Proinflammatory Cytokines Are Involved in the Initiation of the Abnormal Matrix Process in Pseudoexfoliation Syndrome/Glaucoma

Proinflammatory Cytokines Are Involved in the Initiation of the Abnormal Matrix Process in Pseudoexfoliation Syndrome/GlaucomaMatrix Pathobiology
Proinflammatory Cytokines Are Involved in the Initiation of the Abnormal Matrix Process in Pseudoexfoliation Syndrome/Glaucoma
Matthias Zenkel,* Piotr Lewczuk,†
Anselm Junemann,* Friedrich E. Kruse,* Gottfried O.H. Naumann,* and Ursula Schlotzer-Schrehardt* From the Departments of Ophthalmology, * and Psychiatry,†
University of Erlangen-Nurnberg, Erlangen, Germany
Pseudoexfoliation (PEX) syndrome, which is an age- related, generalized elastotic matrix process, cur- rently represents the most common identifiable risk factor for open-angle glaucoma. Dysregulated expres- sion of proinflammatory cytokines has been impli- cated in the initiation of various fibrotic disorders and in the pathophysiology of glaucoma. Here we investigated the presence, expression, regulation, and functional significance of proinflammatory cyto- kines in eyes with early and late stages of PEX syn- drome/glaucoma in comparison with normal and glaucomatous control eyes using multiplex bead anal- ysis, immunoassays, real-time PCR, Western blotting, immunohistochemistry, and cell culture models. Early stages of PEX syndrome were characterized by approximately threefold (P < 0.005) elevated inter- leukin (IL)-6 and IL-8 levels in the aqueous humor and a concomitant approximately twofold (P < 0.001) increase in mRNA expression levels in anterior seg- ment tissues as compared with controls. In contrast, late stages of PEX syndrome/glaucoma did not differ significantly from controls. IL-6, IL-6 receptor, and phospho-signal transducer and activator of transcrip- tion 3 could be mainly localized to walls of iris vessels and to the nonpigmented epithelium of ciliary pro- cesses. IL-6 and IL-8 were significantly up-regulated by ciliary epithelial cells in response to hypoxia or oxidative stress in vitro , whereas IL-6, but not IL-8, induced the expression of transforming growth fac- tor-1 and elastic fiber proteins. These findings sup- port a role for a stress-induced, spatially, and tempo- rally restricted subclinical inflammation in the onset of the fibrotic matrix process characteristic of PEX
syndrome/glaucoma. (Am J Pathol 2010, 176:2868–2879; DOI: 10.2353/ajpath.2010.090914)
Pseudoexfoliation (PEX) syndrome is an age-related, complex, generalized disorder of the extracellular matrix (ECM) characterized by the intraocular and systemic pro- duction of an abnormal fibrillar extracellular material.1
Progressive accumulation of this material (PEX material) in the aqueous humor outflow pathways is considered the primary cause of chronic intraocular pressure elevation and glaucoma development in eyes with PEX syndrome, which is currently the most important single identifiable risk factor for open-angle glaucoma and a leading cause of blindness.2 Previous immunohistochemical and bio- chemical approaches have shown PEX material to repre- sent a highly cross-linked glycoprotein-proteoglycan complex, which is mainly composed of elastic microfibril- lar components, such as fibrillin-1 and latent transforming growth factor binding proteins (LTBP), as well as chap- erone molecules, such as clusterin, and cross-linking enzymes, such as lysyl oxidase-like 1.3–5 Two nonsyn- onymous single nucleotide polymorphisms in lysyl oxi- dase-like 1, coding for a key enzyme of elastic fiber for- mation and stabilization, represent the principal genetic risk factor for PEX syndrome and glaucoma.6 However, the frequent occurrence of the high-risk lysyl oxidase-like 1 haplotype in the general population suggests that ad- ditional genetic and/or exogenous factors are required for manifestation of the PEX-specific matrix processes. Potential comodulating factors include PEX-associated genetic variants in the gene encoding clusterin (CLU),7
elevated concentrations of fibrogenic growth factors, such as transforming growth factor 1 (TGF-1),8 and various external stress factors, such as oxidative stress9
and anterior chamber hypoxia10 in the eyes of PEX pa- tients. Although the exact molecular mechanisms respon-
Supported by grant SFB-539 from the German Research Foundation.
