysis was significant (scores >67 were significant at p < 0.05 and scores >74 were significant at p < 0.01; confidence interval >99% as given by GPS explorer, version 3.6); (ii) the matched peptide masses were abundant in the spectrum; and (iii) the theoretical masses of the significant hit fit the experimentally observed values. We also allowed single MS/MS spectra to contribute to the total MOWSE score in cases where the PMF score alone was already significant for identification of a given protein (p < 0.05). Even though Swiss-Prot protein identifications were unambiguous, in most cases, equine proteins were not found in the database and we had to rely on homologue proteins from other species for identification. The homologue proteins with the highest MOWSE scores were always listed. In cases where the protein was identified by a similar score from different species, the human protein was always listed. Two proteins (immunoglobins 1 and 4) could not be identified in the Swiss-Prot database and were instead identified in the MSDB metadatabase; these proteins are listed with their NCBI identifier. Correct identification of immunoglobin family members was verified by significant MS/MS spectra of family member-specific peptides. If redundant database entries were listed, we selected the most recent entry. Western Blots. Retinal samples were resolved by 10% sodium SDS-PAGE and blotted semidry onto PVDF membranes. Unspecific binding was blocked with 1% PVP in PBS-T (1 h). Blots were subsequently incubated with primary antibodies in PBS-T (overnight at 4 °C; anti-GFAP, Sigma, 1:1000; anti-GS, BDTransduction 1:500; anti-PEDF, Millipore, 1.3 µg/mL; anti-beta actin, Sigma, 1:5000), washed and incubated in horseradish peroxidase-conjugated secondary antibodies (1:15 000; Linaris). Signals were detected with ECL (enhanced chemiluminescence) on Hyperfilm ECL (GE Healthcare) according to manufacturer’s instructions. Image Analysis and Protein Quantification. Quantification of Western blot signals was performed with ImageQuant TL software (GE Healthcare) after scanning the films on a transmission scanner (ImageScanner II, GE Healthcare). Images of 2DE gels (8 bit/600 dpi resolution) were imported to 2DE analysis software (Proteom Weaver; release 2.2.; BioRad) and analyzed using the following spot detection parameters: minimum spot radius: 4, minimum spot intensity (volume above base level): 2000 and minimum contrast (height above base level): 10. Gels from each group (ERU and healthy controls) were processed by pair-match-based normalization, which removes intensity differences of similar spots in different gels due variability of the experimental method (e.g., protein load, silver stain intensity) and not to regulation. Gel images were compared manually, as the patterns between healthy and diseased retinas differed sufficiently to allow proper overlay. Differentially expressed spots were manually selected and spot intensities of respective proteins were included in the statistical analysis. Statistical Significance. Statistical significance was calculated using student’s t-test. Histology and Immunostaining. Bouin (Sigma) fixed eyes were embedded in paraffin (Microm) and sectioned. Antigen retrieval was performed at 99 °C for 15 min in 0.1 M EDTA-

research articles

Retinal Proteome in ERU Table 1. Proteins Identified from Normal Equine Retinaa

theoretical spot

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62

protein name

10-Formyltetrahydrofolate dehydrogenase (EC 1.5.1.6) 14-3-3 protein epsilon (14-3-3E) 14-3-3 protein gamma (KCIP-1) 14-3-3 protein tau (14-3-3 protein theta) (HS1 protein) 14-3-3 protein zeta/delta (KCIP-1) and 14-3-3 protein beta/alpha (KCIP-1) 60 kDa heat shock protein, mitochondrial precursor (Hsp60) 78 kDa glucose-regulated protein precursor (GRP 78) (BiP) Actin, cytoplasmic 2 (gamma actin) Actin, cytoplasmic 2 (gamma actin) Actin, cytoplasmic 2 (gamma actin) Aldose 1-epimerase (EC 5.1.3.3) Aldose reductase (EC 1.1.1.21) Alpha crystallin B chain (Alpha(B)-crystallin) Alpha crystallin A chain Alpha-enolase (EC 4.2.1.11) Alpha-enolase (EC 4.2.1.11) Alpha-enolase (EC 4.2.1.11) Alpha-enolase (EC 4.2.1.11) Alpha-internexin (Neurofilament-66) (NF-66) Alpha-tubulin (Fragment) Annexin A5 (Annexin V) Annexin A6 (Annexin VI) ATP synthase subunit beta, mitochondrial [Precursor] ATP synthase subunit beta, mitochondrial [Precursor] ATP synthase D chain, mitochondrial (EC 3.6.3.14) Beta-synuclein and S-phase kinase-associated protein 1A (Cyclin A/CDK2-associated protein p19) C-1-Tetrahydrofolate synthase, cytoplasmic (C1-THF synthase) Calmodulin (CaM) Calreticulin precursor (CRP55) Calretinin (CR) Carbonic anhydrase II (EC 4.2.1.1) Cellular retinaldehyde-binding protein (CRALBP) Class II beta tubulin isotype Cofilin, non-muscle isoform (Cofilin-1) (p18) Creatine kinase chain B (EC 2.7.3.2) Creatine kinase chain B (EC 2.7.3.2) Cytosolic nonspecific dipeptidase (Glutamate carboxypeptidase-like protein 1) Dihydropyrimidinase related protein-2 (DRP-2) Dihydropyrimidinase related protein-2 (DRP-2) Dihydropyrimidinase related protein-2 (DRP-2) DNA damage binding protein 1 (XAP-1) and 116 kDa U5 small nuclear ribonucleoprotein component (U5 snRNP-specific protein) Elongation factor 1-alpha 1 (EF-1-alpha-1) Elongation factor 2 (EF-2) Endoplasmin precursor (94 kDa glucose-regulated protein) (GRP94) Eukaryotic translation initiation factor 5A (eIF-5A) Fatty acid-binding protein (E-FABP) F-box only protein 2 Fructose-bisphosphate aldolase A (EC 4.1.2.13) Fructose-bisphosphate aldolase C (EC 4.1.2.13) Fructose-bisphosphate aldolase C (EC 4.1.2.13) Fructose-bisphosphate aldolase C (EC 4.1.2.13) Fructose-bisphosphate aldolase C (EC 4.1.2.13) Gamma-enolase (EC 4.2.1.11) Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta 1 (Transducin beta 1) Glutamate dehydrogenase 1, mitochondrial (EC 1.4.1.3) Glutamine synthetase (EC 6.3.1.2) Glutamine synthetase (EC 6.3.1.2) Glutamine synthetase (EC 6.3.1.2) Glutathione S-transferase Mu 5 (EC 2.5.1.18) Glutathione S-transferase Yb-3 (EC 2.5.1.18) and Triosephosphate isomerase (EC 5.3.1.1) Glutathione S-transferase Mu 3 (EC 2.5.1.18) Glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12)

