Molecular Immunology 60 (2014) 14–22

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Tenocyte activation and regulation of complement factors in response to in vitro cell injury Georg Girke, Benjamin Kohl, Catharina Busch, Thilo John, Owen Godkin, Wolfgang Ertel, Gundula Schulze-Tanzil ∗ Department for Orthopaedic, Trauma and Reconstructive Surgery, Charité-University of Medicine, Campus Benjamin Franklin, Berlin, Germany

a r t i c l e

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Article history: Received 14 February 2014 Accepted 19 March 2014 Available online 13 April 2014 Keywords: Complement Scratch assay In vitro cell injury Anaphylatoxin Tendon Tenocytes

a b s t r a c t Inferior tendon healing can lead to scarring and tendinopathy. The role of complement in tendon healing is still unclear. The aim of this study was to understand tenocytes response to mechanical injury and whether complement is regulated by injury. Tenocytes were injured using an optimized automated scratch assay model. Using a self-assembled plotter system, 50 parallel lines of injury were created in a 6 cm diameter tenocyte cell layer. Tenocytes mitotic activity and survival post injury was assessed using FDA/ethidiumbromide assay. Furthermore, this injury model was combined with stimulation of the tenocytes with the complement split fragment C3a. Gene expression of C3aR, C5aR (CD88), CD46, CD55, tumor necrosis factor (TNF)␣, interleukin (IL)-1␤, matrix metalloproteinase (MMP)-1 was analyzed. Immunolabeling for C5aR and CD55 was performed. An enhanced mitotic activity and some dead cells were detected in the vicinity of the scratches. Gene expression of the C3aR was suppressed after 4 h but induced after 24 h post injury. C5aR was downregulated at 24 h, CD46 and CD55 were induced at 24 h in response to injury and CD55 was also elevated at 4 h. MMP-1 was upregulated by injury but both proinflammatory cytokines remained mainly unaffected. Combination of injury with C3a stimulation led to an enhanced C3aR, CD55 and TNF␣ gene expression. According to the gene expression data, the protein expression of C5aR was reduced and that of CD55 induced. In summary, a specific response of complement regulation was found in mechanically injured tenocytes which may be involved in healing responses. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction In various tissues damaged by trauma an increased complement activity has been reported as a typical sequel. It has been suggested that the complement system may play a catabolic role under inflammatory conditions (Hietbrink et al., 2006) and potentially influence the healing outcome (Ignatius et al., 2011). Recently, expression of complement anaphylatoxin receptors and cytoprotective complement regulatory proteins such as CD46, CD55 and CD59 could be shown in tendon and cultured tenocytes (Busch et al.,

Abbreviations: CIS, cell injury system; CRP, complement regulatory proteins; MMP, matrix metalloproteinase. ∗ Corresponding author at: Department for Trauma and Reconstructive Surgery, Charité-University of Medicine, Campus Benjamin Franklin, FEM, Garystrasse 5, 14195 Berlin, Germany. Tel.: +49 30 450 552385; fax: +49 30 450 552985. E-mail address: [email protected] (G. Schulze-Tanzil). http://dx.doi.org/10.1016/j.molimm.2014.03.008 0161-5890/© 2014 Elsevier Ltd. All rights reserved.

2012). Moreover, complement regulation has been demonstrated in response to proinflammatory cytokines and the complement split fragment C3a induced gene expression of the main anaphylatoxin receptors C3aR, C5aR, the proinflammatory cytokines TNF␣ and IL-1␤ and impaired gene expression of CD46 and CD55 in human tenocytes (Busch et al., 2012). During a tendon rupture some tenocytes are directly injured at the rupture site. To discover tenocytes response, in relation to complement activation and regulation during tendon rupture and healing, it can be helpful to start investigation primarily in an in vitro model. Using a simplified in vitro model systemic influences, which may hide effects to be discovered, can be excluded. At present, it is completely unclear how tenocytes might be directly affected by mechanical injuries within an in vitro model. Complement activity and regulatory capacity within tendon and tenocytes is mostly unknown. Generally in response to tendon injuries extracellular matrix and necrotic or apoptotic cell fragments can be delivered, which might lead to an advent of neoepitopes. Complement activity can be

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Fig. 1. Establishment of an automated scratch assay. (a) A cell plotter system was developed. A sterile pipette tip was fixed at the mobile arm of the plotter and used for scratching. The PC-guided arm produces a scratching pattern depicted on the right. (b) The distance between two separate scratches is 1000 ␮m with the width of each scratch of 500 ␮m (b) and illustrated in an injured tenocyte cell layer. (c) 6 cm diameter petri dishes were used. (scale bar 300 ␮m). .

