Changes in Matrix Metalloproteinase Network in a Spontaneous Autoimmune Uveitis Model

Immunology and Microbiology Changes in Matrix Metalloproteinase Network in a Spontaneous Autoimmune Uveitis Model

Florian Hofmaier,1 Stefanie M. Hauck,2 Barbara Amann,1 Roxane L. Degroote,1 and Cornelia A. Deeg1

PURPOSE. Autoimmune uveitis is a sight-threatening disease in ized by repeated episodes of ocular inflammation mediated by which autoreactive T cells cross the blood-retinal barrier. Mo- CD4ϩ T cells targeting retinal antigens, it eventually leads to lecular mechanisms contributing to the loss of eye immune blindness.2–5 Most important, ERU is the only spontaneous privilege in this autoimmune disease are not well understood. animal model for human autoimmune uveitis1,2,5–7 and thus In this study, the authors investigated the changes in the matrix provides the advantage of studying underlying pathomecha- metalloproteinase network in spontaneous uveitis. nisms of a recurrent autoimmune disease on a variable genetic 3,8 METHODS. Matrix metalloproteinase (MMP) MMP2, MMP9, and background, as is the case in human autoimmune uveitis. MMP14 expression and tissue inhibitor of metalloproteinase The transfer value of ERU as a model for human autoimmune (TIMP)-2 and lipocalin 2 (LCN2) expression were analyzed uveitis was already proven because CRALBP, an autoantigen first discovered in the horse model,5 was found to be a fre- using Western blot quantification. Enzyme activities were ex- 9 amined with zymography. Expression patterns of network can- quently targeted autoantigen among humans with uveitis. didates were revealed with immunohistochemistry, comparing In one of our previous studies comparing the vitreous of physiological appearance and changes in a spontaneous recur- healthy and ERU-affected eyes with the use of two-dimensional rent uveitis model. gel electrophoresis, we identified tissue inhibitor of metalloproteinases (TIMP)-2 in healthy vitreous samples.7 RESULTS. TIMP2 protein expression was found to be decreased In general, TIMPs are known as modulators of matrix met- in both the vitreous and the retina of a spontaneous model for alloproteinase (MMP) activity.10 This is important because autoimmune uveitis (equine recurrent uveitis [ERU]), and MMPs play a major role in the degradation of extracellular TIMP2 activity was significantly reduced in ERU vitreous. Func- matrix components and expand their function to modify cyto- tionally associated MMPs such as MMP2, MMP14, and MMP9 kines, protease inhibitors, and cell surface signaling sys- were found to show altered or shifted expression and activity. tems.11–14 Further, migrating immune cells express MMP2 and Although MMP2 decreased in ERU vitreous, MMP9 expression MM9 to overcome blood tissue barriers.15–17 In autoimmune and activity were found to be increased. These changes were diseases such as experimental autoimmune neuritis, MMP2- reflected by profound changes within uveitic target tissue, where and MMP9-mediated protein cleavage is involved in disease.18 TIMP2, MMP9, and MMP14 decreased in expression, whereas Because of this modulation of cell-cell or cell-ECM interactions, MMP2 displayed a shifted expression pattern. LCN2, a potential MMPs and their inhibitors, most notably the TIMPs, inherit stabilizer of MMP9, was found prominently expressed in equine crucial relevance in physiological and pathologic mecha- healthy retina and displayed notable changes in expression pat- nisms.11,13 terns accompanied by significant upregulation in autoimmune Representing a unique member of the TIMP family, TIMP2 conditions. Invading cells expressed MMP9 and LCN2. is not only required for MMP2 inhibition, it also mediates CONCLUSIONS. This study implicates a dysregulation or a change pro-MMP2 activation.19–21 Besides its interaction with MMPs, in functional protein-protein interactions in this TIMP2-associ- TIMP2 is known to function in the inhibition of angiogenesis22 ated protein network, together with altered expression of and in neuroprotection.23 functionally related MMPs. (Invest Ophthalmol Vis Sci. 2011; Given that we found TIMP2 to be downregulated in ERU 52:2314–2320) DOI:10.1167/iovs.10-6475 vitreous through differential proteome analysis, the objective of the present study was to evaluate TIMP2 expression in the quine recurrent uveitis (ERU) is a spontaneous autoim- retina, the target tissue of uveitis. Interactions of TIMP2 and Emune disease in horses that immunopathologically and MMP14 in the process of modulating MMP2 activity24 and 1,2 clinically resembles human autoimmune uveitis. Character- amelioration of