Cell surface and relative mRNA expression of heat shock protein 70 in human synovial cells
Abstract
Heat shock proteins (Hsps) have been repeatedly implicated to participate in the pathogenesis of rheumatoid arthritis (RA). Methods: Herein, Hsp70 cell surface and mRNA expression were studied in human fibroblast-like synovial cells, dermal fibroblasts and peripheral blood leukocytes derived from 24 RA patients, who underwent synovectomy by using flow- cytometric analysis and real-time quantitative reverse-transcriptase polymerase chain reaction. For comparison, peripheral blood leukocytes of 17 healthy controls were tested.
Results: Significantly higher Hsp70 membrane positivity was found on fibroblast-like synovial cells in RA patients (average 18.3%, median 16.5%) than on autologous and healthy control peripheral blood lymphocytes (RA patients: average 4.7%, median 2.9%, p ¼ 0.002; healthy controls: average 6.0%, median 4.5%, p ¼ 0.002) and/or autologous dermal fibroblasts (average 5.1%, median 4.3%, p , 0.001). Strong Hsp70 cell surface expression was also found on peripheral blood monocytes of RA patients (average 53.0%, median 58.1%) and healthy controls (average 49.4%, median 47.5%, p ¼ 0.52). Peripheral blood granulocytes of healthy controls (average 41.8%, median 41.4%) showed significantly increased Hsp70 expression comparing with RA patients (average 10.7%, median 6.4%, p ¼ 0.005).
Significantly higher Hsp70 gene expression was observed in synovial cells of RA patients (average 2.04, median 1.7) when compared with autologous peripheral blood leukocytes (average 0.75, median 0.68; p , 0.001). However, the difference in Hsp70 gene expression between RA-derived synovial cells and healthy control peripheral blood leukocytes (average 1.69, median 1.64) was not observed (p ¼ 0.83). We also found significantly lower relative gene expression in peripheral blood leukocytes of RA patients in comparison with healthy controls (p , 0.001).
Interestingly, we found that Hsp70 gene expression in RA non-affected skin dermis gained from the operation wound was 3.7-fold higher in average (average 7.6, median 8.3) when compared to autologous RA-affected synovial tissue (p , 0.001); 10.1-fold higher in average when compared to autologous peripheral blood leukocytes (p , 0.001) and 4.5-fold higher in average comparing to control peripheral blood leukocytes (p , 0.001).
Conclusion: Hsp70 gene expression in RA-affected synovial tissue is followed by Hsp70 cell surface expression on fibroblast- like synovial cells growing from RA synovial tissue. Hsp70 may be translocated to the cell surface from the cytosol and/or Hsp70 released from inflamed synovial tissue may be captured onto the membrane of synovial cells from the extracellular space via Hsp receptors.
As a physiological response to potentially harmful enviromental stress factors, skin dermis produces higher levels of Hsp70 comparing to the cells of internal organs and tissues.
Keywords: mRNA expression, Hsp70, synovial cells, real-time PCR, cell surface expression, flow cytometry
Introduction
Heat shock proteins (Hsps) are part of a tightly regulated and phylogenetically old biological system that enables organisms to respond adequately to detrimental environmental factors. Based on high interspecies sequence homologies, inducible tissue expression and a strong immunogenicity, Hsps have been repeatedly incriminated to be involved in various autoimmnune conditions, including rheumatoid arthritis (RA) [1– 3].
RA is a chronic systemic autoimmune disease, characterised by inflammation of the synovium and destruction of joint cartilage. Activated cells of the synovium produce pro-inflammatory and matrix- degrading effector molecules, which maintain the inflammation and lead to the destruction of the involved joints. In addition to macrophages, T- and B-cells, fibroblast-like synoviocytes must be con- sidered key cells in driving the pathological processes [4]. Although RA’s aetiology remains unresolved, a combination of inherited and environmental factors participates in disease progression [5,6].