amplified by apoptotic cells, extracellular matrix fragments and neoepitopes (Fishelson et al., 2001). A modified anaphylatoxin receptor expression on tenocytes or an impaired expression of complement inhibiting proteins could be involved in healing processes after cell trauma. Tendon healing is probably influenced by the presence of proinflammatory cytokines such as TNF␣ or IL-1␤ (Schulze-Tanzil et al., 2011; John et al., 2010; Lin et al., 2006). A pronounced proinflammatory response in the healing processes of wounds may result in an increased loss of tissue structure. Structural tissue stability is mainly impaired by the matrix degrading enzymes such as MMP-1 and may offer different therapeutic opportunities (Bedi et al., 2010; Baker et al., 2002). Furthermore, it has been suggested that cytoskeletal alterations associated with a loss of F-actin fibers in tenocytes are detectable after treatment with TNF␣ and might impair the stability of tendons (John et al., 2010). Additionally, IL-1␤ increased the elasticity of human tendon (Qi et al., 2006). This could perhaps be a protective mechanism to prevent tendon ruptures in infectious tissue. However, in the present study the main anaphylatoxin receptors C3aR or C5aR as well as the complement inhibiting proteins CD46 and CD55 were analyzed in cultured tenocytes in response to mechanical cell stress with or without additional stimulation with the chemotactic anaphylatoxin C3a which is released during complement activation. To investigate a possible proinflammatory reaction of the tenocytes after cell injury, TNF␣ and IL-1␤ were analyzed additionally. Finally, to get further information about extracellular matrix remodeling the expression of the matrix degrading enzyme MMP-1 was also included into the analyses.

2. Materials and methods 2.1. Tenocyte isolation and culture Human primary tenocytes were isolated as described previously (Schulze-Tanzil et al., 2004; Stoll et al., 2010) from 15 hamstring tendons (midsubstance of Musculus [M.] semitendinosus, M. semimembranosus, M. gracilis tendons) of healthy middleaged male donors (average age 35.7 years). This study was approved by the Charité review board for experiments with human derived tissues. The peritendineum portion of human tendon

was carefully removed before culturing tendon explants in growth medium for several days. After 1–2 weeks, tenocytes continuously emigrated from these explants and adhered to petri dishes. Three to five days later, when cell density approached confluence, these cells were removed using 0.05% trypsin/1.0 mM EDTA (Biochrom Seromed, Berlin, Germany) and were then multiplied in a monolayer culture to achieve a sufficient quantity of cells for further investigation. Confirmation of the isolated tenocytes was achieved by demonstration of tenomodulin protein and scleraxis gene expression (data not shown). Cultures were grown at 37 ◦ C in a humidified atmosphere with 5% CO2 and the growth medium was changed every three days. Growth medium for tenocytes culture was composed of Ham’s F-12/Dulbecco’s modified Eagle’s medium [50/50, Biochrom-Seromed] containing 10% fetal calf serum [FCS, Biochrom-Seromed], 25 ␮g/mL ascorbic acid [Sigma-Aldrich, Munich, Germany], 50 IU/mL streptomycin, 50 IU/mL penicillin, 2.5 ␮g/mL amphotericin B, essential amino acids, l-glutamine [all: Biochrom-Seromed]. For the experiments tenocytes were cultured at 14,000/cm2 in 6 cm diameter petri dishes [Sarstedt AG, Nümbrecht, Germany] in the growth medium mentioned above for 24 h before injury. 2.2. Cell injury system Tenocytes cultured in 6 cm diameter petri dishes for 24 h were used. Using a self-assembled cell plotter system, 50 parallel lines (width 500 ␮m, distance 1000 ␮m) of injury were created (Fig. 1a and b). The cell injury system (CIS) was based on a common printing plotter system (Haase, Neuss, Germany) for large drawings and was equipped with a very small sterile pipette tip (10 ␮L, Sarstedt AG) on a mobile arm. The mobile arm was able to set cell layer injuries PC-guided (Fig. 1c). Using this cell plotter system, injuries on tenocytes cell cultures could be done in a standardized and reproducible way. Analyses were performed after 4 h and 24 h post cell injury. 2.3. Co-stimulation with cell injury and anaphylatoxin C3a To monitor whether the anaphylatoxin C3a interferes or enhances the cell responses to injury, pre-injured tenocytes were additionally stimulated with the anaphylatoxin C3a. Directly after