Accepted for publication February 18, 2010.
Address reprint requests to Matthias Zenkel, Ph.D., Department of Ophthalmology, University of Erlangen-Nurnberg, Schwabachanlage 6, D-91054 Erlangen, Germany. E-mail: [email protected].
The American Journal of Pathology, Vol. 176, No. 6, June 2010
Copyright © American Society for Investigative Pathology
DOI: 10.2353/ajpath.2010.090914
2868
sible for the excessive production and accumulation of the abnormal PEX material still remain elusive, PEX syn- drome is currently described as a genetically predis- posed elastic microfibrillopathy, which may be triggered by fibrogenic stimuli, such as increased growth factor activity and/or increased stress conditions.
An increased expression of inflammatory markers, such as interleukin (IL)-1, IL-6, IL-8, and vascular endo- thelial leukocyte-adhesion molecule (ELAM)-1, in the aqueous humor and outflow pathways has been linked to glaucoma pathology.11–13 These findings have been sug- gested to reflect the activation of a tissue-specific stress response.13–15 Actually, apart from modulating acute in- flammation and immune responses, inflammatory cyto- kines have been implicated in a multitude of biological processes including cytoprotection, aging, and regula- tion of ECM deposition/remodeling.16,17 In addition, tu- mor necrosis factor (TNF)-, IL-1, and IL-6 have been shown to adopt profibrotic characteristics under certain conditions,18,19 and a series of studies substantiated the involvement of inflammatory cytokines in the pathobiol- ogy of various fibrotic conditions including pulmonary fibrosis and systemic sclerosis.18,20,21 Interestingly, in systemic sclerosis, elevated IL-6 serum levels were mainly related to the early fibrotic phase of the disease.21
Therefore, an involvement of inflammatory cytokines in the abnormal matrix process characteristic of PEX syn- drome may be hypothesized.
The purpose of this study was to investigate the pres- ence and expression of inflammatory cytokines in aque- ous humor and ocular tissues of PEX eyes with special regard to different stages of the aberrant matrix process. Our findings revealed significantly elevated aqueous IL-6 and IL-8 levels, concomitant with an increased mRNA and protein expression of both cytokines in eyes with early stages of PEX syndrome. In contrast, late stages of PEX syndrome without and with glaucoma did not show any significant differences in aqueous levels or expres- sion levels of IL-6 and IL-8 compared with controls. Fur- ther experiments were performed to assess the regula- tion of IL-6 and IL-8 expression and the effect of IL-6 and IL-8 on ECM expression by nonpigmented ciliary epithe- lial (NPE) cells in vitro. The findings suggest that an in- creased expression of IL-6 and IL-8, which may be in- duced by chronic stress conditions, such as oxidative stress14 and hypoxia,22 may act as a triggering factor for the abnormal PEX material production in the early stages of this fibrotic process.
Materials and Methods
Tissues and Samples
Aqueous humor (aspirated during cataract or filtration surgery) and serum samples were collected from pa- tients with cataract (mean age, 75.1 4.8 years; n 26, 13 female, 13 male), a history of resolved (4 months) herpetic uveitis (mean age, 57.9 14.4 years; n 26, 10 female, 16 male), primary open-angle glaucoma (POAG; mean age, 69.5 5.9 years; n 26, 13 female, 13 male),
PEX syndrome including early (mean age, 75.8 8.1 years; n 26, 15 female, 11 male) and late stages of the abnormal matrix process (mean age, 75.5 7.8 years; n 26, 13 female, 13 male), and PEX-associated open- angle glaucoma (mean age, 77.0 7.3 years; n 26, 13 female, 13 male) and were immediately frozen in liquid nitrogen and stored at 80°C.