The synovialtissue of RApatients is characterised by a chronic inflammatory process leading to an alteration of cellular homeostasis and finally to severe tissue damage. Avarietyof different stressors are present in the inflamed synovial tissue of RA patients, each of which has the potential to induce a stress response [7,8].
In RA, physicalineractions between Hsp70 and major histocompatibility complex (MHC) class II shared epitopes suggest that Hsp70 participates in the autoimmune response [9]. Hsp 70-kDa family contains at least eight homologous chaperone proteins consisting of both constitutively expressed and stress-inducible members residing mainly in the cytosol and nucleus which protect the organism from stress-induced cell injury. Our group previously reported strong positivity for membrane-expressed Hsp70 in fibroblast-like synovial cells derived from patients with severe course of RA and juvenile idiopathic arthritis (JIA) [10].
The goal of the current study was to determine cell surface and mRNA expression of stress-inducible Hsp70 in synovial cells derived from synovial tissue of RA patients who underwent synovectomy. Simul- taneously, we studied Hsp70 expression in autologous skin dermis gained from the operation wound and peripheral blood leukocytes of RA patients and healthy controls.
Materials and methods
Patients
Local ethics committee approval and informed consent were obtained prior to testing from all participants in the study. The cohort consisted of
24 rheumatoid factor positive polyarthritis patients (23 females, 1 male; age range 29 – 79 years, median 56.5 years) with disease duration ranging from 3 to 32 years (median 17 years) undergoing the synovectomy at 1st Clinic of Orthopedics, University Hospital Motol in Prague. All of the patients had an active disease at the time of testing. Patients were treated, depending on the stage of the disease, with non-steroidal anti-inflammatory drugs, corticosteroids and/or disease-modifying anti-rheumatic drugs Synovial cells were derived from RA-affected synovial tissue (finger joint n ¼ 1, metacarpophalan- geal joint n ¼ 7, elbow joint n ¼ 1, shoulder joint n ¼ 1, metatarsophalangeal joint n ¼ 6, ankle joint n ¼ 2, knee joint n ¼ 3 and hip joint n ¼ 3) during synovectomy and skin fibroblasts from RA non- affected operation wound.
Healthy controls
Seventeen healthy controls (13 females, 4 males; age range 28 – 69 years, median 56 years) were included in the study. One healthy control was randomly selected to interpret the data of real-time reverse-transcriptase polymerase chain reaction (RT-PCR) analysis using comparative threshold cycle (Ct) method.
Cell cultivation and preparation for fluorescence-activated cell sorting (FACS) analysis
Initially, the skin tissue was trimmed of epidermis, then both synovial and skin tissues were cut to 2 £ 2 mm sections and cultured in AmnioMax medium (Gibco, Invitrogen Corporation, NY, USA) containing 1% antibiotics (Penicillin– Streptomycin, Sigma Bios- ciences, St Louis, MO, USA) and 1% Fungizone (Antimycotic, Gibco) at 378C with 5% CO2 for 1 – 2 months. One section of each sample was used for RNA isolation. The medium was replaced twice a week.
Upon reaching confluence, cells were trypsinised using 0.05% trypsin/0.02% ethylenediamine tetraacetic acid (EDTA) in phosphate-buffered saline (PBS) (PAN Biotech, GmbH, Aidenbach, Germany) for 30 s, after which 1 ml of medium was added. The suspended cells were washed and resuspended again in 1 ml of PBS. The approximate cell number and viability were determined by Trypan blue exclusion method. All cells were viable after trypsinisation. The amount of 0.1 £ 106 cells per tube was the minimal amount used for the FACS analysis.
The adherent synovial cells were defined as fibroblast-like synovial cells (type B) [11]. Cells were identified by their unique growth pattern, morphology and by using monoclonal mouse anti- human vimentin V9 immunoglobulin (IgG1) antibody (Immunotech, Marseille, France) for cells of mesenchymal origin [12].