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CIS-treatment 1 ␮g/mL C3a diluted in growth medium was added to the petri dishes for at least 24 h. The control group was only CIS-treated in the absence of C3a. 2.4. Analysis of mitotic activity For establishment of the scratch assay, the rate of mitosis post scratching was determined using a self-made time lapse microscope (Fig. 2b). The time lapse microscope based on a common binocular microscope (Bresser, Rhede, Germany) and a digital camera for microscope DCM130E (Müller, Erfurt, Germany). To guarantee constant temperature conditions for cell culture growing the microscope light had to be changed to a modified LED light. With this time lapse microscope it was possible to follow the cell culture processes over 24 h by taking a picture of the cell culture every 2 min. These pictures were joined together to a movie file, which affords a time-lapse of cell culture processes. Specifically the mitosis rates were counted within the four quadrants of the petri dishes. The mitotic activity in injured cell cultures was compared to untreated cell cultures over 24 h by counting them manually in defined microscopic fields. 2.5. Live-dead staining Tenocytes seeded on cover slides were incubated in a mixture of 5 ␮L/mL fluorescein diacetate (FDA, Sigma-Aldrich, 3 mg/mL dissolved in acetone [stock solution]) and 1 ␮g/mL ethidiumbromide (Etbr, Carl-Roth, Karlsruhe, Germany) for 10 min at 37 ◦ C. The green (vital cells) or red (dead cells) fluorescence was visualized using fluorescence microscopy (Axioskop 40, Carl Zeiss, Jena, Germany) and digital camera for microscope (Color View II, Olympus, Shinjuku, Japan). 2.6. Messenger RNA analysis by real time detection polymerase chain reaction Real time detection polymerase chain reaction (RTD-PCR) analyses were performed to obtain semiquantitative gene expression data for the anaphylatoxin receptors C3aR and C5aR as well as the regulatory components in the complement cascade: CD46, CD55 and the proinflammatory cytokines TNF␣, IL-1␤ and catabolic enzyme MMP-1. Tenocytes were cultured at 14,000/cm2 in 6 cm diameter petri dishes, rinsed with PBS and stimulated for 4 or 24 h in the scratch assay or were additionally treated with 1 ␮g/mL recombinant C3a. Tenocytes total RNA was isolated using Qiagen RNA isolation mini kit (Qiagen, Hilden, Germany) and the RNA quantity and quality was evaluated using the RNA 6000 Nano assay (Agilent Technologies, Santa Clara, USA). Reverse transcription was performed using the Quanti Tect Reverse Transcription Kit and equal amounts of RNA (500 ng) according to the manufacturer’s instructions (Qiagen). Aliquots of 1 ␮L cDNA (16.7 ng) were amplified using RTD-PCR in a 20 ␮L reaction mixture using the TaqMan Gene Expression Assay (Applied Biosystems [ABI], Foster City, USA) and specific primer pairs for scleraxis, C3aR, C5aR, CD46, CD55, TNF␣, IL-1␤, MMP-1 and the house-keeping gene ␤-actin (␤-actin, ABI; see Tab. 1). All assays were performed in an Opticon 1 – Real-Time-Cycler (OpticonTM RTD-PCR, Biorad, Hercules, USA). The conditions of amplification were as follows 10 min at 95 ◦ C, and then for 40 cycles 15 s at 95 ◦ C, 30 s at 60 ◦ C and followed by 6 ◦ C cooling. The lack of primer dimers and the specifity of amplification were further confirmed by efficiency testing and agarose gel electrophoresis of PCR products all showing a single band of expected size. Relative gene expression levels were normalized and calculated with the 2−CT method (Livak and Schmittgen, 2001).