experimental autoimmune uveitis by selective MMP2 and MMP9 inhibition15–17 were reported. This prompted us to investigate these TIMP2 interactors in the MMP network to 1 From the Institute of Animal Physiology, Department of Veteri- gain further insight into TIMP2 involvement in the pathophysiol- nary Sciences, Faculty of Veterinary Medicine, Ludwig-Maximilians- ogy of spontaneous recurrent autoimmune uveitis. University (LMU) Munich, Munich, Germany; and the 2Department of Protein Science, Helmholtz Zentrum Munich–German Research Center for Environmental Health (GmbH), Neuherberg, Germany. Supported by Deutsche Forschungsgemeinschaft Grant DE 719/2-1 METHODS and 2-2. Submitted for publication August 26, 2010; revised October 27, Vitreous Specimen 2010; accepted November 16, 2010. For this study, a total of 85 vitreous specimens were sampled and Disclosure: F. Hofmaier, None; S.M. Hauck, None; B. Amann, processed. Forty-six were derived from healthy eyes, and 39 were None; R.L. Degroote, None; C.A. Deeg, None 4 Corresponding author: Cornelia A. Deeg, Institute of Animal Phys- derived from eyes diagnosed with ERU. Vitreous samples of control iology, Department for Veterinary Sciences, Faculty of Veterinary Med- horses were obtained from horses euthanatized because of incurable icine, LMU Munich, Veterina¨rstrasse 13, 80539 Munich, Germany; and ERU-unrelated disease; vitreous samples of ERU cases were ob- [email protected]. tained during therapeutic pars plana vitrectomy. Vitreous samples

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were stabilized with EDTA-free protease inhibitor (Roche, Mannheim, a zymogram developing buffer for 30 minutes. The developing buffer Germany), then lyophilized, solubilized in ultrapure water, and dia- was renewed, and the gel was incubated at 37°C for 18 hours, followed lyzed against a 50-mM phosphate buffer (ph 7.6). Protein content was by staining with Coomassie dye. TIMP inhibitory activity appeared as determined using the Bradford assay (Sigma, Deisenhofen, Germany). dark bands against a lighter background. TIMP2 was identified at a All animals were treated according to the ARVO Statement for the Use molecular weight previously described for equine TIMP226 by compar- of Animals in Ophthalmic and Vision Research. All horses were pre- ison with a molecular weight marker (PageRuler Prestained Protein sented as patients to the Equine Clinic of LMU Munich; no experimen- Ladder; Fermentas, St. Leon-Rot, Germany). To ensure the specificity of tal animals were used in this study. the reverse zymography for TIMP activity detection, vitreous samples were electrophoresed on a 12% SDS-gel without substrate. Western Blot Analysis For Western blot analysis, the sample input onto the gels was normal- Immunohistochemistry ized to equal total protein loading. Vitreous samples were resolved by Paraffin-embedded ERU (n ϭ 15) and healthy retinal tissue (n ϭ 10) 8% or 12% SDS-PAGE and blotted semidry on polyvinylidene difluoride samples were sectioned at 8 ␮m. Histopathologic changes were eval- membranes (GE Healthcare, Freiburg, Germany). Unspecific binding uated on sections stained with hematoxylin and eosin. For immuno- was blocked for 1 hour with 1% polyvinylpyrrolidone (PVP) in PBS-T. histochemistry, heat antigen retrieval was performed at 99°C for 15 Blots were incubated with primary antibodies at 4°C overnight (mono- minutes in 0.1 M EDTA-NaOH buffer (ph 8.8). Monoclonal mouse clonal mouse antibody specific for human MMP2 with reported cross- antibody specific for MMP2 (Millipore, Schwalbach, Germany; 1:100) reactivity to horse MMP2 [Millipore, Schwalbach, Germany; 1:500]), and specific for TIMP2 (Biozol, Erding, Germany; 1:25), polyclonal rabbit antibody specific for human MMP9 (Enzo Life Sciences, Lo¨rrach, rabbit antibody specific for MMP-9 (Enzo Life Sciences, Lo¨rrach, Ger- Germany; 1:1000) or monoclonal mouse antibody specific for TIMP2 many; 1:100), polyclonal rabbit antibody specific for human lipocalin (Biozol, Erding, Germany; 1:500), washed, and incubated with horse- 2 (LCN2; Abcam, Cambridge, UK; 1:500), and polyclonal rabbit anti- radish peroxidase-coupled secondary anti-mouse IgG antibody (Sigma- body for human MMP-14 (MT-MMP1; Santa Cruz Biotechnology, Aldrich, Deisenhofen, Germany; 1:5000) or anti rabbit IgG (Sigma- Heidelberg, Germany; 1:100) were used for candidate detection in Aldrich; 1:3000), respectively. Signals were detected with enhanced tissue. For fluorescence labeling, anti-mouse IgG antibody coupled to chemoluminescence on x-ray film (Fuji; Christiansen, Planegg, Ger- Alexa488 or Alexa568, as well as anti-rabbit