Analysis of Hsp70 cell surface expression using flow cytometry
The adherent cells at primary cultures or first passages were analysed as it was previously described by Farkas et al. [13], Nguyen et al. [10] and Hromadnikova et al. [14] using flow cytometry and fluorescein isothio- cyanate(FITC)-conjugated monoclonal antibodies against Hsp70 (cmHsp70.1-FITC, IgG1, multi- mmune GmbH, Munich, Germany) and MHC I (IgG1, Becton Dickinson, San Jose, CA, USA) which was used as a positive control. Briefly, after washing in PBS containing 10% fetal calf serum (FCS) (Sigma Biosciences, St Louis, MO, USA), single-cell suspen- sion of 0.1 £ 106 cells per tube was stained for 30 min on ice.
The percentage of positive stained cells was determined as the number of positively stained cells minus the number of cells stained with an isotype- matched negative control antibody. Only 7-amino- actinomycin D (7-AAD, Becton Dickinson, San Jose, CA, USA) negative, viable cells with intact cell membranes were gated and analysed.
The cut-off value for Hsp70 of 10% was chosen based on the results from previous screening of normal cells and tissues (peripheral blood lymphocytes and skin fibroblasts) by flow cytometry [10,13].
RNA isolation
Total RNA was extracted from the whole blood of 12 healthy controls and 21 RA patients immediately after collecting into EDTA containing tubes using QIAamp RNA Blood Mini Kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions. Patients’ whole blood was sampled shortly before the synovectomy.
Twenty-two RA-derived synovial tissues and 20 RA- derived skin dermis stored in RNAlater w (Ambion, Applied Biosystems, Foster City, CA, USA) at 2208C were later homogenised and disrupted using rotor– stator homogeniser (Polytron PT 1200 E, Kinematica AG, Lucerne, Switzerland). RNA was subsequently isolated from supernatant using QIAamp RNA Blood Mini Kit (Qiagen, Hilden, Germany) and the protocol for isolation of total RNA from tissues.
To obtain DNA-free RNA samples, deoxyribonu- clease I (DNase I, Fermentas, Ontario, Canada) treat- ment was carried out to remove any contaminating DNA.
RNA concentration and purity were determined by ultraviolet spectrophotometry (NanoDrop Spectro- photometer ND-1000, NanoDrop Technologies, Wil- mington, DE, USA). All samples had an optical density (OD) 260/280 nm ratio .1.9, indicating high quality and purity. RNA samples were stored at 2208C until use.
Analysis of relative Hsp70 gene expression using real-time quantitative RT-PCR
All PCR reactions were performed on the ABI PRISM 7300 Sequence Detection System (Applied Biosystem, Foster City, CA, USA) as we previously described [15]. To perform one-step real-time quantitative RT-PCR, human Hsp70 and b-actin primers and probes were designed using Primer Express version 2.0 (Applied Biosystems, Foster City, CA, USA) from the human Hsp70 (NCBI, Entrez Gene, HSPA1A, GeneID 3303) and b-actin (NCBI, Entrez Gene, ACTB, GeneID 60) gene sequences. Primer and probe sequences are shown in Table I.
To amplify the Hsp70 gene and endogenous control b-actin, PCR reactions were set up in the same tube in a reaction volume of 25 ml consisting of 1 £ TaqMan One-Step RT-PCR Master Mix, Taq- Man probes (100 nM FAM-labelled human Hsp70 probe and 200 nM VIC-labelled b-actin probe), forward and reverse primers (200 nM for human Hsp70 and 300 nM for b-actin) and 1.0 ng of extracted total RNA. The minimum primer and probe concentrations giving the lowest Ct and maximum DRn were used. Each sample was analysed in triplicate. The analysis of each sample was repeated two times in two different days.
The thermal profile used for the one-step real-time RT-PCR included reverse transcription at 488C for 40 min, denaturation at 958C for 10 min, followed by 50 cycles of PCR with denaturation at 958C for 15 s and annealing/extension at 608C for 1 min.