All individuals underwent standardized ophthalmo- logic examination for signs of PEX syndrome in mydriasis and were classified as early- or late-stage PEX syndrome according to a semiquantitative grading score based on ocular structural changes associated with PEX, ie, PEX material deposits on anterior segment structures and various pigment-related signs (1, mild; 2, moderate; 3, marked; and 4, heavy manifestation).23 Early stages with mild disease (scores 1–2) were defined by diffuse pre- cipitation of PEX material on the anterior lens capsule (pregranular stage or incomplete intermediate zone) or small flakes of PEX material on the pupillary margin, pigment liberation after pupillary dilation, mild pigment deposition on iris or lens, mild chamber angle deposition, beginning pupillary ruff atrophy, and slightly reduced mydriasis as compared with the contralateral eye. Late stages with severe disease (scores 3–4) were character- ized by massive PEX material deposits on pupillary mar- gin and/or lens revealing the classic pattern with a com- plete clear intermediate zone, heavy pigment deposition on anterior segment structures, extensive angle pigmen- tation and Sampaolesi line, extensive pupillary ruff atro- phy, and highly restricted mydriasis. PEX glaucoma was defined, if elevated intraocular pressure (IOP 20 mmHg), an open chamber angle, reproducible visual field defects in computed perimetry, and characteristic glaucomatous optic disk damage were found in the pres- ence of manifest PEX material deposits. PEX patients with a history of previous trauma or intraocular surgery were excluded from the study.
Ocular tissues were obtained from six donor eyes with early PEX syndrome without glaucoma (mean age, 80.2 5.9 years; four female, two male), six donor eyes with late PEX syndrome without glaucoma (mean age, 81.3 5.3 years; two female, four male), and six normal appearing donor eyes (mean age, 77.3 6.6 years; three female, three male) without any known ocular disease. Donors were classified as early or late stage PEX syn- drome according to the amount of macroscopically visi- ble PEX material deposits on ocular structures. Because PEX deposits can be detected earliest on zonular fibers,2
early stages were defined by a frosted appearance of the zonules, whereas late stages revealed prominent PEX material deposits on lens, iris, ciliary processes, and zonules. The presence of PEX material was confirmed by electron microscopic analysis of small tissue sectors, and the absence of glaucoma was confirmed by microscopic analysis of optic nerve cross-sections. These eyes were obtained at autopsy and were processed within 8 hours after death.
In addition, tissues of three eyes with PEX-associated open-angle glaucoma (mean age, 79.3 1.7 years; two female, one male), three eyes with POAG (mean age, 76.7 7.8 years; two female, one male), three eyes with
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PEX-associated angle closure glaucoma (mean age, 83.3 3.1 years; two female, one male), and three eyes with angle closure glaucoma without evidence of PEX (mean age, 80.0 3.3 years; two female, one male) were used. These eyes had to be surgically enucleated be- cause of painful absolute glaucoma and were processed immediately after enucleation; their medical history has been documented previously.5
Informed consent to tissue donation was obtained from the patients or, in case of autopsy eyes, from their rela- tives, and the protocol of the study was approved by the local Ethics Committee and adhered to the tenets of the Declaration of Helsinki for experiments involving human tissues and samples.
Multiplex Bead Immunoassay
Aqueous humor samples (50 l, undiluted) were simulta- neously analyzed for 20 different cytokines using the Beadlyte Human Multiplex Cytokine Detection kit (Up- state Biotechnology, Buckingham, U.K.) and the Luminex 100 flow cytometry system (Luminex, Austin, TX) accord- ing to the manufacturer’s instructions. The sensitivity (pi- cograms per milliliter) of the assay was as follows: IL-1, 1.5; IL-1, 0.6; IL-2, 1.0; IL-3, 4.0; IL-4, 0.7; IL-5, 0.2; IL-6, 0.3; IL-7, 3.9; IL-8, 0.5; IL-10, 0.3; IL-12, 1.5; IL-13, 1.5; IL-15, 0.5; TNF-, 0.8; IFN-, 2.0; monocyte chemoattrac- tant protein-1, 8.5; macrophage-inflammatory protein-1, 22.5; regulated on activation normal T cell expressed and secreted, 3.0; Eotaxin, 9.0; and granulocyte-macrophage colony-stimulating factor, 1.0. Samples were run as sin- gle measurements, whereas standard curves of known concentrations of recombinant human cytokines (in- cluded in the kit) were run in duplicate. Aqueous protein concentrations were determined using the Bradford pro- tein assay kit (Bio-Rad, Munich, Germany) with bovine serum albumin as a standard.