IgG coupled to Alexa488 many). or Alexa568 or Alexa 647 (all from Invitrogen, Karlsruhe, Germany; Films were scanned on a transmission scanner, and quantification 1:500), were used, respectively. Cell nuclei were counterstained with of Western blot signals by densitometry was performed (Im- DAPI (Invitrogen, Karlsruhe, Germany; 1:1000). Multiple labeling was ageQuantTL software; GE Healthcare). TIMP2 and MMP2 abundances performed consecutively, with blocking steps (ProteinBlock; Dako, between ERU cases and controls were statistically analyzed using the Hamburg, Germany) between single antibody incubations. Fluores- Mann–Whitney U test. The differences were considered significant if cence staining was photographed (Axio Imager M1; Zeiss, Go¨ttingen, Ͻ P 0.05. Germany) and visualized with digital microscopy software (Axio Vision 4.6; Zeiss). Gelatin Zymography Gelatinolytic activity of MMP2 and MMP9 in vitreous samples was determined using gelatin zymography. Ten micrograms of vitreous RESULTS protein per specimen were resolved by 8% SDS-PAGE containing gelatin (1 mg/mL; Sigma-Aldrich) under denaturing, but nonreducing, TIMP2 Activity Is Significantly Reduced in conditions. Gels were washed in 2.5% Triton X-100 (Sigma-Aldrich) for ERU Vitreous 45 minutes at room temperature, then transferred to zymogram devel- MMPs are inhibited by specific endogenous TIMPs, a group of oping buffer, pH 7.6, containing 50 mM Tris, 0.2 M NaCl, 5 mM CaCl 2 peptidases involved in degradation of the extracellular matrix. (all from Applichem, Darmstadt , Germany), and 0.02% surfactant (Brij TIMP2 is constitutively expressed in healthy horse vitreous.7 35; Sigma-Aldrich) and incubated for 30 minutes. The developing TIMP2 has a unique role among TIMP family members because buffer was renewed, and gels were incubated at 37°C for 18 hours. of its ability to directly suppress the proliferation of endothelial Gels were stained with Coomassie dye (Coomassie Blue R250; Sigma- 25 cells. As a result, the encoded protein may be critical to the Aldrich). Gelatinolytic activity was detected as transparent bands on maintenance of tissue homeostasis by suppressing the prolif- a blue background of undegraded substrate. Control gels were incu- eration of quiescent tissues in response to angiogenic factors bated in the presence of 50 mM EDTA or 2 mM phenylmethylsulfonyl and by inhibiting protease activity in tissues undergoing re- fluoride (PMSF) to ensure the MMP specificity of the observed gela- modeling of the extracellular matrix. Because of these unique tinolytic bands. Recombinant human pro-MMP2 and pro-MMP9 (both functions, we were interested in changes in TIMP2 expression from ProteaImmune GmbH, Berlin, Germany) were used as positive in association with spontaneous uveitis. Expression of TIMP2 controls. was determined in healthy controls (Fig. 1A, left) and com- To compare MMP activity between controls and ERU samples, gels pared with uveitic vitreous specimens (Fig. 1A, right). A uni- were scanned and inverted to give black bands on a light background. form, significant, downregulation of TIMP2 protein expression Bands were analyzed by densitometry, as already described for West- to 5.7% (Ϯ6.3%) was determined in ERU cases. Next, we ern blot. The mean densitometry software value of all control samples analyzed TIMP activity by reverse zymography (Fig. 1B). Gela- on one gel was set at 100% and put into relation to the measured values tin degradation was inhibited only at 21 kDa in all vitreous of the ERU samples run on the same gel. Pro-MMP9 and pro-MMP2 samples tested, indicating only TIMP2 to be active in vitreous abundances between ERU cases and controls were statistically ana- (Fig. 1B, blue band). In uveitis cases, TIMP2 activity was re- lyzed using the Mann-Whitney U test. The differences were considered duced, which was evident in a markedly decreased area of Ͻ significant if P 0.05. substrate protected by vitreous TIMP2 activity in uveitic samples when compared with controls (Fig. 1B). In addition, in the Detection of TIMP2 by Reverse Zymography upper part of the gel vitreous-derived MMP activity appeared. Vitreous samples were separated on a 12% SDS-gel containing 1 mg/mL Gelatin digestion was obvious at only one band size in control gelatin and 0.8 ␮g recombinant MMP2. Ten micrograms of protein per vitreous (Fig. 1B, white band at 70 kDa) but at one additional sample was loaded onto the gel. Gels were washed in 2.5% Triton band exclusively in ERU cases (Fig. 1B, bands at 70 kDa in ERU X-100 for 45 minutes at room temperature and afterward incubated in cases and additional band at 95 kDa).