accession no.

MWb

pIc

scored

Homo sapiens Homo sapiens Homo sapiens Homo sapiens

FTHFD_HUMAN 1433E_HUMAN 1433G_HUMAN 1433T_HUMAN

99622 29326 28325 28032

5.63 4.63 4.80 4.68

166 558 387 383

Homo sapiens Homo sapiens Rattus norvegicus

1433Z_HUMAN 1433B_HUMAN CH60_RAT

27899 28048 61088

4.73 4.76 5.91

546 141 625

Homo sapiens

GRP78_HUMAN

72402

5.07

810

Bos taurus Bos taurus Bos taurus Mus musculus Sus scrofa Bos taurus Equus caballus Homo sapiens Homo sapiens Homo sapiens Mus musculus Homo sapiens Macaca mulatta Pan troglodytes Homo sapiens Bos taurus Bos taurus Homo sapiens Homo sapiens Homo sapiens

ACTG_BOVIN ACTG_BOVIN ACTG_BOVIN GALM_MOUSE ALDR_PIG CRYAB_BOVIN CRYAA_HORSE ENOA_HUMAN ENOA_HUMAN ENOA_HUMAN ENOA_MOUSE AINX_HUMAN Q9GLW6 ANXA5_PANTR ANXA6_HUMAN ATPB_BOVIN ATPB_BOVIN ATP5H_HUMAN SYUB_HUMAN SKP1_HUMAN

41793 41793 41793 37799 36056 20024 19762 47038 47038 47038 47010 55528 49662 35840 76037 56284 56284 18405 14279 18686

5.31 5.31 5.31 6.26 5.77 6.76 5.78 6.99 6.99 6.99 6.36 5.34 4.90 4.94 5.42 5.15 5.15 5.22 4.41 4.40

414 664 465 79 121 383 157 557 497 657 108 526 256 536 317 601 199 119 190 165

Homo sapiens

C1TC_HUMAN

102049

6.94

119

Homo sapiens Cricetulus griseus Rattus norvegicus Homo sapiens Homo sapiens Homo sapiens Homo sapiens Canis familiaris Canis familiaris Mus musculus

CALM_HUMAN CRTC_CRIGR CALB2_RAT CAH2_HUMAN CRAL_HUMAN Q8IWR2 COF1_HUMAN KCRB_CANFA KCRB_CANFA CPGL1_MOUSE

16696 48384 31499 29154 36548 50283 18588 42701 42701 53190

4.09 4.33 4.94 6.86 4.98 4.82 8.26 5.47 5.47 5.43

325 361 355 159 264 281 371 720 532 276

Bos taurus Bos taurus Rattus norvegicus Homo sapiens Homo sapiens

DPYL2_BOVIN DPYL2_BOVIN DPYL2_RAT DDB1_HUMAN U5S1_HUMAN

62638 62638 62278 128142 110336

5.95 5.95 5.95 5.14 4.84

731 577 325 178 132

Bos taurus Homo sapiens Sus scrofa

EF1A1_BOVIN EF2_HUMAN ENPL_PIG

50451 96115 92698

9.10 6.42 4.75

278 538 534

Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Bos taurus

IF5A_HUMAN FABPE_HUMAN FBX2_HUMAN ALDOA_HUMAN ALDOC_HUMAN ALDOC_HUMAN ALDOC_HUMAN ALDOC_HUMAN ENOG_HUMAN GBB1_BOVIN

16918 15497 33706 39720 39325 39325 39325 39325 47137 37246

5.08 6.60 4.29 8.39 6.46 6.46 6.46 6.46 4.91 5.60

399 184 198 483 130 171 582 523 656 207

Homo sapiens Bos taurus Homo sapiens Canis familiaris Homo sapiens Rattus norvegicus Pan troglodytes Homo sapiens Sus scrofa

DHE3_HUMAN GLNA_BOVIN GLNA_HUMAN GLNA_CANFA GSTM5_HUMAN GSTM4_RAT TPIS_PANTR GSTM3_HUMAN G3P_PIG

61398 41900 41933 41897 25716 25704 26807 26428 35910

7.66 6.41 6.42 6.30 7.30 7.27 6.51 5.37 8.52

259 142 249 141 300 214 151 143 533

species

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Table 1. (Continued) theoretical spot