IL-6 Enzyme-Linked Immunosorbent Assay
The concentrations of IL-6 were assessed in aqueous humor (50 l, diluted 1/2) and serum (50 l, undiluted) samples with a commercially available sandwich enzyme immunoassay kit (Quantikine; R&D Systems, Minneapo- lis, MN), according to the manufacturer’s instructions. All measurements were performed in duplicate. The sensi- tivity of the assay for IL-6 was 0.04 pg/ml. Aqueous protein concentrations were determined as described above.
Real-Time RT-PCR
Ocular tissues were prepared under a dissecting micro- scope and shock frozen in liquid nitrogen. Total RNA was extracted from ocular tissues and cultured cells using the RNeasy kit (Qiagen, Hilden, Germany), including an on- column DNase I digestion step. First-strand cDNA syn- thesis from 0.5 g of total RNA and quantitative real-time PCR were performed using the MyIQ thermal cycler and software (Bio-Rad) as described previously.4 PCRs (25 l) were run in duplicate and contained 2 l of the 1/5 diluted first-strand cDNA, 0.4 mol/L each of upstream- and downstream-primer, and IQ SYBR Green Supermix (Bio-Rad). Exon-spanning primers (MWG Biotech, Anz- ing, Germany), designed by means of Primer 3 software (http://fokker.wi.mit.edu/primer3/input.htm), and PCR con- ditions are summarized in Table 1. For quantification, serially diluted standard curves of plasmid-cloned cDNA were run in parallel, and amplification specificity was checked using melt curve and sequence analyses using the Prism 3100 DNA Sequencer (Applied Biosystems, Foster City, CA). For normalization of gene expression levels, mRNA ratios relative to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were calculated.
Table 1. Primers Used for Quantitative Real-Time PCR
Gene Accession no. Product (bp) Tan (°C) MgCl2 (mmol/L) Sequences
IL-6 NM_000600 129 64 3.5 5-CACACAGACAGCCACTCACCTC-3 5-GTGCCTCTTTGCTGCTTTCACAC-3
IL-8 NM_000584 110 62 3.0 5-CCACCGGAAGGAACCATCTCAC-3 5-GGCAAAACTGCACCTTCACACAG-3
Fibrillin-1 NM_000138 184 64 3.5 5-GAATGCAAGAACCTCATTGGCAC-3 5-TGGCGGTAAACCCATCATTACAC-3
LTBP-1 NM_000627 92 64 4.0 5-AGGAGTTACAGGCTGAGGAATG-3 5-ACATCGACACAGGTCATCTTGG-3
Fibulin-1 NM_006487 231 62 3.5 5-CTGCGAGTACAGCCTCATGG-3 5-AAGCAGGAGCAGACCACCTC-3
Col-II-1 NM_001844 163 64 3.5 5-GCAGGAATTCGGTGTGGACATAG-3 5-GGATGAATGGACATCAGGTCAGG-3
TGF-1 NM_000660 75 64 4.0 5-CGAGCCTGAGGCCGACTACTAC-3 5-CATAGATTTCGTTGTGGGTTTCC-3
TGF-2 NM_003238 102 64 4.0 5-CCTGCTGCACTTTTGTACCATC-3 5-TGGTATATGTGGAGGTGCCATC-3
TGF-3 NM_003239 193 65 3.0 5-TTGGAGGAGAACTGCTGTGTGC-3 5-AGGCAGATGCTTCAGGGTTCAG-3
GAPDH NM_002046 117 64 3.5 5-AGCTCACTGGCATGGCCTTC-3 5-ACGCCTGCTTCACCACCTTC-3
Tan, annealing temperature; Col, collagen.