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FIGURE 1. Detection of TIMP2 in vitreous samples. (A) TIMP2-speciﬁc Western blot signal, quantiﬁed by densitometry (***P Ͻ 0.001). 94.3% decrease in TIMP2 expression in ERU vitreous (n ϭ 16) compared with control samples (n ϭ 19). Insets above columns: representative West- ern blot bands; left: controls; right: ERU. (B) Reverse zymography gel demonstrating decreased TIMP2 activity in uveitic vitreous compared with control samples.

MMP Activity Changes in Autoimmune Condition next evaluated the expression patterns of TIMP2 and MMP2 in uveitis target tissue, the retina itself. Differential interference con- Because TIMP2 inhibits MMPs, especially MMP2, we next eval- trast (DIC) image of the normal equine retina (Fig. 3A) compared uated MMP activity in the vitreous of controls and ERU cases using gelatin zymography (Fig. 2A2, representative zymogram). with the uveitic retina (Fig. 3B) revealed destruction of the retinal Pro-MMP2 activity was detected in 78 of 79 vitreous specimens structure in uveitic eyes. TIMP2 (Fig. 3C, red) and MMP2 (Fig. 3E, but with higher activity in controls (Fig. 2A, lanes 1–4) than in green) are expressed at photoreceptor inner and outer segments uveitic specimens (Fig. 2A, lanes 5–8). Gelatinolytic activity of of normal equine retinas, which is consistent with the expression 12 MMP2 was reduced in ERU cases to 70% (Ϯ43%) of physiolog- described in other species. TIMP2 expression almost disappears ical pro-MMP2 values (Fig. 2B). Compared with healthy vitre- in the ERU condition (Fig. 3D), whereas MMP2 is strongly upregu- ous, only 11% (Ϯ4%) of MMP2 protein was still expressed in lated in ERU (Fig. 3F). Further, MMP2 expression pattern changes autoimmune uveitis, as revealed by Western blot analysis (Fig. from one restricted to photoreceptor inner segments and weak 2C). Further, MMP9 gelatinolytic activity was evident in 76% of expression in the outer limiting membrane (ONL) to strong ex- ERU vitreous samples, but was mostly undetectable in controls pression at the ONL and additional dispersed expression through- (Fig. 2D). MMP9 activity was 12.65- (Ϯ20.48)-fold increased in out retinal tissue (Fig. 3F). MMP2 seems also to be coexpressed at diseased eyes (Fig. 2D). Accordingly, MMP9 protein expression cell nuclei in the ERU diseased retina (Fig. 3F; turquoise staining increased 17-fold (16.96 Ϯ 14.66) in ERU vitreous (Fig. 2E). pattern at DAPI (blue)-stained cell nuclei, MMP2 expression is green). MMP Network Changes Expression in Target TIMP2 has been also described as a positive regulator of Tissue during Autoimmune Uveitis MMP14 by promoting the availability of the enzyme at the cell Because TIMPs have the capacity to inhibit protease activity in surface and supporting pericellular proteolysis after forming the tissues undergoing remodeling of the extracellular matrix, we trimolecular complex of MMP14, TIMP2, and pro-MMP2. Hence,

FIGURE 2. Analysis of MMP activity in vitreous samples. (A) Representa- tive gelatin zymography of vitreous samples. Lanes 5–8: detection of pro-MMP9 gelatinolytic activity as clear band against blue background at 95 kDa in ERU. Lane 7: active MMP9 at 85 kDa. Lane 10: pro-MMP9 standard. Lanes 1–8: detected pro- MMP2 gelatinolytic activity at 70 kDa in ERU and control samples. Lanes 1, 3, 4, 7: active MMP2 at 62 kDa. Lane 9: pro-MMP2 standard. Proof of MMP-dependent gelatinase activity in vitreous samples. Activity inhibited by EDTA (lanes 11–13) but not by PMSF (lanes 14–16). (B) 30% decline in observed pro-MMP2 gelatinolytic activity in ERU vitreous samples compared with controls and quantification of gelatinolytic bands by densitometry (**P Ͻ 0.01). (C) Reduced pro-MMP2 expression to an average of 11% in ERU vitreous samples and quantification of Western blot signal by densitometry. Insets: representative Western blot signals. (D) Aver- age 12.65-fold increase of pro-MMP9 gelatinolytic activity in ERU vitreous samples and quantification of gelatinolytic band by densitometry (***P Ͻ 0.001). (E) Average 16.96-fold increase of MMP9 protein expression in ERU vitreous as detected by Western blot analysis (*P Ͻ 0.05).

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expression pattern in the equine retina (Figs. 4A, 4C, hematoxylin and eosin staining and DIC image of healthy control retina; 4B, 4D, ERU retina). Among the MMP family, MMP9 is the most important angiogenic factor.27 MMP9 was found to be expressed in ganglion cell layer, inner plexiform layer, and photoreceptor segments in physiological conditions of the equine retina (Fig. 4E, red). In contrast, MMP9 was no longer expressed in uveitic retinal tissue itself (Fig. 4F); rather, inﬁl- trating cells stained positive for MMP9 (Fig. 4F).

