Immunoassay für Matrix-Metalloproteinase-9 in der Tränenflüssigkeit bei Patienten mit Pseudoexfoliationssyndrom – eine Pilotstudie

Zusammenfassung

Hintergrund: Matrix-Metalloproteinasen (MMPs) sind proteolytische Enzyme, die u. a. durch gereizte Epithelzellen der Augenoberfläche freigesetzt werden. Ein unspezifischer Entzündungsparameter im Tränenfilm ist die Unterform MMP-9, der auch ein Einfluss bei der PEX-Glaukomentstehung zugeschrieben wird. Neuerdings gibt es einen Schnell-Immunoassay zum Nachweis von MMP-9 in der Tränenflüssigkeit. Ziel dieser Studie war die Analyse der MMP-9-Konzentration im Tränenfilm beim PEX-Syndrom. Zusätzlich erfolgte eine Einschätzung der Durchführbarkeit, Auswertbarkeit und Zuverlässigkeit des Tests.

Methoden: Es wurden zufällig jeweils 10 Patienten mit PEX-Syndrom sowie allgemein- und augengesunde Kontrollpatienten ausgewählt. Es wurde der Schnell-Immunoassay RPS InflammaDry Detector™ von Rapid Pathogen Screening Inc. verwendet. Der Schnelltest wurde jeweils an einem Auge durchgeführt.

Ergebnis: Im Tränenfilm beim PEX-Syndrom sind erhöhte MMP-9-Konzentrationen nachweisbar. 80 % der PEX-Patienten und 20 % der Kontrollen zeigten ein positives Testergebnis (>/= 40 ng/ml MMP-9), woraus eine Test-Sensitivität und -Spezifität von jeweils 80 % resultiert. Dies entspricht ungefähr den publizierten Werten für das Trockene Auge (Sensitivität: 87 %, Spezifität: 92 %). Die Durchführung des Tests ist einfach und verständlich. Die Patienten tolerierten die Aufnahme des Tränenfilms mittels Teststreifen gut. Allerdings ist es trotz Färbeindikator schwierig einzuschätzen, ob genügend Tränenfilm gewonnen wurde. Beim Ablesen des Teststreifens ist die Färbung der Resultatlinie schwach.

Schlussfolgerung: Der MMP-9-Schnell-Immunoassay ist ein neuartiger, sinnvoller Ansatz zum Nachweis einer Entzündungsaktivität der Augenoberfläche. Wir zeigen eine Hochregulation des unspezifischen Entzündungsmarkers MMP-9 im Tränenfilm beim PEX-Syndrom und vermuten eine Assoziation mit einer Tränenfilmstörung. Allerdings ist eine Verbesserung der Einschätzung der gewonnenen Tränenmenge und Ablesbarkeit wünschenswert.

Abstract

Introduction: Matrix-metalloproteinases (MMPs) are proteolytic enzymes released by irritated epithelial cells of the ocular surface. It has been established that the subtype MMP-9 can serve as an inflammatory marker within the tear film. MMP-9 is also attributed to have an effect on the PEX-glaucoma development. Recently, a rapid immunoassay for detection of MMP-9 in the tear film was developed to estimate inflammatory extent during dry eye disease. The aim of this study was to analyse the MMP-9 concentration in tear film in PEX-syndrome. In addition, an assessment of the feasibility, reliability and readability of the test was done.

Methods: We randomly selected 10 patients with PEX-syndrome and 10 healthy control patients and measured tear film MMP-9 of one eye with the RPS InflammaDry Detector™ (Rapid Pathogen Screening Inc., USA).

Results: We detected increased levels of MMP-9 in tear film in PEX-syndrome. 80 % of the PEX-patients and 20 % of the controls showed a positive test result (>/= 40 ng/mL MMP-9) indicating a test specificity and sensitivity of 80 %. This corresponds approximately to the published values for the dry eye (sensitivity 87 %, specificity: 92 %). The performance of the test is simple. The patients tolerated the inclusion of the test strips well. However, it is difficult to estimate whether enough tear film was used and in many cases, the intensity of the “indicator line” was weak.