2.7. Immunofluorescence labeling for C5aR and CD55 Tenocytes were cultured on cover slips for 24 h, before fixing in 4% paraformaldehyde solution freshly prepared from paraformaldehyde (USB, Cleveland, USA) in PBS solution for 15 min. Cells were washed with Tris buffered saline (TBS: 0.05 M Tris, 0.015 M NaCl, pH 7.6) and incubated with protease-free donkey serum (5%, diluted in TBS) for 10 min at room temperature (RT). Subsequently, cells were permeabilized using 0.1% Triton X-100 for 6 min, rinsed and incubated with the primary antibodies in a humid chamber for 1 h at RT. The following primary antibodies were used: mouse-anti-human CD5aR (GeneTex Inc., Biozol, Eching, Germany), goat-anti-human CD55 (R&D systems, Minneapolis, US), rabbit-anti-human type II collagen (Acris, Hiddenhausen, Germany). Negative controls included replacement of primary antibodies with respective isotype mouse or goat (Invitrogen, Carlsbad, USA) used at the same concentration as that for the primary antibodies. Chondrocytes were subsequently washed with TBS before incubation with donkey-anti-mouse-, anti-rabbit- or -anti-goat-Alexa-Fluor® 488 (Invitrogen) coupled secondary antibodies (diluted 1:200 in TBS), respectively, for 1 h at RT. Cell nuclei were counterstained using 4 ,6 -diamidino-2phenylindol (DAPI) (Roche Diagnostics GmbH). Labeled cells were washed several times with TBS, before mounting with fluoromount mounting medium (Southern Biotech, Biozol Diagnostica, Eching, Germany) and examined under a fluorescence microscope (Axioskop 40, Carl Zeiss, Jena, Germany). The immunofluorescence images were obtained and evaluated under standardized conditions. Three images were generated for each staining, in each image eight cells (at 200× magnification) were analyzed for fluorescence intensity using the software ImageJ (Rasband, National Institute of Health, USA) per consumption. The maximum number of countable cells was determined for each field based on the cell density before starting the assessment. According to the average value of the fluorescence intensities measured in each case, the background fluorescence was subtracted. The statistical analyses were performed from the calculated means of each image.

2.8. Statistical analysis Normalized data were expressed as the mean and error of mean (mean ± SEM). Differences between experimental groups were considered significant at p < 0.05 as determined by student’s paired two-tailed t test, based on the assumption of a Gaussian distribution (Prism 5, GraphPad software inc, San Diego, California, USA).

3. Results 3.1. Dead tenocytes in response to scratching Live-death-staining were subsequently undertaken to investigate the rate of surviving cells after treatment in the cell plotter system (Fig. 2d). Dead cells could be identified along the cutting line by a few small red marks. But the majority of cells were still alive, shown by a green staining. Even cells which appear to be injured survived the treatment (yellow marked cell in Fig. 2d). However, some cells which might have detached could not be shown by this assay. Live-death-stainings were conducted again after 4 h, 24 h and 48 h. At all time points only few dead cells could be observed. Few dead cells could also be found in the control staining [Fig. 2e]. An indication of a time depending cell death after treatment could not be detected.

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Fig. 2. Characterization of the automated scratch assay. (a) Shown are 16 images taken over a time frame of 24 h after cell injury, with time-lag between two images of 45 min, scale bar 200 ␮m. (b) Analysis of mitosis in the scratch assay is summarized. Depicted is a single mitosis over 8 min. The time-lag between two separate images is 2 min. Scale bar 50 ␮m. The number of mitoses within 24 h in three independent scratch assays (injury) was compared to non-injured tenocyte cultures (co). (c) Statistical analysis shows a significant induction of mitoses after scratching. (d) An exemplary live-death assay of an injured culture performed directly after injury is shown. Only few dead cells (red) are discernible. Yellow arrow: viable cell which cellular process was damaged by scratching. Scale bar: 200 ␮m. (e) An episode of live-death-staining of different time points (0.5 h, 2 h, 4 h) with an untreated control stain after 4 h. Scale bar: 200 ␮m. (For interpretation of the references to color in this text, the reader is referred to the web version of the article.)

Table 1 Oligonucleotides used for RTD PCR analysis.

␤-Actin (ACTB) C3aR C5aR ␤-Actin (ACTB) CD46 CD55 TNF␣ IL-1␤ MMP-1 scleraxis * **

Company

Forward/reverse primer

Probe/reference

bp

ABI* ABI* Qiagen Qiagen ABI* ABI* ABI* ABI* ABI* ABI*

**

NM NM NM NM NM NM NM NM NM NM

171 82 95 147 94 62 94 80 133 63

ABI: applied biosystems. sequence is not provided by ABI.