FIGURE 3. Shift in retinal expression of TIMP2 and functional associated proteins in uveitis. DIC image of a healthy retina (A) compared with a uveitic retina (B). TIMP2 expression (red) in healthy retina showing an accumulation at the photoreceptor inner and outer segments (C) compared with reduced TIMP2 expression (red) in ERU- affected retinal tissue (D). Expression of MMP2 (green) in healthy retina, where the main immunoﬂuorescence signal is visible at photoreceptor inner and outer segments (E). MMP2 expression pattern (green) is shifted in the uveitic state toward an enhanced signal at the outer limiting membrane and in the outer nuclear layer (F). MMP14 (magenta) is predominantly detected at the inner photoreceptor segments of healthy eyes (G). Decrease in MM14 expression in ERU (H). TIMP2 (red), MMP2 (green), and MMP14 (magenta) are coexpressed at the inner photoreceptor segments of healthy eyes (I, overlay, white), whereas in the uveitic state MMP2 is predominant (J; TIMP2, red; MMP2, green; MMP14, magenta; overlay, white). Cell nuclei are stained with DAPI (C–J, blue).

we also examined MMP14 expression in healthy and uveitic retinal tissue. MMP14 is similar to MMP2 expressed at photore- FIGURE 4. Changes in MMP9 and LCN2 expression patterns in uveitic ceptor inner segments in physiological conditions (Fig. 3G, ma- retina. Hematoxylin and eosin staining of a healthy retina (A) com- genta) but is undetectable in the autoimmune diseased retina (Fig. pared with a retina affected by uveitis (B). The same specimen but 3H). Therefore, in ERU, only an upregulated and changed expres- different sections (DIC images: C, healthy; D, uveitis) is shown stained with antibodies against MMP9 and LCN2. Predominant expression of sion pattern of MMP2 remains, whereas expression of the other MMP9 (red) in photoreceptor segments of healthy retina (E). Uveitic interactors of this network, TIMP2 and MMP14, is no longer retina showing reduced MMP9 expression (red) confined to well- evident in ERU target tissue (Fig. 3I; triple staining, overlay of defined areas (F). LCN2 expression (green) in healthy retina (G) com- expression of TIMP2, MMP2, and MMP14 results in white stain- pared with shifted LCN2 expression pattern in ERU-affected retinal ing; Fig. 3J, only MMP2 (green) expression remains in ERU). tissue (H). Double staining of LCN2 (green) and MMP9 (red), detection of overlay in the inner plexiform layer and at the outer limiting Coexpression of MMP9 and LCN2 Is Limited to membrane of healthy retina (I, overlay, yellow). Coexpression of both Infiltrating Cells in ERU proteins at the site of the remaining MMP9 expression in the retina affected by uveitis (J, overlay, yellow). Cell nuclei are stained with Considerable MMP9 activity was detected in vitreous of spon- DAPI (E–J, blue). Insets: enlargements of representative cells reflecting taneous uveitis cases; therefore, we next evaluated MMP9 the staining pattern of large infiltrating cells in ERU.

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We added analysis of LCN2 expression because both pro- MMP9 ameliorated IRBP-induced experimental autoimmune teins strongly interact and in vitro data suggest a possible role uveitis in mice.17 Interestingly, elevated MMP2 and MMP9 for LCN2 in the protection of MMP9 against autolysis.28 Fur- levels have been described in aqueous humor samples of pa- ther, we anticipated a role of LCN2 in autoimmune uveitis tients with active uveitis.37–39 Despite these reports focusing because in mouse models of retinal degeneration Mu¨ller glia on aqueous humor expression, little is known about the ex- cells were reported to express LCN2 in response to photore- pression of MMP2 or MMP9 in uveitic vitreous. ceptor damage.29 Immunohistochemistry revealed LCN2 ex- In this context, the observed decrease of pro-MMP2 gela- pression in healthy retina in the nerve fiber layer, the inner tinolytic activity and protein expression, combined with a very plexiform layer, the outer nuclear layer, and the outer limiting notable increase in MMP9 gelatinolytic activity in the vitreous membrane (Fig. 4G, green). In contrast, LCN2 was markedly of horses with spontaneous uveitis (Fig. 2), is an important upregulated in ERU (Fig. 4H), and expression patterns shifted finding. Given that MMP2 is known to be a modulator of toward diffuse LCN2 expression throughout whole retinal tis- chemokines, as is the case for monocyte chemoattractant pro- sue in the uveitic state and infiltrating cells (Fig. 4H). MMP9 tein D3, which, after cleavage by MMP2, acts as a general and LCN2 were coexpressed only in the inner plexiform layer chemokine antagonist,40 