63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118

2124

protein name

Heat shock 70 kDa protein 1 Heat shock 70 kDa protein 4 Heat shock cognate 71 kDa protein and Vacuolar ATP synthase catalytic subunit A, (EC 3.6.3.14) Heat shock protein HSP 90-alpha (HSP 86) (Fragment) Hexokinase, type I (EC 2.7.1.1) (Brain form hexokinase) Lactoylglutathione lyase (EC 4.4.1.5) Lamin B2 L-lactate dehydrogenase A chain (EC 1.1.1.27) L-lactate dehydrogenase B chain (EC 1.1.1.27) Malate dehydrogenase, cytoplasmic (EC 1.1.1.37) Mitochondrial inner membrane protein (Mitofilin) (p87/89) Mu-crystallin NADH-ubiquinone oxidoreductase 75 kDa subunit, mitochondrial precursor (EC 1.6.5.3) Neurofilament triplet L protein (68 kDa neurofilament protein) (66 kDa neurofilament protein) and Alpha-internexin Neurofilament triplet H protein [Fragment] Neutral alpha-glucosidase AB precursor (EC 3.2.1.84) and Heat shock 70 kDa protein 4L (Osmotic stress protein 94) NG,NG-dimethylarginine dimethylaminohydrolase 1 (EC 3.5.3.18) (Dimethylargininase 1) NG,NG-dimethylarginine dimethylaminohydrolase 2 (EC 3.5.3.18) (Dimethylargininase 2) Peroxiredoxin-2 (EC 1.11.1.15) (Thioredoxin peroxidase 1) Peroxiredoxin 6 (EC 1.11.1.15) (Antioxidant protein 2) Phosphatidylethanolamine-binding protein (PEBP) (HCNPpp) Phosphoglycerate kinase 1 (EC 2.7.2.3) Phosphoglycerate mutase 1 (EC 5.4.2.1) Protein disulfide-isomerase A3 (EC 5.3.4.1) Protein disulfide-isomerase precursor (EC 5.3.4.1) (PDI) and Vimentin (Fragment) PRP19/PSO4 homolog (Neuronal differentiation-related gene protein) Puromycin-sensitive aminopeptidase (EC 3.4.11.-) (PSA) Pyruvate kinase, isozyme M1 (EC 2.7.1.40) Pyruvate kinase, M1 isozyme (EC 2.7.1.40) Recoverin Retinol-binding protein I, cellular (Cellular retinol-binding protein) (CRBP) S-arrestin (retinal S-antigen) S-arrestin (retinal S-antigen) Secernin 1 Selenium-binding protein 2 (56 kDa acetaminophen-binding protein) (AP56) Serine/threonine protein phosphatase 2A, 65 kDa regulatory subunit A, alpha isoform (PP2A, subunit A) Serotransferrin [Precursor] Serotransferrin [Precursor] Serotransferrin [Precursor] Serum albumin [Precursor] Serum albumin [Precursor] and Annexin A6 (Annexin VI) Serum albumin [Precursor] Serum albumin [Precursor] and Dihydropyrimidinase-related protein 2 (DRP-2) Superoxide dismutase [Cu-Zn] (EC 1.15.1.1) Superoxide dismutase [Mn], mitochondrial (EC 1.15.1.1) Superoxide dismutase [Mn], mitochondrial (EC 1.15.1.1) Superoxide dismutase [Mn], mitochondrial (EC 1.15.1.1) Syntaxin binding protein 1 (Unc-18 homolog) (N-Sec1) T-complex protein 1, alpha subunit (TCP-1-alpha) (CCT-alpha) T-complex protein 1, epsilon subunit (TCP-1-epsilon) (CCT-epsilon) T-complex protein 1, eta subunit (TCP-1-eta) (CCT-eta) Transitional endoplasmic reticulum ATPase (TER ATPase) (VCP) Translationally controlled tumor protein (TCTP) Transthyretin precursor (Prealbumin) (TBPA) (TTR) (ATTR) Tubulin alpha-1 chain (Alpha-tubulin 1) Tubulin beta chain (T beta-15)

Journal of Proteome Research • Vol. 6, No. 6, 2007

accession no.

MWb

pIc

scored

Canis familiaris Homo sapiens Homo sapiens Sus scrofa Equus caballus Rattus norvegicus Homo sapiens Homo sapiens Bos taurus Homo sapiens Sus scrofa Homo sapiens

HSP71_CANFA HSP74_HUMAN HSP7C_HUMAN VATA1_PIG HS90A_HORSE HXK1_RAT LGUL_HUMAN LAM2_HUMAN LDHA_BOVIN LDHB_HUMAN MDHC_PIG IMMT_HUMAN

70312 95096 71082 68617 83445 103540 20803 67762 36785 36507 36585 84026

5.48 5.18 5.37 5.42 5.00 6.29 5.25 5.29 8.18 5.72 6.15 6.08

626 301 629 103 540 250 180 171 471 150 379 250

Macropus fuliginosus Mus musculus

CRYM_MACFL NUAM_MOUSE

33931 80724

6.25 5.51

98 348

Bos taurus

NFL_BOVIN

62534

4.59

639

Homo sapiens Sus scrofa Mus musculus Homo sapiens Bos taurus

AINX_HUMAN NFH_PIG GA2A_MOUSE HS74L_HUMAN DDH1_BOVIN

55528 16204 107300 95453 31423

5.34 5.99 5.67 5.63 5.67

108 559 152 78 232

Homo sapiens

DDAH2_HUMAN

29911

5.66

278

Homo sapiens Rattus norvegicus Macaca fascicularis

PRDX2_HUMAN PRDX6_RAT PEBP_MACFA

21761 24729 20868

5.67 5.65 7.42

431 283 268

Equus caballus Mus musculus Homo sapiens Rattus norvegicus Cricetulus griseus Rattus norvegicus