To eliminate the need for standard curves, the comparative Ct method [16] was used to interpret the data. The difference (DCt) between the Ct values of the Hsp70 and the endogenous control was calculated for each sample. RNA isolated from the whole blood of the healthy control (# 17) was chosen as the reference for each comparison which had been made. The comparative DDCt calculation involved finding the difference between each sample’s DCt and the reference’s DCt. Finally, the DDCt values were transformed to absolute values using the formula 2DDCt. Multiple negative water blanks (no template control, NTC) were included in every analysis. Samples were also tested to ensure that they were negative for DNA using a complete master mix without the reverse transcriptase (no amplification control, NAC). No amplification was observed for these controls, indicating the specificity of the assays for the respective mRNAs. To minimise the risk of contamination, RNA extraction and prep- aration of real-time RT-PCR reaction were performed in laminar air-flow and aerosol-resistant tips were used, entirely.
Statistics
All data were analysed with Excel (Microsoft Windows 2000) and statistical software KyPlot version 2.0 beta 15. Relative mRNA expression was compared between patients and healthy controls by using Mann– Whitney U test. Correlation between variables including cell surface and relative mRNA expression was calculated using Spearman’s rank correlation coefficient (r). If the correlation coefficient value is 21 or 1, there is a perfect negative or positive correlation. If it ranges within (21; 0.5) or (0.5; 1), there is a strong negative or positive correlation. If it varies from 20.5 to 0 or from 0 to 0.5, there is a weak negative or positive correlation. If the r value reaches 0, there is no correlation. Significance level was established at a p value of ,0.05.
Results
Analysis of cell surface Hsp70 expression on synovial cells, dermal fibroblasts and peripheral blood leukocytes.Consistently with our previous studies [10,14] Hsp70 membrane positivity was detected on fibro- blast-like synovial cells in 13 out of 18 RA patients (range 2.1 – 44.7%, average 18.3%, median 16.5%) in contrast to autologous or healthy control peripheral blood lymphocytes (RA patients: average 4.7%, median 2.9%; healthy controls: average 6.0%, median 4.5%) and/or autologous dermal fibroblasts (average 5.1%, median 4.3%), which showed in most cases no Hsp70 membrane expression. Increased Hsp70 expression on fibroblast-like synovial cells when compared with autologous and/or healthy control peripheral blood lymphocytes ( p ¼ 0.002, p ¼ 0.002) as well as autologous dermal fibroblasts ( p , 0.001) was statistically significant. Healthy control peripheral blood lymphocytes were used as a negative control for Hsp70 cell surface expression studies (Tables II and III).
We observed strong Hsp70 cell surface expression on monocytes in all tested patients (10/10, average 53.0%, median 58.1%) and healthy controls (10/10, average 49.4%, median 47.5%, p ¼ 0.52). A signifi- cantly higher Hsp70 membrane expression was found on granulocytes of healthy controls (9/10, average 41.8%, median 41.4%) when compared with gra- nulocytes of RA patients (3/10, average 10.7%, median 6.4%, p ¼ 0.005; Tables II and III). Statistical analysis of the data is shown in Table IV.
Analysis of relative Hsp70 gene expression in synovial tissue, skin dermis and peripheral blood leukocytes
Significantly higher relative Hsp70 gene expression in RA-derived synovial cells (range 0.75 – 7.54, average 2.04, median 1.7) was observed when compared with autologous peripheral blood leukocytes (range 0.23 – 1.44, average 0.75, median 0.68, p , 0.001; Tables II and III). However, we found significantly lower relative gene expression in peripheral blood leukocytes of RA patients in comparison with healthy controls (range 1.05 – 3.04, average 1.69, median 1.64, p , 0.001). On the other hand, Hsp70 gene expression compared between RA-derived synovial cells and peripheral blood leukocytes of healthy controls did not reach any statistical significance (p ¼ 0.83).
Interestingly, we found that Hsp70 gene expression in RA non-affected skin dermis gained from the operation wound was 3.7-fold higher in average (range 0.97 – 16.43, average 7.6, median 8.3) when compared to autologous RA-affected synovial tissue (p , 0.001); 10.1-fold higher in average when compared to autologous peripheral blood leukocytes (p , 0.001) and 4.5-fold higher in average comparing to control peripheral blood leukocytes (p , 0.001).