**

TGCCATCTGGTTCCTCAA GCCAGTGGTGATGCTGTA TGGGACGACATGGAGAA GAAGGTCTCAAACATGATCTGG ** ** ** ** ** **

001101.2 004054.2 001736.3 001101.2 172351.1 000574.2 000594.2 000576.2 001145938.1 001080514.1

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3.2. Results of tenocytes time lapse and mitosis analysis

4. Discussion

Morphologically the wound in the cell layer produced by the scratching has been closed within 24 h. Thus, tenocytes migrated from the margin to the middle of the scratch track to close the defect in the cell layer. The comparison of injured and untreated cell layers over 24 h showed a significantly elevated mitotic activity in the injured cell layer (Fig. 2c). Cell injury led to a significant induction of mitoses in tenocytes. The visual aspect suggested an augmented mitotic activity especially in tenocytes, which were in close association with the injury. Furthermore, clearly marked augmentation of mitotic activity could be observed three hours after cell injury.

Several clinical studies characterized conclusive perspectives following tendon rupture and surgical or non-surgical intervention within in vivo situations (Porter et al., 2013; Amendola, 2014). In some cases healed tendons did not retrieve their complete functionality but the underlying mechanisms and the mediators involved remained unclear. To study selected aspects of changed gene expression due to tendon disorders, e.g. driven by particular mediators, cell culture experiments are quite convenient and well established (Busch et al., 2012; Schulze-Tanzil et al., 2011; John et al., 2010). However, a reproducible experimental setting is necessary to allow the investigations of cell injury in vitro. This study presents an optimized in vitro model, which enables injuries of tenocyte cultures in a reproducible, standardized and specific way. Our own foregone experiments have shown that manually performed cell culture injuries by using a scalpel or pipette tip, led to slight and hardly reproducible results in gene expression analyses. To achieve stronger effects and to standardize the method applied, we designed an optimized CIS based on an automatized PC-guided plotter system. This model allows injuring many cells in cell culture and generating highly reproducible treatments. An important precondition for applicability of CIS was to ensure the survival of sufficient treated cells. Results of live–death-staining confirmed the survival of the majority of cells, interestingly also some directly damaged cells remained viable. It must be methodologically considered that only adherent cells could be shown in the present live–death-staining. In addition to direct cell damage and stretching, which may lead to necrotic cell death, induction of apoptosis should also be taken into account (Wu et al., 2010). However, the live–death assay used here could not distinguish between both types of cell death. In view of the fact that only a minority of dead cells were detected so far, no further analysis was undertaken to prove whether complement activation is directly involved in tenocyte death due to mechanical injury. Interestingly, the live–death-staining showed singular tenocytes, which lost their cytoplasmic extension, but still remained viable (Fig. 2d, yellow arrow). These cells would either die at a later observation time point or they were able to activate cytoprotective mechanisms possibly also by increasing complement inhibiting proteins. Overall, these mechanisms of cell damage mimicked in the CIS could be utilized, and to some degree compared with effects of tendon ruptures on tenocytes in vivo. Nevertheless, substantial peculiarities of tendon tissue in vivo compared to the tenocytes monolayer culture used here should be strongly kept in mind such as the presence of an abundant extracellular matrix in vivo, and the particular order of the few cells embedded in this matrix in communicating cell rows directly interconnected by gap junctions (Maeda et al., 2012). The capacity of cell reorganization in CIS-treated cell cultures could be studied by analyzing the mitotic cell activity. Thereby, changes of tenocyte proliferation rates after CIS-treatment could be observed. This finding indicates an induced cell proliferation after direct cell trauma. Obviously, not only the direct damage of tenocytes, but also the newly occurred empty place in the scratch track might be an important stimulus for enhanced cell proliferation. However, the direct impact on cell cycle machinery remains undiscovered. Increased in vivo fibroblast proliferation during wound healing, following soft tissue trauma, has been widely reported (Zeichen et al., 2000; Buckley et al., 1988; Skutek et al., 2001; Banes et al., 1985) and also for healing tendon tissue (Wu et al., 2010). The fact that increased cell proliferation was identified, after treatment, is a further argument to use CIS as an in vitro cell culture to mimic aspects of tendon rupture. Furthermore, it might be very interesting for future studies to investigate cell signaling pathways, which stimulate tenocytes to migrate into the cell layer defect.