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Immunohistochemistry
Indirect immunofluorescence labeling was performed on cryosections of ocular tissues as previously described24
using antibodies against IL-6 (clone B-E8; Abcam, Cam- bridge, U.K.), IL-6 receptor (IL-6R, clone B-R6; Abcam), phospho-signal transducer and activator of transcription 3 (p-STAT3) (pY705; Abcam), CD11b (clone ICRF44; Abcam), and CD68 (clone EBM11, DakoCytomation, Glostrup, Denmark). Antibody binding was detected by Alexa 488-conjugated secondary antibodies (Molecular Probes, Eugene, OR), and nuclear counterstaining was performed with propidium iodide (Sigma-Aldrich, Mu- nich, Germany). In negative control experiments, the pri- mary antibody was replaced by PBS or equimolar con- centrations of an irrelevant primary antibody.
Western Blot Analysis
Ocular tissue specimens were homogenized in extraction buffer (50 mmol/L Tris-HCl, pH 8.0, 150 mmol/L NaCl, 1% Nonidet P-40, 0.5% deoxycholic acid, and 0.1% SDS) containing protease and phosphatase inhibitor mixtures (Merck Biosciences, Nottingham, U.K.) using a rotor- stator homogenizer. Protein concentrations were deter- mined by the Micro BCA Protein Assay kit (Thermo Sci- entific, Bonn, Germany). Ten micrograms of total protein was separated by SDS-polyacrylamide gel electrophore- sis under reducing conditions and transferred onto nitro- cellulose membranes (Hybond ECL; GE Healthcare, Mu- nich, Germany) with a semidry blotting unit (Bio-Rad). Membranes were blocked with Superblock (Thermo Sci- entific) for 1 hour and incubated overnight at 4°C with mouse monoclonal antibodies against human p-STAT3 (clone 4; BD Biosciences, Heidelberg, Germany) or STAT3 (clone 84; BD Biosciences) diluted 1/500 or 1/2500 in Superblock, respectively. Equal loading was verified with mouse anti-human -actin antibody (clone AC-15; Sigma-Aldrich) 1/5000 in Superblock. In negative control experiments, the primary antibody was replaced by PBS. Immunodetection was performed with an affinity- purified horseradish peroxidase-conjugated secondary antibody (Thermo Scientific) diluted 1/10,000 in Super- block, according to the manufacturer’s instructions. Sig- nals were analyzed using the Chemi-Smart 5000 chemi- luminescence detection system and software (Vilber Lourmat, Eberhardzell, Germany).
Cell Culture
To study the regulation of IL-6 and IL-8 expression, the immortalized human NPE cell line ODM-225 was used at passage 18. Cells were maintained in Dulbecco’s modi- fied Eagle’s medium (Invitrogen, Grand Island, NY) con- taining 10% fetal calf serum and 50 g/ml gentamicin in a 95% air–5% CO2 humidified atmosphere at 37°C. Cells were grown to subconfluence (90%), kept in serum-free medium for 24 hours, and then exposed to either hypoxia (2% oxygen), oxidative stress (50 mol/L of hydrogen peroxide, H2O2), 5 ng/ml TGF-1 (R&D Systems; Wies-
baden, Germany), 10 ng/ml IL-6 (PeproTech, Hamburg, Germany), or 10 ng/ml IL-8 (PeproTech) for up to 72 hours under serum-free conditions. Parallel cultures maintained under normoxic (21% oxygen) and serum- free conditions without addition of H2O2, TGF-1, IL-6, or IL-8 served as controls.