PGK1_HORSE PGAM1_MOUSE PDIA3_HUMAN PDIA1_RAT VIME_CRIGR PRP19_RAT

44842 28797 56782 57315 51874 55670

8.64 6.75 5.98 4.82 4.94 6.17

546 610 232 431 115 306

Homo sapiens Felis catus Homo sapiens Canis familiaris Bos taurus

PSA_HUMAN KPYM_FELCA KPY1_HUMAN Q8MIH6 RET1_BOVIN

103895 58391 58339 23382 15860

5.49 7.23 7.95 5.14 5.12

455 397 522 211 237

Canis familiaris Canis familiaris Mus musculus Mus musculus

ARRS_CANFA ARRS_CANFA SCRN1_MOUSE SBP2_MOUSE

45177 45377 46924 53165

6.14 6.14 4.67 5.78

194 139 254 174

Sus scrofa

2AAA_PIG

65948

5.00

518

Equus caballus Equus caballus Equus caballus Equus caballus Equus caballus Homo sapiens Equus caballus Equus caballus Bos taurus Equus caballus Homo sapiens Homo sapiens Homo sapiens Homo sapiens Cricetulus griseus

TRFE_HORSE TRFE_HORSE TRFE_HORSE ALBU_HORSE ALBU_HORSE ANXA6_HUMAN ALBU_HORSE ALBU_HORSE DPYL2_BOVIN SODC_HORSE SODM_HUMAN SODM_HUMAN SODM_HUMAN STXB1_HUMAN TCPA_CRIGR

80268 80268 80268 70550 70550 76037 70550 70550 62638 16101 24722 24722 24722 67925 60814

6.83 6.83 6.83 5.95 5.95 5.42 5.95 5.95 5.95 6.04 8.35 8.35 8.35 6.49 5.71

281 555 694 547 244 112 673 166 187 296 73 101 70 545 318

Homo sapiens

TCPE_HUMAN

60089

5.45

480

Mus musculus Mus musculus

TCPH_MOUSE TERA_MOUSE

60127 89936

7.95 5.14

362 557

Sus scrofa Homo sapiens Meriones unguiculatus Rattus norvegicus

TCTP_PIG TTHY_HUMAN TBA1_MERUN TBB1_RAT

19697 15991 50804 50387

4.84 5.52 4.94 4.79

228 104 659 711

species

research articles

Retinal Proteome in ERU Table 1. (Continued)

theoretical spot

protein name

119

Ubiquinol-cytochrome-c reductase complex core protein I, mitochondrial precursor (EC 1.10.2.2) Ubiquitin carboxyl-terminal hydrolase isozyme L1 (EC 3.4.19.12) (UCH-L1) Ubiquitin-activating enzyme E1 Ubiquitin-activating enzyme E1 (A1S9 protein) Vacuolar ATP synthase subunit B, brain isoform (EC 3.6.3.14) (V-ATPase B2 subunit) Vacuolar ATP synthase subunit B, brain isoform (EC 3.6.3.14) (V-ATPase B2 subunit) Vesicle-fusing ATPase (EC 3.6.4.6) (Vesicular-fusion protein NSF)

120 121 122 123 124 125

accession no.

MWb

pIc

scored

Bos taurus

UQCR1_BOVIN

53444

5.94

248

Equus caballus

UCHL1_HORSE

25245

5.14

377

Oryctolagus cuniculus Homo sapiens Bos taurus

UBE1_RABIT UBE1_HUMAN VATB2_BOVIN

118697 118858 56882

5.51 5.49 5.66

227 224 550

Bos taurus

VATB2_BOVIN

56882

5.66

585

Homo sapiens

NSF_HUMAN

83115

6.38

547

species

a Proteins listed have been identified with a probability score that is significant with p < 0.05. b Theoretical molecular weight of the identified protein (Dalton). c Theoretical isoelectric point of the identified protein (pI). d Probability based MOWSE score as given by Mascot scores >67 are significant (p < 0.05) for this analysis.

NaOH (pH 8.8) buffer. We used monoclonal mouse antibodies specific for vimentin (Sigma, 1:400) and PEDF (Millipore, 15 µg/mL) for candidate detection in tissues. Additionally, goat anti-equine interferon-gamma (R&D Systems, 15 µg/mL) was used to detect the expression pattern of this pro-inflammatory cytokine typical for autoimmune uveitis. For fluorescence labeling, vimentin and PEDF were stained with anti-mouse IgG antibodies coupled to Alexa 488 or 565 (1:200, Invitrogen), respectively, and IFN-gamma was detected with an anti-goat IgG antibody coupled to Alexa 546 (1:200, Invitrogen).

Results Proteomic Map of Normal Equine Retina. We first obtained a proteomic map of normal equine retinal proteins (Figure 1) by 2DE separation. Retinal 2DE patterns were highly reproducible between experiments and among sampled cases. Major landmark proteins could be subsequently and conclusively identified by mass spectrometry (Table 1). Typical proteins representing distinct retinal cell types, e.g., as carbonic anhydrase II (spot number 31), glutamine synthetase (56-58), and cRALBP (32) for Mueller glial cells or S-antigen (94-95) for photoreceptors, could be also detected reliably. The latter two proteins, as well as recoverin (spot number 92), are also known

as major autoantigens in several uveitis models.2,6,8 We also identified key components of nerve fiber layers, such as neurofilament triplet L (76) and H (77) proteins or alpha internexin (19). Uveitis is Associated with Breakdown of Blood-Retinal Barrier. Comparison of the healthy retinal proteome with two different stages of uveitic disease revealed marked differences between overall protein patterns (Figure 2, A-C). As expected, the breakdown of blood-retinal barrier was evident by release of classical serum proteins into retinal tissue (boxed areas 1: alpha-1-antitrypsin; boxed areas 2: albumin; boxed areas 3: transferrin; boxed areas 4: immunoglobins 1 and 4 heavy chain constant region; boxed areas 5: hemoglobin) whose levels increased with disease progression, as seen in the comparisons between the healthy proteome (A), intermediate developed disease ((B), 4 uveitic attacks) and advanced disease stage ((C), >10 attacks). A significant amount of hemoglobin was only detected in the advanced disease stages ((C), boxed area 5). Upregulated Proteins in Uveitic Condition. Twelve proteins could be identified by 2DE in ERU cases showing upregulation in diseased retina (Figures 2 and 3; Table 2). Seven of these comprised classical plasma proteins: alpha-1-antitrypsin, albumin, transferring, immunoglobin gamma 1 heavy chain

