Establishment of a matrix-associated transepithelial resistance invasion assay to precisely measure the invasive potential of synovial fibroblasts.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 60, No. 9, September 2009, pp 2606–2611 DOI 10.1002/art.24782 © 2009, American College of Rheumatology Establishment of a Matrix-Associated Transepithelial Resistance Invasion Assay to Precisely Measure the Invasive Potential of Synovial Fibroblasts Christina Wunrau,1 Eva-Maria Schnaeker,2 Katharina Freyth,1 Noreen Pundt,1 Doreen Wendholt,1 Katja Neugebauer,1 Uwe Hansen,1 Thomas Pap,1 and Berno Dankbar1 led to a measurable decrease and subsequent breakdown of electrical resistance. Unlike in the assay with OASFs, which did not achieve a breakdown of resistance up to 72 hours, RASFs exhibited a pronounced invasiveness in this assay, with a 50% breakdown after 42 hours. Treatment of fibroblasts with either a matrix metalloproteinase inhibitor or antibodies against ␤1 integrin significantly reduced the invasiveness of RASFs. Conclusion. The MATRIN assay is a valuable and sensitive biologic assay system that can be used to determine precisely the invasive potential of RASFs in vitro, and thus would be suitable for screening antiinvasion compounds. Objective. Synovial fibroblasts (SFs) contribute to several aspects of the pathogenesis of rheumatoid arthritis (RA) and have been implicated most prominently in the progressive destruction of articular cartilage. Targeting the invasive phenotype of RASFs has therefore gained increasing attention, but the precise measurement of their invasive capacity and the evaluation of potential treatment effects constitute a challenge that needs to be addressed. This study used a novel in vitro invasion assay based on the breakdown of transepithelial electrical resistance to determine the course of fibroblast invasion into extracellular matrix. Methods. A matrix-associated transepithelial resistance invasion (MATRIN) assay was used to assess SFs from patients with RA in comparison with SFs from patients with osteoarthritis (OA). The SFs were grown on a commercially available collagen mix that was placed onto the upper side of a Transwell polycarbonate membrane. In addition, freshly isolated cartilage extracts were studied to assess the conditions in vivo. Under this membrane, a monolayer of MDCK-C7 cells was seeded to create a high electrical resistance. Results. Invasion of fibroblasts into the matrix affected the integrity of the MDCK-C7 monolayer and The extracellular matrix (ECM) is a network of structural proteins, including collagens, glycoproteins, and proteoglycans. The excessive proteolysis of the ECM plays an essential role in conditions such as rheumatoid arthritis (RA). The hyperplastic synovial membrane is a key feature of this chronic systemic inflammatory disease (1), and synovial fibroblasts (SFs) of the lining layer are key effectors of cartilage degradation (2,3). RASFs exhibit features of stable cellular activation, resulting in up-regulation of both adhesion molecules and matrix-degrading enzymes, especially matrix metalloproteinases (MMPs) (4). Quantification of the invasive potential of RASFs, as well as the assessment of treatment strategies with respect to matrix destruction, has been a major challenge. Until now, invasion has been measured by methods such as the Boyden chamber assay or in vivo in the SCID mouse model (5,6). However, all of these assays have several disadvantages, the most striking of which is the imprecise nature of the readout data that serve to quantify the invasive potential of RASFs. Supported in part by SET Stiftung, Germany. Dr. Schnaeker’s work was supported by DFG SCHN 1071/1-1. 1 Christina Wunrau, MSc, Katharina Freyth, Dipl Biol, Noreen Pundt, MSc, Doreen Wendholt, PhD, Katja Neugebauer, PhD, Uwe Hansen, PhD, Thomas Pap, MD, Berno Dankbar, PhD: University Hospital Muenster, Muenster, Germany; 2Eva-Maria Schnaeker, MD: St. Josef Hospital, Cloppenburg, Germany. Drs. Pap and Dankbar contributed equally to this work. Address correspondence and reprint requests to Thomas Pap, MD, Institute of Experimental Musculoskeletal Medicine, University Hospital Muenster, Domagkstrasse 3, D-48149 Muenster, Germany. E-mail: firstname.lastname@example.org. Submitted for publication August 15, 2008; accepted in revised form June 1, 2009. 