Conclusion: The rapid MMP-9-immunoassay is a novel, meaningful approach for the detection of inflammatory activity of the ocular surface. We have shown an up-regulation of the non-specific inflammatory marker MMP-9 in tear film in PEX-syndrome and suggest an association with a tear film disorder. However, an improvement in the estimation of the amount of collected tears and readability is desirable.

Schlüsselwörter

MMP-9 - PEX - Schnell-Immunoassay

Key words

MMP-9 - PEX - rapid immunoassay

• Literatur

• 1 Li DQ, Meller D, Liu Y et al. Overexpression of MMP-1 and MMP-3 by cultured conjunctivochalasis fibroblasts. Invest Ophthalmol Vis Sci 2000; 41: 404-410
• 2 Chotikavanich S, de Paiva CS, Li de Q et al. Production and activity of matrix metalloproteinase-9 on the ocular surface increase in dysfunctional tear syndrome. Invest Ophthalmol Vis Sci 2009; 50: 3203-3209
• 3 Borden P, Heller RA. Transcriptional control of matrix metalloproteinases and the tissue inhibitors of matrix metalloproteinases. Crit Rev Eukaryot Gene Expr 1997; 7: 159-178
• 4 Woessner jr. JF. Matrix metalloproteinases and their inhibitors in connective tissue remodeling. FASEB J 1991; 5: 2145-2154
• 5 Murphy G, Docherty AJ. The matrix metalloproteinases and their inhibitors. Am J Respir Cell Mol Biol 1992; 7: 120-125
• 6 Sternlicht MD, Werb Z. How matrix metalloproteinases regulate cell behavior. Annu Rev Cell Dev Biol 2001; 17: 463-516
• 7 Mossbock G, Weger M, Faschinger C et al. Role of functional single nucleotide polymorphisms of MMP1, MMP2, and MMP9 in open angle glaucomas. Mol Vis 2010; 16: 1764-1770
• 8 Li DQ, Chen Z, Song XJ et al. Stimulation of matrix metalloproteinases by hyperosmolarity via a JNK pathway in human corneal epithelial cells. Invest Ophthalmol Vis Sci 2004; 45: 4302-4311
• 9 Acera A, Rocha G, Vecino E et al. Inflammatory markers in the tears of patients with ocular surface disease. Ophthalmic Res 2008; 40: 315-321
• 10 Lema I, Sobrino T, Duran JA et al. Subclinical keratoconus and inflammatory molecules from tears. Br J Ophthalmol 2009; 93: 820-824
• 11 Leonardi A, Brun P, Abatangelo G et al. Tear levels and activity of matrix metalloproteinase (MMP)-1 and MMP-9 in vernal keratoconjunctivitis. Invest Ophthalmol Vis Sci 2003; 44: 3052-3058
• 12 Solomon A, Dursun D, Liu Z et al. Pro- and anti-inflammatory forms of interleukin-1 in the tear fluid and conjunctiva of patients with dry-eye disease. Invest Ophthalmol Vis Sci 2001; 42: 2283-2292
• 13 Hadassah J, Bhuvaneshwari N, Rao U et al. Evaluation of succinylated collagen bandage lenses in corneal healing by the expression of matrix metalloproteinases (MMP-2 and MMP-9) in tear fluid. Ophthalmic Res 2009; 42: 64-72
• 14 Sobrin L, Liu Z, Monroy DC et al. Regulation of MMP-9 activity in human tear fluid and corneal epithelial culture supernatant. Invest Ophthalmol Vis Sci 2000; 41: 1703-1709
• 15 Luo L, Li DQ, Doshi A et al. Experimental dry eye stimulates production of inflammatory cytokines and MMP-9 and activates MAPK signaling pathways on the ocular surface. Invest Ophthalmol Vis Sci 2004; 45: 4293-4301
• 16 Honda N, Miyai T, Nejima R et al. Effect of latanoprost on the expression of matrix metalloproteinases and tissue inhibitor of metalloproteinase 1 on the ocular surface. Arch Ophthalmol 2010; 128: 466-471