Table 2. Differentially Expressed Proteins in ERU Retinaa protein name

species

accession number

Alpha-1-antitrypsin Serum albumin Serotransferrin Immunoglobin gamma 1 heavy chain constant region Immunoglobin gamma 4 heavy chain constant region Hemoglobin, subunit alpha Hemoglobin, subunit beta Apolipoprotein A-I Aldose 1-epimerase Selenium-binding protein 1 (SP56) Alpha crystallin A chain SET protein (Phosphatase 2A inhibitor I2PP2A) Glial fibrillary acidic protein Vimentin Glutamine synthetase Pigment epithelium-derived factor Interferon gamma

Equus caballus Equus caballus Equus caballus Equus caballus Equus caballus Equus caballus Equus caballus Canis familiaris Bos taurus Mus musculus Equus caballus Mus musculus Homo sapiens Cricetulus griseus Bos taurus Mus musculus Equus caballus

A1AT2_HORSE ALBU_HORSE TRFE_HORSE CAC44760 CAC44762 HBA_HORSE HBB_HORSE APOA1_CANFA GALM_BOVIN SBP1_MOUSE CRYAA_HORSE SET_MOUSE GFAP_HUMAN VIME_CRIGR GLNA_BOVIN PEDF_MOUSE IFNG_HORSE

expression in uveitis

v v v v v v v v v v v v v vb Vc Vc vb

a Proteins were identified from 2DE gels by mass spectrometry as specified in the Supporting Information. Regulated proteins identified by western blot and/or immunofluorescence labeling are indicated (b mode of regulation found in retinal sections by immunodetection; c mode of regulation found on 1D and 2D western blots and by immunodetection on retinal sections). Protein upregulation in ERU compared to normal retinas is indicated as v; downregulation is indicated as V.

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Figure 1. Healthy equine retinal proteome. Representative silver-stained two-dimensional gel electrophoresis (2DE) gel of the healthy equine retina (pH gradient from 3 to 11). Molecular masses of standards (MW) and pI are indicated. Protein spot numbers refer to mass spectrometric identifications, as given in Table 1.

Figure 2. Increased abundance of serum-derived proteins indicates break down of blood-retinal barrier. Representative silver-stained 2DE of healthy retina (A) compared to spontaneous uveitis cases with different severity of disease ((B) intermediate developed disease, 4 uveitic attacks; (C) advanced disease stage, > 10 attacks); pH gradient from 3 to 11. Boxed areas indicate localization of serumderived proteins increasing with ERU disease severity, 1: alpha-1-antitrypsin; 2: albumin; 3: transferring; 4: immunoglobin gamma 1 heavy chain constant region and immunoglobin gamma 4 heavy chain constant region; 5: hemoglobin, alpha and beta chain. Detailed informations on respective mass spectrometric identifications are listed in supplemental Table 2 and Supplemental figure (see Supporting Information).

constant region and immunoglobin gamma 4 heavy chain constant region, hemoglobin, alpha and beta chain (Figure 2) and apolipoprotein A1 (Figure 3A). Further, significantly higher abundant proteins in diseased stage were aldose 1-epimerase (Figure 3B), selenium-binding protein 1 (Figure 3C), alpha crystallin A chain (Figure 3D), SET protein (Phosphatase 2A inhibitor I2PP2A; Figure 3E) and glial fibrillary acidic protein (GFAP; Figure 3F). Differentially Expressed Mueller Glial Proteins Indicate Critical Role for RMG in Uveitis. First we confirmed the upregulation of GFAP found on 2D gels by 1D and 2D Western 2126

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blots and found significant upregulation (Figure 4A). Because the prominent upregulation of GFAP, a class III intermediate filament, suggested for a critical role of Mueller glial cells in the pathogenesis of recurrent uveitis, we also examined the regulation of other Mueller glia-specific proteins (Figure 4). Glutamine synthetase (GS) was downregulated with disease progression (Figure 4B). Upregulation of GFAP/vimentin accompanied by GS downregulation is typical for activated Mueller glial cells.15 Pigment epithelium-derived factor (PEDF), a neurotrophic factor and a potent angiogenesis inhibitor, which we previously detected as significantly downregulated

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glial cells during activation (Figure 4H). Double staining for vimentin (green) and interferon-gamma (red) in normal (Figure 4I) and uveitic retinas (Figure 4J) confirmed that differential expression for these candidate proteins is co-localized to Mueller glial cells (yellow). In the uveitic condition, retinal Mueller glial cells upregulate vimentin and interferon-gamma, an event associated with a significant reduction in PEDF expression.