Cell viability was assessed using a fluorescent kit (Live/Dead Viability/Cytotoxicity kit; Molecular Probes) and a plate reader (Fluoroscan Ascent 2.4; Thermo Sci- entific), according to the manufacturer’s instructions, to count viable cells stained with calcein-AM and dead cells stained with ethidium homodimer-1 dye. The ratio of living to dead cells was calculated for the different treatment conditions and compared with parallel con- trol cultures under serum-free and normoxic conditions without treatment.
Statistics
Data are presented as mean SD and were analyzed using SPSS statistical software (SPSS, Chicago, IL). Sta- tistical evaluation of differences between groups was performed using the Mann-Whitney U-test. Pearson’s cor- relation analysis was used to assess the significance of correlations. A value of P 0.05 was considered statis- tically significant.
Results
Cytokine Profiles in Aqueous Humor
As a first step to explore the role of inflammatory media- tors in the pathogenesis of PEX syndrome/glaucoma, we used a multiplex bead immunoassay to screen aqueous levels of 20 inflammatory cytokines in eyes with early and late stages of PEX syndrome and PEX glaucoma as well as in control eyes with cataract, POAG, or a history of resolved ocular inflammation (n 14 for each group). Whereas IL-1, IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-15, IFN-, monocyte chemoattractant protein-1, macrophage-inflammatory protein-1, regulated on acti- vation normal T cell expressed and secreted, eotaxin, and granulocyte-macrophage colony-stimulating factor could be detected in all sample groups, aqueous levels of IL-1, IL-3, IL-5, and TNF- were consistently below the sensitivity of the assay. Aqueous cytokine concentra- tions are summarized in Table 2.
Whereas the majority of detectable cytokines dis- played equal levels between the various groups of pa- tients, significantly increased levels of IL-6 (3.5-fold; P 0.001) and IL-8 (2.5-fold; P 0.005) could be measured in aqueous samples of eyes with early stages of PEX syndrome (IL-6, 186 99 pg/ml; IL-8, 25.5 23.5 pg/ml) as compared with control eyes with cataract (IL-6, 52.0 34.0 pg/ml; IL-8, 10.1 6.6 pg/ml). In contrast, samples from eyes with late stages of PEX syndrome and PEX glaucoma did not display any significant differences as compared with controls. Patients with POAG displayed a tendency to increased IL-8 levels, which, however, did not reach statistical significance (1.8-fold; P 0.065).
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Patients with a history of inflammatory ocular disease, serving as positive controls, displayed a significant in- crease of aqueous IL-1 (1.8-fold; P 0.01), IL-6 (3.7- fold; P 0.001), IL-8 (2.6-fold; P 0.01), and regulated on activation normal T cell expressed and secreted (2.4- fold; P 0.005) levels compared with cataract controls. Aqueous IL-6 and IL-8 levels in the different patient groups are illustrated in Figure 1.
Aqueous levels of IL-6 and IL-8 were positively corre- lated in both patients with early stages of PEX syndrome (r 0.937; P 0.01) and in patients with previous ocular inflammation (r 0.914; P 0.01). The latter showed further positive correlations between IL-6 and IL-1 (r 0.848; P 0.01) as well as between IL-8 and IL-1 (r 0.875; P 0.01). However, no correlations between cy- tokine levels and aqueous protein levels could be established.
To validate and supplement the results obtained by multiplex analysis, concentrations of IL-6 were measured in aqueous humor and serum samples by enzyme-linked immunosorbent assay (n 12 for each patient group). This approach confirmed a significant increase in IL-6 levels (2.4-fold; P 0.001) in early stages of PEX syn- drome (162.0 90.1 pg/ml) as compared with cataract controls (68.8 21.9 pg/ml) (Figure 2A). Again, late stages of PEX syndrome and PEX glaucoma did not differ significantly from the control group. Serum levels of IL-6 measured 3.1 1.3 pg/ml in patients with cataract and
were only elevated in patients with resolved ocular inflam- mation (6.5 3.1 pg/ml; P 0.0001) but not in patients with PEX syndrome/glaucoma or POAG (data not…