• 17 [no authors listed] The definition and classification of dry eye disease: report of the Definition and Classification Subcommittee of the International Dry Eye WorkShop (2007). The ocular surface 2007; 5: 75-92
• 18 Pflugfelder SC. Antiinflammatory therapy for dry eye. Am J Ophthalmol 2004; 137: 337-342
• 19 Li DQ, Chen Z, Song XJ et al. Hyperosmolarity stimulates production of MMP-9, IL-1β and TNF-by human corneal epithelial cells via a c-Jun NH2-terminal kinase pathway (Abstract). Invest Ophthalmol Vis Sci 2002; 43:
• 20 Luo L, Li D. Experimental dry eye induced expression of inflammatory cytokines (IL-1β and TNF-), MMP-9 and activated MAPK by the corneal epithelium. Invest Ophthalmol Vis Sci 2003; 44:
• 21 De Paiva CS, Corrales RM, Villarreal AL et al. Corticosteroid and doxycycline suppress MMP-9 and inflammatory cytokine expression, MAPK activation in the corneal epithelium in experimental dry eye. Exp Eye Res 2006; 83: 526-535
• 22 Pflugfelder SC, Farley W, Luo L et al. Matrix metalloproteinase-9 knockout confers resistance to corneal epithelial barrier disruption in experimental dry eye. Am J Pathol 2005; 166: 61-71
• 23 Matsubara M, Zieske JD, Fini ME. Mechanism of basement membrane dissolution preceding corneal ulceration. Invest Ophthalmol Vis Sci 1991; 32: 3221-3237
• 24 Ye HQ, Azar DT. Expression of gelatinases A and B, and TIMPs 1 and 2 during corneal wound healing. Invest Ophthalmol Vis Sci 1998; 39: 913-921
• 25 Fini ME, Parks WC, Rinehart WB et al. Role of matrix metalloproteinases in failure to re-epithelialize after corneal injury. Am J Pathol 1996; 149: 1287-1302
• 26 Kaufman HE. The practical detection of MMP-9 diagnoses ocular surface disease and may help prevent its complications. Cornea 2013; 32: 211-216
• 27 Gartaganis SP, Georgakopoulos CD, Mela EK et al. Matrix metalloproteinases and their inhibitors in exfoliation syndrome. Ophthalmic Res 2002; 34: 165-171
• 28 Ho SL, Dogar GF, Wang J et al. Elevated aqueous humour tissue inhibitor of matrix metalloproteinase-1 and connective tissue growth factor in pseudoexfoliation syndrome. Br J Ophthalmol 2005; 89: 169-173
• 29 Erdogan H, Arici DS, Toker MI et al. Conjunctival impression cytology in pseudoexfoliative glaucoma and pseudoexfoliation syndrome. Clin Exp Ophthalmol 2006; 34: 108-113
• 30 Detorakis ET, Koukoula S, Chrisohoou F et al. Central corneal mechanical sensitivity in pseudoexfoliation syndrome. Cornea 2005; 24: 688-691
• 31 Kozobolis VP, Christodoulakis EV, Naoumidi II et al. Study of conjunctival goblet cell morphology and tear film stability in pseudoexfoliation syndrome. Graefes Arch Clin Exp Ophthalmol 2004; 242: 478-483
• 32 Oncel BA, Pinarci E, Akova YA. Tear osmolarity in unilateral pseudoexfoliation syndrome. Clin Exp Optom 2012; 95: 506-509
• 33 Roedl JB, Bleich S, Reulbach U et al. Homocysteine in tear fluid of patients with pseudoexfoliation glaucoma. J Glaucoma 2007; 16: 234-239
• 34 Roedl JB, Bleich S, Schlotzer-Schrehardt U et al. Increased homocysteine levels in tear fluid of patients with primary open-angle glaucoma. Ophthalmic Res 2008; 40: 249-256
• 35 Gurdal C, Genc I, Sarac O et al. Topical cyclosporine in thyroid orbitopathy-related dry eye: clinical findings, conjunctival epithelial apoptosis, and MMP-9 expression. Curr Eye Res 2010; 35: 771-777
• 36 Li DQ, Zhou N, Zhang L et al. Suppressive effects of azithromycin on zymosan-induced production of proinflammatory mediators by human corneal epithelial cells. Invest Ophthalmol Vis Sci 2010; 51: 5623-5629