MMP2 might inherit an anti-inflamma- and at the outer limiting membrane (Fig. 4I) in physiological tory function in healthy vitreous. Therefore, decreases in conditions. Only LCN2 expression was left in ERU retinas (Fig. MMP2 might favor chemotactic attraction of T cells or macro- 4H), but MMP9 coexpression occurred in infiltrating cells (Fig. phages, the predominant cells infiltrating the eyes in ERU.2,41 4J; LCN2, green; overlay of MMP9 and LCN2, yellow). MMP9 is the most important angiogenic factor of the MMP family,27 switching vessels from vascular quiescence to angiogenesis by rendering VEGF more available to its receptors. DISCUSSION Further, in vitro chemical hypoxia of cultured human RPE cells led to increases in VEGF gene expression and secretion of TIMPs play an important role in physiological and pathologic MMP9.42 This hypoxic expression of MMP9 was mediated by mechanisms because they influence matrix metalloproteinases, autocrine VEGF signaling. We have already demonstrated a role which are known to modulate cell-cell or cell-ECM interac- for VEGF in ERU retinas7; therefore, we were interested in tions.11,13,20 The results of this study demonstrate a notable unraveling the meaning of MMP9 upregulation in ERU vitreous. decrease in TIMP2 expression and an altered expression of Surprisingly, MMP9 expression in the uveitic retina was de- several members of the MMP family in target tissues of spon- creased (Fig. 4F), indicating that this might not have been the taneous recurrent uveitis. source of the elevated MMP9 level in uveitic vitreous. T cells, TIMP2 was first identified in healthy horse vitreous in an preferentially Th1 cells, express MMP9 and MMP2 to facilitate initial proteomic experiment, analyzing vitreous proteins cell migration, as is known from in vitro studies on human T changed in ERU by two-dimensional gel electrophoresis.7 In cells derived from patients with multiple sclerosis.43 Further, the present study, we further investigated the expression of MMP9 is secreted by macrophages,15 and inflammatory cells TIMP2 in eyes from horses affected by spontaneous recurrent were thought to be a major provider of MMP9 in experimental uveitis compared with controls. Reduced TIMP2 expression in choroidal neovascularization of mice.44 As mentioned earlier, ERU could be quantified to 94.3% (Ϯ6.31%), revealing a signif- both cell types are the major infiltrating cells in ERU retinas.2,41 icant decrease in uveitic vitreous compared with controls (Fig. They are, therefore, likely to contribute to the alteration of 1A). Subsequently, decreased TIMP2 activity in an ERU vitreous MMP9 expression during ERU, especially because the only specimen was confirmed through reverse zymography (Fig. remaining MMP9 expression in the uveitic retina was detected 1B). Additionally, significant reduction of TIMP2 expression on infiltrating cells (Fig. 4F). In a mouse model of choroidal could be confirmed for the target tissue of recurrent uveitis, neovascularization, neutrophil granulocytes were demon- the retina (Fig. 3D). Despite a report of increased TIMP2 strated to contain MMP9 in intracellular granules that were expression in the aqueous humor of uveitis-related secondary recruited to the neovascular area.44 This could have been the glaucoma,30 to our knowledge no one has yet monitored case in ERU retinas (Fig. 4F insert; high resolution of invading changes in ocular TIMP2 expression in autoimmune uveitis, cell shows MMP9-positive granules); hence, these cells ex- though TIMP2 occurrence has previously been reported in pressing MMP9 in ERU should be further characterized in healthy human retina31,32 and vitreous.33 additional studies. In ERU, activated peripheral T lymphocytes cross the blood In the healthy human retina, MMP2 is reported to be ex- retinal barrier, targeting autoantigens in the retina.2 TIMP2 has pressed in ganglion cell bodies and their axons.32 An increase not only been assigned neuroprotective properties,23 it was in retinal MMP2 expression is known from mouse models of also reported to be capable of preventing in vitro migration of diabetic retinopathy,45 but to date there are no reports on CD3ϩ T cells derived from a diabetic mouse model through an MMP2 expression changes in the retina caused by autoimmune endothelial cell layer.34 In the same study, TIMP2 treatment uveitis. Retinas of horses affected by ERU showed notably also restored insulin levels in an in vitro organ culture model elevated MMP2 expression and altered expression patterns for type 1 diabetes mellitus, which the authors suggested was (Fig. 3F), possibly because of the active involvement of MMP2 caused