Discussion

Figure 3. Quantification of significantly upregulated proteins in spontaneous uveitis. ERU proteome profiles demonstrated several significantly upregulated proteins in the diseased state ((AF) shaded bars; arbitrary units) in contrast to healthy controls (white bars) as quantified by image analysis software (( standard deviation). Inserts in each panel depict the appearance of the respective protein spots. Significance levels are: *** p < 0.001; ** p < 0.01. Protein identifications by mass spectrometry are listed in Supplemental Table 2 (see Supporting Information). APO-AI: Apolipoprotein A-I precursor; GALM: Aldose 1-epimerase; SBP1: Selenium-binding protein 1; CRYAA: Alpha crystallin A chain; SET: SET protein (Phosphatase 2A inhibitor I2PP2A); GFAP: Glial fibrillary acidic protein.

in uveitic vitreous specimens (Deeg et al., Proteomics, in press), was also comparatively and significantly downregulated in retinal tissue in our studies (Figure 4C). Activation of Mueller Glial Cells is Associated with Upregulation of Pro-Inflammatory Cytokine IFN Gamma. To validate the potential of regulated candidates pointing to a fundamental role of Mueller glial cells, the expression of GFAP, vimentin, GS, and PEDF was assessed by immunohistochemistry in both normal and ERU cases as shown in Figure 5. Similar protein regulation patterns as previously shown by Western blot quantification could be also detected directly in uveitis target tissue. Whereas activated Mueller glial cells disorganize and upregulate vimentin (Figure 4E and F), PEDF, which is co-localized partly with vimentin expression in healthy retina (data not shown), is significantly downregulated in uveitis (Figure 4A and B). We further characterized Mueller glial activation by staining for the pro-inflammatory cytokine interferon-gamma, a classical T-helper 1 cell cytokine, where presence was not detected in normal retinal tissue (Figure 4G). Interestingly, interferon-gamma was upregulated by Mueller

Analysis of protein expression profiles of targeted tissue in diseased and normal state by a nonhypothesis driven approach enables the identification of key molecules involved in the disorder and subsequently, pathogenesis-associated biological pathways. Although many reports have revealed the altered function of immune components in autoimmune diseases, the portion and involvement of the damaged organ itself remains unclear. In the present proteomic study, healthy equine retinal control samples were compared to animals presenting with clinical signs of uveitis. We could identify 17 differentially regulated proteins in the uveitic retinas (Table 2). As expected, several proteins belong to the high-abundant plasma proteins, and therefore, their appearance reflects the damage of the blood-retinal barrier that is associated with the disease. One such protein detected in ERU cases is alpha-1-antitrypsin (Figure 2, inset 1). Alpha-1-antitrypsin belongs to the classical high abundance plasma proteins16 and is the major serine proteinase inhibitor in plasma.17 Circulating levels of alpha-1antitrypsin rise 2-3-fold during inflammation, mostly being produced in the liver.17 It is therefore unclear whether increased levels of alpha-1-antitrypsin in the uveitic retina are indicative of a permeable blood-retinal barrier allowing passive transfer of plasma components18 or directly associated to retinal inflammation. In contrast, high expression of classical plasma proteins such as serum albumin (Figure 2, inset 2), serotransferrin (Figure 2, inset 3), and hemoglobin subunits alpha and beta (Figure 2, inset 5) clearly indicate blood-retinal barrier permeability. Amounts of these proteins with very high plasma abundance increased with severity of disease (Figure 2A, B, and C, controls, ERU-intermediate, and ERU-advanced, respectively) and therefore directly mirror extent of tissue leakage.16 The appearance of immunoglobulin gamma heavy chains (Figure 2, inset 4) could also just display a transfer of high abundant plasma proteins through leaky inner or outer bloodretinal barriers. However, it could also be related to the immune pathogenesis of recurrent uveitis.19 Although uveitis is a T-cell mediated autoimmune disease driven by T-helper 1 cells,20 autoantibodies are detectable in the sera of human uveitis patients21,22 and equine cases with spontaneous uveitis.6,18 Since intraocular tetanus-specific antibodies can also be detected in intraocular specimens of ERU cases,18 this points to an unspecific transfer of all IgGs after blood-retinal barrier disruption. Here, the level of immunoglobulin heavy chains present in retina is proportional to disease severity (Figure 2, inset 4). We also observed that apolipoprotein A1, a classical and high abundant serum protein, increased in the uveitic retinal proteome16 (Figure 3A, Apo A1). Apo A1, is a lipoprotein expressed in retinal pigment epithelium cells23 with important function in lipid transportation. Although altered expression of Apo A1 has not been reported so far in autoimmune uveitis, this protein is elevated in the vitreous of patients with diabetic macular edema24 and in age-related extramacular drusen.25 Since the endothelial membrane deposition of complement Journal of Proteome Research • Vol. 6, No. 6, 2007 2127

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Figure 4. 2DE Western blots and quantification of 1D Western blots of retinal Mueller glial cell-derived protein expression during uveitis. Proteins from ERU retinas and healthy controls were separated by 2DE or 1D PAGE, blotted and probed for GFAP (A), glutamine synthetase (B), or PEDF (C). Signal intensities on 1D blots were quantified with image analysis software (( standard deviation). Inserts in each histogram show the appearance of the respective protein bands and anti-actin Western blot signals as loading control. Significance levels are: ** p < 0.01; * p < 0.05.

complexes induces increased binding of Apo A1 to the surface of retinal pigment epithelium (RPE) cells,26 elevated levels in the pathological condition may be directly related to a functional role in disease pathogenesis involving RPE cells rather than simply reflecting blood-retinal barrier breakdown. Aldose 1-epimerase (GALM), another protein upregulated in ERU cases (Figure 3B), belongs to the aldose epimerase family and acts as mutarotase in the hexose metabolism pathway. This enzyme has been isolated from bacteria, plants, and animals and is present in the cytoplasm of most cells.27 However, the meaning of its elevated expression in uveitis is not known, and so far there is no description of GALM in association with ocular diseases. Probably the increased level of an ubiquitously expressed cytoplasmic enzyme reveals the grade of infiltration of the retina by inflammatory cells.1,28 Also expressed at higher levels in the retinal ERU proteome is R-A-crystallin (Figure 3C; CRYAA), a main physiological constituent of the lens.29 Originally considered to be a lens specific protein providing transparency, extra-lenticular expression of crystallins was also found.30 Many crystallins are expressed in adult mouse retina, and R-A-crystallin is distributed to the ganglion cell layer nuclei and the inner and outer photoreceptor nuclear layers.31 R-Crystallins, which belong to the small heat shock protein family, also act as molecular chaperones and provide an efficient defense mechanism under stress conditions,31 preventing the nonspecific aggregation of denatured proteins and thus playing a possible role in cell survival and genomic stability.31 As such, R-crystallins may function in the protection of retinal neurons from damage by environmental or metabolic stress. Due to these attributes, the elevation of R-A-crystallin observed here can be explained in 2128