2606 MATRIN ASSAY FOR SYNOVIAL FIBROBLAST INVASIVENESS The purpose of this study was to establish an in vitro assay that determines precisely the invasive potential of SFs as well as the kinetics of matrix degradation of SFs. The assay is based on the detection of a decrease in and subsequent breakdown of the transepithelial electrical resistance (TEER) generated by an epithelial monolayer. Presently, TEER assays are used to measure the invasive potential of tumor cells, especially melanoma cells (7–9). In these assays, the breakdown of electrical resistance indicates the opening of tight junctions prior to cell invasion (9). This system was recently modified by adding a collagen matrix between the cell layer and the invasive RASFs, in order to obtain an assay that comes closer to the in vivo situation of human RA. This matrix-associated transepithelial resistance invasion (MATRIN) assay offers the possibility of comparing precisely the invasive potential of RASFs with that of other cells, and facilitates the measurement of effects of pharmacologic modifications in a low volume with a high throughput and high reproducibility. PATIENTS AND METHODS Tissue preparation and cell culture. Samples of synovial membrane tissue were obtained from patients with RA whose diagnosis met the American College of Rheumatology (formerly, the American Rheumatism Association) criteria (10) and from patients with osteoarthritis (OA) at the time of joint replacement surgery. Fibroblasts were isolated by enzymatic digestion of synovial tissue, and the cells were then cultured in Dulbecco’s modified Eagle’s medium. The human melanoma cell line A7 was grown in RPMI 1640. The highresistance C7 subclone of Madin-Darby canine kidney cells (MDCK-C7) was cultured in minimum essential medium. The properties of these cells have been described in detail previously (11). All media (PAA Laboratories, Pasching, Austria) were supplemented with 10% fetal calf serum (Biochrom, Berlin, Germany), 100 units/ml penicillin, and 10 g/ml streptomycin (PAA Laboratories). MATRIN assay. A collagen matrix consisting of 97% type I and 3% type III collagen (Inamed Biomaterials, Fremont, CA) was coated onto the upper side of a polycarbonate membrane of a filter cup (area 4.2 cm2, pore diameter 0.4 m) mounted in a 6-well dish (BD, Heidelberg, Germany). In an additional experimental setting, pieces of human cartilage sections were homogenized and the suprastructural cartilage components were extracted. Cartilage extracts (200 l; protein concentration 0.25 mg/ml) were coated onto the membranes with a heterobifunctional crosslinker (2 mM sulfo-SANPAH; Pierce, Rockford, IL) and ultraviolet light (2 lamps at 4 watts each). In all settings, MDCK-C7 cells were seeded onto the basal side of the coated filter cups. Both the upper and lower chambers contained 2.5 ml of medium, and 1.0 ml from each compartment was replaced by fresh medium every day to maintain stable metabolic conditions. 2607 Electrical resistance across the MDCK-C7 monolayer was measured with a set of 2 circular Ag/AgCl electrodes connected with an ohm-meter (World Precision Instruments, Sarasota, FL). These electrodes were assembled in a flexible clamp, which facilitates the positioning of one electrode into the insert and of the other electrode into the well, always with the same immersion depth into the medium. Following full establishment of the MDCK-C7 monolayer, 7 ⫻ 105 fibroblasts were seeded onto the collagen matrices. In some experiments, RASFs were treated with 200 ng/ml MMP Inhibitor III (Calbiochem, Nottingham, UK) or incubated with 20 g/ml ␤1 integrin function–blocking antibody (Chemicon, Billerica, MA) to specify measurable alterations of the invasive behavior. To avoid an artificial decrease in resistance due to alterations in temperature or CO2, the 6-well plate was measured within 3 minutes, allowing measurements of kinetics. All measurements were performed in quadruplicate and were terminated after total