by the potential TIMP2 capability of preventing auto- in the destruction of retinal tissue or at least in the inflam- reactive T cells from destroying insulin-producing ␤ cells.34 matory process, especially considering the different modu- Therefore, the significant reduction of TIMP2 in spontaneous latory capabilities of MMPs.11,13,14 Another member of the recurrent uveitis target tissue is an important finding because it MMP family, MMP14, which has been detected by Western might contribute to disease pathogenesis, either by loss of the blot analysis in healthy human retinas,46 was expressed at eye’s immune privilege or by lack of neuroprotection. the inner photoreceptor segments of healthy horse eyes One major effect of TIMP2 is its ability to modulate MMP2 (Fig. 3G), but expression decreased notably in retinas af- activity, either supporting MMP2 activation or inhibiting this fected by ERU (Fig. 3H). protease in a dose-dependant manner.19,35 This might be an Additionally, we could for the first time demonstrate the important function in the context of autoimmune uveitis be- coexpression of TIMP2, MMP2, and matrix metalloprotei- cause MMP inhibition by the synthetic inhibitor BB-1101 re- nase-14 (MMP14) in retinas under physiological conditions duced the incidence of S-Ag–induced experimental uveoretini- (Fig. 3I). Because TIMP2 is an important modulator of MMP2 tis in Lewis rats,36 and the specific inhibition of MMP2 and activity19,20 and this effect is also reported to involve interac-

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tion with MMP14,24 coexpression of these three proteins in References healthy retinas might point to an involvement in maintaining tissue integrity by controlling MMP2 activation. In all ERU cases 1. Deeg CA, Hauck SM, Amann B, et al. Equine recurrent uveitis—a investigated, TIMP2 and MMP14 expression was almost unde- spontaneous horse model of uveitis. Ophthalmic Res. 2008;40: 151–153. tectable, whereas MMP2 expression increased and presented 2. Deeg CA, Ehrenhofer M, Thurau SR, Reese S, Wildner G, Kaspers changes in protein expression patterns (Fig. 3J). It has been B. Immunopathology of recurrent uveitis in spontaneously dis- shown in cell culture experiments that the phosphorylation eased horses. Exp Eye Res. 2002;75:127–133. status of MMP2 significantly affects its activity and probably 47 3. Deeg CA, Hauck SM, Amann B, Kremmer E, Stangassinger M, also its substrate specificity. A possible role of caveolin 1 in Ueffing M. Major retinal autoantigens remain stably expressed the modification of MMP2 at the cell membrane was discussed, during all stages of spontaneous uveitis. Mol Immunol. 2007;44: because MMP2 was phosphorylated by protein kinase C (PKC) 3291–3296. in vitro.47 PKC localizes to caveolin-1 at the cell membrane, 4. Deeg CA, Kaspers B, Gerhards H, Thurau SR, Wollanke B, Wildner where MMP2 has been found to associate with caveolin-1,48 G. Immune responses to retinal autoantigens and peptides in suggesting a possible role for this kinase in the phosphoryla- equine recurrent uveitis. Invest Ophthalmol Vis Sci. 2001;42:393– tion of secreted MMP2. Interestingly, we were able recently to 398. demonstrate the increased expression of caveolin-1 at retinal 5. Deeg CA, Pompetzki D, Raith AJ, et al. Identification and functional membranes of ERU cases.49Therefore, we suggest further in- validation of novel autoantigens in equine uveitis. Mol Cell Pro- depth investigations of MMP pathway and protein modifica- teomics. 2006;5:1462–1470. tions of participating candidates in the future for greater un- 6. Deeg CA. Ocular immunology in equine recurrent uveitis. Vet Ophthalmol. 2008;(Suppl 1):61–65. derstanding of the interaction within the network. 7. Deeg CA, Altmann F, Hauck SM, et al. Down-regulation of pigment With respect to MMP9 activity, we believe LCN2 warrants epithelium-derived factor in uveitic lesion associates with focal further exploration. LCN2 is a protein that protects MMP9 from vascular endothelial growth factor expression and breakdown of 28,50 degradation, thereby preserving MMP9 activity. Interest- the blood-retinal barrier. Proteomics. 2007;7:1540–1548. ingly, we could demonstrate LCN2 expression in healthy reti- 8. Deeg CA, Thurau SR, Gerhards H, Ehrenhofer M, Wildner G, nas (Fig. 4G) and, for the first time, evaluate notable changes in Kaspers B. Uveitis in horses induced by interphotoreceptor reti- its expression pattern in spontaneous autoimmune uveitis ret- noid-binding protein is similar to the spontaneous disease. Eur inas (Fig. 4H). This is an important finding, because in autoim- J Immunol. 