3.3. Gene expression of tenocytes subjected to the scratch assay The influence of the cell plotter system assisted cell scratching on tenocyte gene expression was investigated at two different time points, 4 h and 24 h after cell injury. The gene expression of the anaphylatoxin receptor C3aR in tenocytes was slightly, but significantly suppressed after 4 h, whereas 24 h after cell injury a significantly elevated gene expression of the C3aR was found (Fig. 3a). In contrast to the C3aR, the C5aR showed a significantly reduced gene expression after 24 h. Although mechanical injuries led to no significant change after 4 h, a tendency of augmented gene expression could be observed (Fig. 3b). The complement regulating protein (CRP) CD46 showed a significantly increased gene expression after 24 h (Fig. 3c). Accordingly, CD55 as a gene with complement inhibiting and cytoprotective properties, presented a significantly elevated gene expression for both investigation time points (Fig. 3d). The gene expression of proinflammatory cytokines TNF␣ and IL-1␤ were not significantly affected by this mechanical treatment (Fig. 3e and f). The extracellular matrix degrading enzyme MMP-1 was highly and significantly upregulated after 24 h, although no significant changes could be detected after 4 h (Fig. 3g). 3.4. Gene expression of tenocytes subjected to the scratch assay and treated with C3a This experimental setup was chosen in order to investigate the behavior of injured cells under the influence of the anaphylatoxin C3a. Tenocytes were injured by the use of CIS and additionally stimulated with anaphylatoxin C3a, while the control group was only injured. Interestingly, the C3aR revealed a significantly elevated gene expression 24 h after co-treatment consisting of CIS and C3a stimulation (Fig. 4a). However, after 24 h the C5aR showed only a tendency of an increased gene expression (Fig. 4b, not significant). A significantly augmented gene expression could also be demonstrated for the complement inhibiting protein CD55 24 h after co-treatment (Fig. 4c). Furthermore, the proinflammatory cytokine TNF␣ showed a significantly elevated gene expression at both 4 h and 24 h time points (Fig. 4d). The matrix degrading enzyme MMP-1 revealed no significant gene expression changes (Fig. 4e). 3.5. Immunofluorescence labeling of tenocytes subjected to scratch assay To investigate influences of mechanical damage on the cells the protein expression in tenocytes was detected, using immunofluorescence staining. C5aR-immunolabeled tenocytes showed 24 h after mechanical injury a significantly reduced expression of C5aR (Fig. 5a–e). The CD55-immunolabeled tenocytes displayed a significantly enhanced protein expression of the complement regulating protein CD55 (Fig. 6a–e).

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Fig. 3. Gene expression analysis of injured tenocyte cultures using scratch assay. Regulation of C3aR, C5aR, CD46, CD55 as well as TNF␣, IL-1␤ and MMP-1 gene expression under CIS-injury was analyzed in cultured tenocytes using RTD-PCR. Data were normalized versus the housekeeper ␤-actin and then versus the non-treated controls ((a) C3aR, (b) C5aR, (c) CD46, (d) CD55, (e) TNF␣ and (f) IL-1␤, (g) MMP-1). Five (C3aR, CD46, CD55, TNF␣, IL-1␤ and MMP-1) or four (C5aR) independent experiments were performed using tenocytes derived from five or four different healthy donors (hamstring tendon). Equal amounts of RNA were reverse transcribed and triplicates of 16.7 ng cDNA of each sample were used for analysis.

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Fig. 4. Gene expression analysis of injured tenocyte cultures, additionally treated with recombinant C3a using a scratch assay. Regulation of C3aR, C5aR, CD46, CD55, TNF␣, IL-1␤ and MMP-1 gene expression under CIS-injury and stimulation with 1 ␮g/mL C3a was analyzed in cultured tenocytes using RTD-PCR. Data were normalized versus the housekeeper ␤-actin and then versus the CIS-treated controls ((a) C3aR, (b) C5aR, (c) CD46, (d) CD55, (e) TNF␣ and (f) IL-1␤, (g) MMP-1). Five independent experiments were performed using tenocytes derived from five different healthy donors (hamstring tendon). Equal amounts of RNA were reverse transcribed and triplicates of 16.7 ng cDNA of each sample were used for analysis.

Tenocytes, which were treated by CIS exhibit different changes in their complement gene expression. The C3aR revealed 24 h after treatment an elevated gene expression. Basically, this might be an indication of increased sensitivity of tenocytes for complement activation. In cases of complement activation the anaphylatoxin C3a is able to induce C3aR, but also the proinflammatory cytokines such as TNF␣ upregulated this anaphylatoxin receptor (Busch et al., 2012). Furthermore, after 24 h C5aR was slightly suppressed. Up to now little is known about the differential regulation and activity of C3aR and C5aR in tenocytes, so that no general statement can be made. From other tissues it is known that treatment with C5aRantibodies may improve the outcome of particular inflammatory diseases, e.g. in periodontitis (Breivik et al., 2011). Both CD46 and CD55, acting as complement inhibiting and cytoprotective proteins on tenocytes (Busch et al., 2012), were found to be upregulated by cell injury at the gene expression level. Interestingly the CD55 was induced already after 4 h, whereas both CD46 and CD55 exhibited an increase in gene expression after 24 h. The immunofluorescence staining confirmed an elevated protein expression of CD55. This can be considered as a strong indication that mechanically damaged tenocytes are able to inhibit complement activation by inducing their own complement regulating proteins. Nevertheless, complement activation is well known as a typical sequel of trauma in various human tissues (Bellander et al., 2001; Hietbrink et al., 2006; John et al., 2007). As part of the healing process after the tendon rupture MMPs, such as MMP-1 are of particular importance. They are largely