breakdown of the electrical resistance. MMP enzyme-linked immunosorbent assays (ELISAs). Supernatants were collected from the MATRIN assay at appropriate time intervals. MMP ELISAs were performed according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN). Statistical analysis. Data are expressed as the mean ⫾ SD. Statistical analysis was performed using GraphPad Prism software (version 4.0; GraphPad Software, San Diego, CA). Correlations were determined by Spearman’s rank correlation test for bivariate analysis. Differences between groups were examined for statistical significance using the Mann-Whitney test. P values less than 0.05 were considered statistically significant. RESULTS The experimental setting and procedure for the MATRIN assay are illustrated in Figure 1A. After the MDCK-C7 cells had reached confluence (Figure 1B) and had established an electrical resistance up to 3,500⍀, the SFs were added onto the collagen matrix in the upper compartment of the Transwell system. The decrease and subsequent breakdown of the electrical resistance in the course of invasion were determined as a way to evaluate the invasiveness of the fibroblasts. The first set of experiments determined the invasive potential of RASFs in comparison with that of OASFs (using invasive A7 melanoma cells as a positive control). As shown in Figure 2A, MDCK-C7 cells were able to keep the initially established electrical resistance for more than 144 hours, demonstrating that the lack of cells in the upper compartment preserved the integrity of the monolayer over prolonged periods of time. Addition of RASFs led to a 50% breakdown of the TEER within 46 hours, followed by a complete breakdown after ⬃72 hours. In contrast, addition of OASFs resulted in a 50% breakdown of the TEER only after 112 hours 2608 WUNRAU ET AL Figure 1. Matrix-associated transepithelial resistance invasion (MATRIN) assay for measurement of synovial fibroblast (SF) invasion. A, The schematic representation of the MATRIN assay shows a filter cup coated apical with collagen, which was mounted in a culture dish. An MDCK-C7 monolayer was grown basal on the insert, and rheumatoid arthritis SFs or control cells were added on top of the collagen matrix. Electrical resistance was measured with a set of electrodes. B, Assessment of the monolayer of MDCK-C7 cells shows that MDCK-C7 cells constitute a cell clone with particularly “tight” junctional complexes. The high transepithelial electrical resistance of the C7 subclone of MDCK cells can be measured. (⌬t50 ⫽ 66 hours; P ⬍ 0.01 versus RASFs), indicating that RASFs exhibit a significantly higher invasive capacity than OASFs. As expected, A7 cells, which were used as a positive control, displayed a high degree of invasiveness, as demonstrated by a 50% breakdown of the electrical resistance within 18 hours and a subsequent complete breakdown after ⬃24 hours. In order to determine whether the MATRIN assay involving coating with a type I and type III collagen matrix is an accurate and reliable setting for measuring fibroblast invasion, we compared the results in this matrix with the observations in isolated human cartilage extracts. Neither the human cartilage extracts nor the crosslinking procedure itself had effects on the TEER. Moreover, there were no differences with respect to the invasive potential of RASFs between the different coating components (Figure 2B). To understand this process in further detail, we assessed whether matrix degradation by RASFs, as measured by the extent of breakdown of electrical resistance, was associated with the secretion of MMPs. Supernatants obtained from the MATRIN assay were analyzed for the levels of MMPs 1, 3, and 13. As shown in Figure 3A, the expression of all investigated MMPs was found to be increased more than 4-fold over the time course of 72 hours. Analysis of the invasion of Figure 2. Invasive capacity of rheumatoid arthritis synovial fibroblasts (RASFs) in the matrix-associated transepithelial resistance invasion (MATRIN) assay, and testing of surface coatings in the MATRIN assay. A, RASFs from 4 patients exhibited a strong invasiveness in the MATRIN assay, as demonstrated by the