2002;32:2598–2606. mune uveitis little is known about LCN2 function in the retina. 9. Deeg CA, Raith AJ, Amann B, et al. CRALBP is a highly prevalent Thus far, LCN2 has been identified only in thee aqueous humor autoantigen for human autoimmune uveitis. Clin Dev Immunol. of patients with active uveitis, forming a complex with 2007;2007:39245. MMP9.37 Additionally, LCN2 was found to be linked to other 10. Brew K, Dinakarpandian D, Nagase H. Tissue inhibitors of autoimmune diseases such as autoimmune myocarditis, and to metalloproteinases: evolution, structure and function. Biochim Biophys Acta. 2000;1477:267–283. be expressed in cardiomyocytes, fibroblasts, and neutrophils of 11. Nagase H, Visse R, Murphy G. Structure and function of matrix an induced rat model and in the myocardium of human pa- 51 metalloproteinases and TIMPs. Cardiovasc Res. 2006;69:562–573. tients with myocarditis. The authors suspected an induction 12. Sivak JM, Fini ME. MMPs in the eye: emerging roles for matrix of LCN2 by proinflammatory cytokines such as IL-1 and sug- 51 metalloproteinases in ocular physiology. Prog Retin Eye Res. 2002; gested a cytoprotective role for LCN2. Double staining of the 21:1–14. uveitic retina with MMP9 revealed areas of LCN2/MMP9 coex- 13. Murphy G, Nagase H. Progress in matrix metalloproteinase re- pression restricted to infiltrating cells (Fig. 4J). It has been search. Mol Aspects Med. 2008;29:290–308. described that LCN2 forms a complex with MMP9 in human 14. Malla N, Sjoli S, Winberg JO, Hadler-Olsen E, Uhlin-Hansen L. neutrophil granulocytes,52 but the heterogeneous cell types Biological and pathobiological functions of gelatinase dimers and expressing LCN2/MMP9 in ERU (Fig. 4J) might point to an complexes. Connect Tissue Res. 2008;49:180–184. additional involvement of other cell populations and warrant 15. Goetzl EJ, Banda MJ, Leppert D. Matrix metalloproteinases in further characterization. As mentioned earlier, not only was immunity. J Immunol. 1996;156:1–4. LCN2 expression restricted to infiltrating cells, it was also 16. Wu B, Crampton SP, Hughes CC. Wnt signaling induces matrix strongly expressed in retinal tissue. In line with our observa- metalloproteinase expression and regulates T cell transmigration. tions, LCN2 expression was evident in retinal Mu¨ller glial cells Immunity. 2007;26:227–239. in a rat model of diabetes53 and has been interpreted as a 17. El-Shabrawi Y, Walch A, Hermann J, Egger G, Foster CS. Inhibition reaction of Mu¨ller glial cells to photoreceptor damage in mouse of MMP-dependent chemotaxis and amelioration of experimental 29 autoimmune uveitis with a selective metalloproteinase-2 and -9 models of retinal degeneration. The obvious changes in the inhibitor. J Neuroimmunol. 2004;155:13–20. retinal LCN2 expression pattern of horses affected by ERU are 18. Zhao XL, Li GZ, Sun B, et al. MMP-mediated cleavage of beta- thus an interesting finding and may be linked to the activation dystroglycan in myelin sheath is involved in autoimmune neuritis. of Mu¨ller glia cells, which we can already identify as key Biochem Biophys Res Commun. 2010;392:551–556. 54 players in ERU. 19. Caterina JJ, Yamada S, Caterina NC, et al. Inactivating mutation of This study provides the first report on the simultaneous the mouse tissue inhibitor of metalloproteinases-2(Timp-2) gene decrease of TIMP2 protein expression in the vitreous and alters proMMP-2 activation. J Biol Chem. 2000;275:26416–26422. retina in a spontaneous model of autoimmune uveitis, whereas 20. Kudo T, Takino T, Miyamori H, Thompson EW, Sato H. Substrate functionally associated matrix metalloproteinases showed al- choice of membrane-type 1 matrix metalloproteinase is dictated by tered or shifted expression. These findings implicate a funda- tissue inhibitor of metalloproteinase-2 levels. Cancer Sci. 2007;98: mental change in functional protein-protein interactions in the 563–568. MMP-associated protein network, highlighting the importance 21. Wang Z, Juttermann R, Soloway PD. TIMP-2 is required for efficient of further studies targeting the identification of other func- activation of proMMP-2 in vivo. J Biol Chem. 2000;275:26411– 26415. tional protein aspects in the pathophysiology of autoimmune 22. Seo DW, Li H, Guedez L, et al. TIMP-2 mediated inhibition of uveitis. angiogenesis: an MMP-independent mechanism. Cell. 2003;114: 171–180. Acknowledgments 23. Magnoni S, Baker A, Thomson S, et al. Neuroprotective effect of adenoviral-mediated gene transfer of TIMP-1 and -2 in ischemic The authors thank Sieglinde Hirmer for excellent technical assistance. brain injury. Gene Ther. 2007;14:621–625.

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