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two ways. First, this could be a sign of structural damage to the lens, as this protein is expressed in the lens cup, in the posterior half of the lens vesicle, and in a high concentration in the lens fiber cells.32 Because cataract formation is a complication of advanced recurrent uveitis,33 raised R-Acrystallin expression in the retina could point toward lens fiber disruption. Second, the higher amount of R-A-crystallin in the retinal proteome of ERU cases could be a direct reaction to metabolic stress induced by retinal inflammation. An important finding is the as of yet unaddressed association of the SET-protein (Figure 3D) with recurrent uveitis. This multitasking protein is involved in apoptosis, transcription, nucleosome assembly, and histone binding and is a potent inhibitor of protein phosphatase 2A.34 We observed an upregulation of SET in ERU retinas (Figure 3D), and although the functional significance of this upregulation remains speculative, interesting roles of SET protein in association with uveitis pathogenesis arise. The SET complex is involved in cytotoxic T-lymphocyte (CTL)-induced apoptosis, when the T-cell protease granzyme A cleaves SET and thus initiates a caspase independent cell death pathway characterized by singlestranded DNA nicking.35 Granzyme A activates a DNAase (GzmA-activated DNAase) within the SET complex to produce a novel form of DNA damage during cytotoxic T lymphocytemediated death. Because the inflammation in autoimmune uveitis is mainly T-cell driven, SET upregulation possibly points to a critical role of cytotoxic T-cells at all stages of uveitis. Experimental evidence has shown that cytotoxic T-cells participate in early phases of the immune response of active Behcet’s disease, constitutively expressing perforin and granzymes in agreement with their cytolytic pontential.36 The

Retinal Proteome in ERU

research articles dysfunctional in schizophrenia.37 In this region, SBP1 expression increased intraglially but decreased intraneuronally, and further, SBP1 was upregulated in peripheral blood samples of patients. The authors proposed SBP1 as a leading schizophrenia biomarker.37 We plan to examine precise SBP1 expression patterns of different retinal cell types in spontaneous recurrent uveitis.

Figure 5. Evaluation of regulated Mueller glial cell associated candidates in retinal tissue. Nomarski image of normal equine retina (C) compared to uveitic retina (D) shows a disorganization of retinal structure with loss of photoreceptor outer segments in the diseased state. Vimentin expression (green) in the normal (E) and uveitic (F) retinal Mueller glial cells demonstrated an upregulation in the diseased state. PEDF expression is high in morphological structures that resemble RMG in normal equine retina (A). In contrast, in ERU condition (B) PEDF is downregulated throughout the retina. Interferon-gamma is not detectable in healthy retina (G). However, clear upregulation occurs in RMG in the diseased state (H). Double labeling of vimentin (green) and interferon-gamma (red) in normal retina (I) and uveitic retina (J) clearly demonstrates colocalization (yellow) of interferon-gamma and vimentin in RMG in recurrent uveitis (J).

possible role of CD8+ T-cells in autoimmune uveitis involving both granzyme A and SET complex therefore also merits closer examination in the context of autoimmune uveitis. We also observed a higher expression of selen binding protein 1 (SBP1) in uveitic retinas (Figure 3E) compared to healthy controls. Little is known about the physiological function of SBP1 beyond its clear role in binding the antioxidant selenium.37 Interestingly, an altered SBP1 expression in the dorsolateral prefrontal cortex of the brain of schizophrenic patients has been described, a brain region that has also been

Interestingly, we observed significant upregulation of two Mueller glial cell (RMG) markers, glial fibrillary acidic protein (GFAP; Figures 3E, 4A), and vimentin (Figure 5F) in retinal ERU proteomes. The increased expression of these two class-III intermediate filaments points to an altered function of RMG in association with uveitis. Differential changes in GFAP expression in RMG is the most sensitive nonspecific response to retinal diseases and injuries and can be used as a universal early cellular marker for retinal injury.38 Additionally, during the specific gliotic responses of RMG, the expression of glutamine synthetase (GS) is also reduced. This RMG-specific enzyme is normally involved in neurotransmitter recycling and ammonia detoxification.38 Retinal detachment induced photoreceptor degeneration with consequent loss of glutamatereleasing neurons leads to reduced GS expression levels in RMG.39 Because activated RMG typically upregulate both GFAP and vimentin with a concomitant GS downregulation, we additionally examined GS regulation in uveitic retinas, to evaluate RMG responses in the diseased state. GS is significantly downregulated in ERU (Figure 4B). As RMG are the dominant type of macroglial cells in the retina, they play a crucial role in neuronal support.38 From the early stages of retinal development, RMG are essential in creating and maintaining the neuroretinal architecture40 and support neuronal survival and fundamental information processing.41 The importance of RMG in maintaining retinal structure and function is elucidated by the observation that selective RMG destruction causes retinal dysplasia, photoreceptor apoptosis, and, at a final state, retinal degeneration and proliferation of the RPE.42,43 Because the protein expression profile of major RMG activation markers points to a crucial role of this cell type in uveitic state, we next examined the RMG-derived neurotrophic factor pigment epithelium-derived protein (PEDF). PEDF occurs natively in the eye where it counteracts the stimulatory activity of angiogenesis inducers, thus preventing ocular neovascularization under normal conditions.44 We have previously observed significant downregulation of PEDF in ERU vitreous (Deeg et al., Proteomics, in press). In this study, we now also found a distinct PEDF downregulation (Figure 4C, Figure 5B) in ERU retinas. Because we also have recently demonstrated VEGF upregulation in uveitic retinas (Deeg et al., Proteomics, in press), we were further interested in the direct linkage of RMG activation and their modulation of immune and inflammatory responses. It has been speculated that RMG can act as antigen-presenting cells in inflammatory condition and upregulate pro-inflammatory cytokines.45 We studied interferon-gamma (IFN-γ) as typical T-helper-1 cytokine46 and could establish a direct link between activated RMG and IFN-γ expression by immunohistochemical colocalisation of both vimentin and IFN-γ (Figure 5J, coexpression (yellow) of vimentin (green) and IFN-γ (red) in spontaneous uveitis). Retinal IFN-γ expression was restricted to inflamed tissues only (Figure 5H) and could not be detected in the normal retina (Figure 5G). These findings suggest a critical role for RMG in the pathogenesis of autoimmune uveitis. Journal of Proteome Research • Vol. 6, No. 6, 2007 2129