percent decrease in the transepithelial electrical resistance (TEER) over a total of 144 hours, whereas osteoarthritis SFs (OASFs) from 5 patients showed a significantly reduced invasiveness. The A7 cells and the MDCK-C7 layer alone served as internal assay controls, and the broken line indicates the 50% resistance breakdown level. B, The invasive capacity of RASFs was similar regardless of whether the membrane was coated with fresh human cartilage extract (indicated by asterisk) or commercial collagen (indicated by pound sign). An untreated MDCK-C7 monolayer served as the control in each culture condition, and a layer treated with crosslinker also served as a control. Bars show the mean ⫾ SD of triplicate cultures. MATRIN ASSAY FOR SYNOVIAL FIBROBLAST INVASIVENESS Figure 3. Detection of treatment effects by matrix-associated transepithelial resistance invasion (MATRIN) assay. A, Secretion of matrix metalloproteinase 1 (MMP-1), MMP-3, and MMP-13 was detected in the MATRIN assay, in supernatants of 700,000 rheumatoid arthritis synovial fibroblasts (RASFs) from 4 different patients. A stepwise increase in the levels of MMPs 1 and 3 is evident after 24, 48, and 72 hours. Bars show the mean and SD. B, MMP inhibition experiments were carried out by treating RASFs with 200 ng/ml MMP inhibitor III, and the invasive potential of the RASFs was then examined by measuring the percent transepithelial electrical resistance (TEER) in RASFs treated with inhibitor in comparison with that of untreated RASFs. The effect was particularly evident at early phases of invasion. An untreated MDCK-C7 monolayer and a MDCK-C7 layer treated with inhibitor served as assay controls. Results are the mean of quadruplicate experiments. Broken lines indicate the 50% and 90% resistance breakdown levels. C, Fibroblast adhesion was assessed by treating RASFs with 20 g/ml of a function-blocking antibody for human ␤1 integrin, which reduced the invasive potential (the percent TEER) of the RASFs as compared with that of untreated RASFs. An untreated MDCK-C7 monolayer served as assay control. Results are the mean ⫾ SD of quadruplicate experiments. Broken line indicates the 50% resistance breakdown level. RASFs showed a significant correlation with the levels of all MMPs measured. For MMP-1, the correlation coefficient was 0.9940 (P ⬍ 0.05), while that for MMP-3 was 0.9715 (P ⬍ 0.05) and that for MMP-13 was 0.9824 (P ⬍ 0.05). To confirm the involvement of MMPs in RASF- 2609 mediated breakdown of resistance, as well as to assess the applicability of this method for the measurement of treatment effects, we incubated RASFs with the broadspectrum MMP inhibitor III, which blocks collagenases (MMPs 1 and 13) as well as gelatinase A (MMP-2), stromelysin (MMP-3), and matrilysin (MMP-7). As demonstrated in Figure 3B, this inhibitor had no effects on the integrity of the MDCK-C7 monolayer itself, but caused a significant delay in the resistance breakdown by the treated RASFs. This effect was particularly evident early after the addition of the inhibitor. The 20% breakdown value was delayed by 14 hours (⌬t80 ⫽ 14 hours; P ⬍ 0.01 versus untreated RASFs). The inhibitory effect decreased somewhat over time and resulted in a difference of 9 hours before breakdown occurred at the 50% resistance level (⌬t50 ⫽ 9 hours; P ⬍ 0.01 versus untreated RASFs). To further determine the mechanisms by which breakdown of the resistance occurs, we performed experiments in which we blocked the adhesion of RASFs to the ECM with the use of anti–␤1 integrin antibodies. These experiments revealed that blocking of ␤1 integrin resulted in a significant delay in matrix invasion by RASFs. As shown in Figure 3C, blocking of ␤1 integrin caused a delay of 11 hours before breakdown at 50% resistance (⌬t50 ⫽ 11 hours; P ⬍ 0.01 versus untreated RASFs). DISCUSSION Cell invasion is a complex process in which the attachment of cells to the ECM and secretion of MMPs are key steps, leading to digestion of the surrounding ECM, cell migration, and penetration of the ECM (4,12). To date, in vitro measurement of cell invasion, especially of tumor cells, has been performed mainly by Boyden chamber experiments