responsible for degradation of tendon tissue extracellular matrix (Del Buono et al., 2012; Lakemeier et al., 2011; Bedi et al., 2010; Fu et al., 2002). MMP-1 was detected as strongly increased after CIStreatment. Several references for a comparable increase of MMP-1 after tendon rupture can be found in the literature (Fu et al., 2002; Lakemeier et al., 2011). Therefore, the present study is able to point out that directly harmed cells are one possible reason for well reported increase of MMP-1 after tendon rupture. Interestingly, the proinflammatory cytokines TNF␣ and IL-1␤ were not impaired by CIS-treated tenocytes, although a few in vivo trials showed an increase in TNF␣ after cyclic strain or after rotator cuffs ruptures (Skutek et al., 2001; Millar et al., 2009). Probably, the expression of TNF␣ and IL-1␤ in CIS-treated tenocytes remains unaffected due to the absence of other exogenous inductive proinflammatory cytokines, anaphylatoxins or immune competent cells. This is supported by the observation that a combination of CIS with stimulation of the tenocytes with C3a led to a detectable increase in the gene expression of these cytokines. Previous works have characterized the influence of anaphylatoxin C3a on healthy tenocytes in vitro, concerning the expression of anaphylatoxin receptors and complement inhibiting proteins on tenocytes as well as proinflammatory cytokines in tenocytes (Busch et al., 2012). It is still unclear however, what influence anaphylatoxin C3a on injured tenocytes might have. In light of the fact that tendon rupture stimulates local complement activation and therefore release of complement split fragments such as C3a and C5a, it is reasonable to assume an enhanced effect

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Fig. 5. Immunofluorescence labeling for C5aR of injured and non-injured control cultures. Protein expression of C5aR in cultured tenocytes after treatment in CIS is shown. Primary human tenocytes, grown in monolayer culture, were injured with CIS or remained untreated (co, control). C5aR was immunolabeled with specific antibodies and the immunofluorescence intensity was evaluated. Tenocytes derived from three healthy donors (hamstring tendons) were analyzed in independent experiments. (a) A control staining was performed by omitting the primary antibody, (b) for the isotype control cells were labeled by a unspecific binding anti-mouse primary antibody, (c) a staining of untreated tenocytes was added as experimental control, (d) immunofluorescence labeling was performed by using monoclonal mouse-anti-C5aR 24 h after injury by CIS, (e) statistical analysis of immunofluorescence intensity; p < 0.05, paired students t-test). Scale bar: 50 ␮m.

under the proinflammatory conditions of the first healing phase. The influence of C3a on injured tenocytes was further addressed. In injured and C3a-stimulated tenocytes a significantly augmented gene expression of C3aR after 24 h could be observed. Tenocytes, which were treated only by C3a showed a comparable induction of C3aR gene expression (Busch et al., 2012). These results could indicate that a posttraumatic activated complement system might sensitize tenocytes against anaphylatoxin C3a. Furthermore, it is known that CD55 is still expressed on tenocytes and is able to inhibit the alternative pathway of complement activation (Wang et al., 2010; Busch et al., 2012; McGregor et al., 2012). Co-treatment with CIS and C3a revealed a significantly increased expression of CD55. This concurred with the increased expression of CD55 after sole CIS-treatment, however a sole stimulation with C3a induced a decreased expression of CD55 (Busch et al., 2012). Hence, an injuryinduced expression of the complement inhibiting protein CD55 can be regarded as possible in vivo. Probably, cytoprotective CD55

might improve the outcome after tendon ruptures. This hypothesis is supported by the observation that an induced expression of CD55 is playing a central role in the recovery outcome after brain injury (VanLandingham et al., 2007). The removal of extracellular matrix fragments in damaged tissue and later the remodeling process of the novel extracellular matrix are executed by degradative enzymes such as MMPs. The gene expression of MMP-1 was induced by CIS after 24 h but did not show any significant changes in response to an additional C3a treatment. Moreover, the individual treatment of tenocytes with C3a alone led to an early reduction in gene expression (own unpublished data). Both mechanisms, mechanical injury and complement activation, therefore show some interference on tenocyte MMP gene expression. For the interpretation of the MMP-1 expression analyzes it must be taken into account that an induced gene expression of MMP-1 will not automatically lead to an increased activity of MMP-1. MMPs in general are substantially regulated