research articles Conclusions Systematic exploration of target tissue proteomes for spontaneous uveitis in comparison to healthy controls enabled the identification of several differentially regulated proteins participating in pathways underlying immune response and maintenance of the blood-retinal barrier. Importantly, Mueller glial cells come into focus such that additional studies to unravel their involvement in the pathogenesis of uveitis deserve attention.

Abbreviations ApoA1, apolipoprotein A1; CFA, complete Freund’s adjuvans; cRALBP, cellular retinaldehyde binding protein; CRYAA, R-crystallin; ERU, equine recurrent uveitis; GALM, aldose 1-epimerase; GFAP, glial acidic fibrillary protein; GS, glutaminsynthetase; IRBP, interphotoreceptor retinoid binding protein; PBL, peripheral blood-derived lymphocytes; PEDF, pigment epithelium derived factor; PP2A, protein phosphatase 2A; RMG, retinal Mueller glial cell; S-Ag, S-antigen; SBP1, selenium-binding protein 1; Th cell, T-helper cell;

Acknowledgment. The authors would like to thank Dr. Ursula Olazabal for critical discussions. This work was supported by the Deutsche Forschungsgemeinschaft (DFG) SFB 571 and DE 719/2-1; EU grant EVI-GENORET LSHG-CT-2005512036; and the German Federal Ministry of Education and Research: BMBF-QuantPro 0313865A. Supporting Information Available: Supplementary Figure 1 and Supplementary Tables 1 and 2. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) de Smet, M. D.; Chan, C. C. Regulation of ocular inflammationwhat experimental and human studies have taught us. Prog. Retin. Eye Res. 2001, 20(6), 761-797. (2) Deeg, C. A.; Amann, B.; Raith, A. J.; Kaspers, B. Inter- and intramolecular epitope spreading in equine recurrent uveitis. Invest. Ophthalmol Vis. Sci. 2006, 47(2), 652-656. (3) Gilger, B. C.; Malok, E.; Cutter, K. V.; Stewart, T.; Horohov, D. W.; Allen, J. B. Characterization of T-lymphocytes in the anterior uvea of eyes with chronic equine recurrent uveitis. Vet. Immunol. Immunopathol. 1999, 71(1), 17-28. (4) Verma, A.; Artiushin, S.; Matsunaga, J.; Haake, D. A.; Timoney, J. F. LruA and LruB, novel lipoproteins of pathogenic Leptospira interrogans associated with equine recurrent uveitis. Infect. Immun. 2005, 73(11), 7259-7266. (5) Martin, T. M.; Smith, J. R.; Rosenbaum, J. T. Anterior uveitis: current concepts of pathogenesis and interactions with the spondyloarthropathies. Curr. Opin. Rheumatol. 2002, 14(4), 337341. (6) Deeg, C. A.; Pompetzki, D.; Raith, A. J.; Hauck, S. M.; Amann, B.; Suppmann, S.; Goebel, T. W.; Olazabal, U.; Gerhards, H.; Reese, S.; Stangassinger, M.; Kaspers, B.; Ueffing, M. Identification and functional validation of novel autoantigens in equine uveitis. Mol. Cell. Proteomics 2006, 5(8), 1462-1470. (7) Caspi, R. R.; Roberge, F. G.; Chan, C. C.; Wiggert, B.; Chader, G. J.; Rozenszajn, L. A.; Lando, Z.; Nussenblatt, R. B. A new model of autoimmune disease. Experimental autoimmune uveoretinitis induced in mice with two different retinal antigens. J. Immunol. 1988, 140(5), 1490-1495. (8) de Smet, M. D.; Bitar, G.; Mainigi, S.; Nussenblatt, R. B. Human S-antigen determinant recognition in uveitis. Invest. Ophthalmol. Vis. Sci. 2001, 42(13), 3233-3238. (9) Jungblut, P. R.; Zimny-Arndt, U.; Zeindl-Eberhart, E.; Stulik, J.; Koupilova, K.; Pleissner, K. P.; Otto, A.; Muller, E. C.; SokolowskaKohler, W.; Grabher, G.; Stoffler, G. Proteomics in human disease: cancer, heart and infectious diseases. Electrophoresis 1999, 20(10), 2100-2110.

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Journal of Proteome Research • Vol. 6, No. 6, 2007 2131