in which the migrated cells were counted at a prespecified end point (13). This method is generally accepted and widely used, but there are a number of disadvantages associated with this assay; e.g., the technique of counting the cells is imprecise, and determination of the invasiveness is limited to only one time point in one setting. With our novel MATRIN assay, we designed a truly quantitative method that permits us to assess cell invasion in a time-dependent manner by measuring the same parameter several times. Moreover, our novel MATRIN assay is highly sensitive, since minor decreases of the TEER could be measured at early time points, reflecting marginal collagen degra- 2610 dation. Therefore, this assay is suitable not only for the evaluation of cells exhibiting a low invasive potential but also for the analysis of invasion kinetics and treatment effects. With the use of the MATRIN assay, we were able to investigate the invasive potential of SFs, which we had not been able to evaluate with the Matrigel assay. We could clearly demonstrate that RASFs exhibit a highly invasive phenotype that differs markedly from that of OASFs both in its extent and in its kinetics. This is in accordance with the notion that invasion of SFs into articular structures is an important feature of joint destruction in RA and that activated RASFs are involved prominently in this process through their attachment to the cartilage surface and the release of matrixdegrading enzymes (14). In accordance with the prominent role of MMPs in cartilage degradation, we found increased MMP levels over the time course of invasion and a significant retardation of RASF invasion in the presence of an MMP inhibitor. Moreover, we demonstrated that blocking of ␤1 integrin, an integrin subunit that has been shown to be involved prominently in adhesion of cells to collagen (15), again results in a delayed invasion, indicating that, in addition to the release of MMPs, adhesion of RASFs to the collagen matrix is an important step in cartilage invasion. From a technical point of view, we could clearly show that our assay is sensitive enough to investigate treatment-mediated alterations of the invasive potential of these cells. Another advantage of our assay is the separation of the cells by a collagen matrix, which avoids undesirable cell–cell interactions. Whereas MDCK-C7 cells are cultured on the basal side of the cell culture insert, serving as a measuring instrument, SFs are seeded on the apical side of the insert onto the collagen matrix. Thus, SFs are in direct contact with the collagen matrix, while, conversely, direct interactions of MDCK-C7 cells and SFs can be excluded. We could also clearly demonstrate that there is no measurable difference when the membrane is coated with human cartilage extract or when the membrane is coated with the commercially available collagen mixture used in these experiments, supporting the use of the easily accessible collagen mixture. Our MATRIN assay is obviously an improvement over commonly used invasion assays in terms of sensitivity, precision, and handling. This assay allows assessment of not only the overall invasiveness of the cells, but WUNRAU ET AL also the kinetics of the invasive process and, thus, of distinct molecular events associated with the different steps of cell invasion. Therefore, the MATRIN assay is a novel tool for analyzing new therapeutic strategies aimed at modulating the invasive potential of RASFs. ACKNOWLEDGMENTS We thank Borna Truckenbrod and Vera Eckervogt for technical assistance, and Jennifer Gerding for reading the manuscript. We also thank the Department of Dermatology (University Hospital Muenster, Muenster, Germany) for kindly providing the A7 and MDCK-C7 cells. AUTHOR CONTRIBUTIONS All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Pap had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study conception and design. Wunrau, Schnaeker, Freyth, Neugebauer, Hansen, Pap, Dankbar. Acquisition of data. Wunrau, Schnaeker, Freyth, Pundt, Dankbar. Analysis and interpretation of data. Wunrau, Schnaeker, Wendholt, Pap, Dankbar. 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