Fig. 6. Immunofluorescence labeling for CD55 of injured and non-injured control cultures. Protein expression of CD55 in cultured tenocytes after treatment in CIS is shown. Primary human tenocytes, grown in monolayer culture, were injured with CIS or remained untreated (co, control). CD55 was immunolabeled with specific antibodies and analyzed regarding the immunofluorescence intensity. Tenocytes derived from three healthy donors (hamstring tendons) were analyzed in independent experiments. (a) A control staining was performed by omitting the primary antibody, (b) for the isotype control cells were labeled by a unspecific binding anti-goat primary antibody, (c) a staining of untreated tenocytes was added as experimental control, (d) immunofluorescence labeling was performed by using polyclonal rabbit-anti-C5aR 24 h after injury by CIS, (e) statistical analysis of immunofluorescence intensity; p < 0.05, paired students t-test). Scale bar: 50 ␮m.

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by tissue inhibitors of metalloproteinases (TIMPs), which are also expressed in tenocytes (Baker et al., 2002; Del Buono et al., 2012). To investigate the implication of C3a and mechanical injuries on tenocytes extracellular matrix remodeling in vivo, further studies are needed to be conducted. In vivo studies revealed that in ruptured M. supraspinatus tendons a higher expression of proinflammatory cytokines such as TNF␣ and IL-6 was detectable (Millar et al., 2009). As a marker of tenocyte proinflammatory response, TNF␣ gene expression was significantly increased after CIS and C3a co-treatment. The upregulatory influence of C3a on tenocyte TNF␣ gene expression has already been observed in tenocytes solely stimulated with C3a (Busch et al., 2012). These findings might characterize anaphylatoxin C3a as a potent inductor of the expression of proinflammatory cytokines in tenocytes whereas mechanical injury alone could not elevate TNF␣ expression in vitro. If tendon ruptures in vivo led to an activation of complement, released anaphylatoxins could probably provoke a proinflammatory response in tenocytes. This has to be further discovered and validated by in vivo studies. 5. Conclusion In the present study a new PC-guided technique was performed to treat cell cultures with standardized and reproducible cell injuries. We could confirm the survival of the majority of cells after treatment and validate an augmented proliferation of tenocytes post cell injury. Substantially, an increased expression of complement inhibiting proteins, such as CD46 and CD55 could be observed. Although an attended proinflammatory reaction by tendon ruptures in vivo is well known, elevated gene expression of proinflammatory cytokines, such as TNF␣ and IL-1␤ could not be found after cell trauma but was detectable in response to additional complement split fragment treatment. Acknowledgements The authors would like to acknowledge the support of the study by the AFOR foundation Olten, Switzerland Additionally, funding for equipment used in this study was provided by the Sonnenfeld foundation Berlin. References Amendola, A., 2014. Outcomes of open surgery versus nonoperative management of acute Achilles tendon rupture. Clin. J. Sport Med. 24 (1), 90–91. Baker, A.H., Edwards, D.R., et al., 2002. Metalloproteinase inhibitors: biological actions and therapeutic opportunities. J. Cell Sci. 115 (Pt 19), 3719–3727. Banes, A.J., Gilbert, J., Taylor, D., Monbureau, O., 1985. A new vacuum-operated stress-providing instrument that applies static or variable duration cyclic tension or compression to cells in vitro. J. Cell Sci. 75, 35–42. Bedi, A., et al., 2010. The effect of matrix metalloproteinase inhibition on tendonto-bone healing in a rotator cuff repair model. J. Shoulder Elbow Surg. 19 (3), 384–391. Bellander, B.M., Singhrao, S.K., et al., 2001. Complement activation in the human brain after traumatic head injury. J. Neurotrauma 18 (12), 1295–1311.

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Tenocyte activation and regulation of complement factors in response to in vitro cell injury.

Inferior tendon healing can lead to scarring and tendinopathy. The role of complement in tendon healing is still unclear. The aim of this study was to...
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