Statistical analysis of the data is shown in Table IV.
Correlation of Hsp70 cell surface and relative gene expression in synovial cells
Spearman’s rank correlation was used to discover the strength of a link between two variables—Hsp70 cell surface and relative gene expression in synovial cells derived from 16 RA-affected joints. Weak positive correlation (r ¼ 0.24) was observed, however the data did not reach the statistical significance (p ¼ 0.36).
Discussion
In recent years, it has become clear that the fibroblast- like synovial cells play an important role in the pathogenesis of RA. They contribute to the synovial cell hyperplasia in RA and mediate autonomously cartilage destruction after their activation [4]. The resistance of synovial cells to stress is an important observation and might be associated with the expression of Hsps in the synovial tissue, which can protect the cells from apoptotic death [17].
The aim of our work was to study Hsp70 cell surface and relative mRNA expression in synovial cells derived from synovial tissue of RA patients who underwent synovectomy. Simultaneously, we studied Hsp70 expression in peripheral blood leukocytes of RA patients and healthy controls. Since no control synovial tissues from non-inflammatory joint diseases like traumatic joint diseases or degenerative joint disease (osteoarthritis) were available to make comparison to chronic inflammatory arthritides (RA), the study was expanded for the data concerning autologous dermal fibroblasts derived from the operation wound of patients with RA. The skin was processed under the same conditions as synovial tissue. Peripheral blood lymphocytes and skin fibroblasts were used as negative controls for Hsp70 cell surface expression on the basis of results from previous screening by flow cytometry, where a cut-off value for Hsp70 of 10% was chosen [10,13,14].
We found Hsp70 membrane positivity on fibroblast- like synovial cells in RA patients in contrast to autologous and/or healthy control peripheral blood lymphocytes and autologous dermal fibroblasts showing in most cases no Hsp70 membrane expression. This observation was consistent with the data of Schett et al. [18], who previously reported enhanced cytoplasmic expression of inducible Hsp70 in RA synovial tissue using Western blotting, immunohistochemistry and immunofluorescence. However, in another study the overexpression of constitutive heat shock cognate 70 (Hsc70) but not inducible Hsp70 was detected in RA synovial lining cells using immunochemistry and Western blotting [19].
In contrast to peripheral blood lymphocytes, which were found not to express membrane-bound inducible Hsp70 at all, we found positive cell surface expression on peripheral blood monocytes in both cohorts of RA patients and healthy controls. Interestingly, peripheral blood granulocytes of healthy controls showed significantly increased Hsp70 expression comparing with RA patients. Since many cell surface receptors like cluster of differentiation 14 (CD14), scavenger receptors (CD36, lectin-like oxidized low-density lipoprotein receptor (LOX-1), etc.), CD40, a2-macroglobulin/ low-density lipoprotein receptor (LRP/CD91), toll- like receptors (TLR-2 and TLR-4) and c-type lectins (CD94, etc.) that can bind Hsps [20] were described, we presume that we might detect extracellular Hsp70 bound to these receptors.
Relative Hsp70 gene expression was studied on synovial tissue gained after synovectomy and skin dermis gained from RA non-affected operation wound unlike Hsp70 cell surface expression which was investigated on fibroblast-like synovial cells and dermal fibroblasts growing from synovial tissue and skin dermis in culture. Even if monolayer expansion is often used as a technique to increase cell number to achieve a large population of cells for various studies, the gene expression in cultured cells may be significantly different from the primary population. This, for example, has been shown in case of articular cartilage, where gene expression studies performed on primary and passaged chondrocytes revealed change of collagen II into collagen I after one passage [23]. These observations led us to study Hsp70 gene expression directly in synovial tissue and skin dermis and not in synovial and dermal cell cultures.
Significantly higher Hsp70 gene expression was observed in synovial cells of RA patients when compared with autologous peripheral blood leuko- cytes. This could support the observation of Hsp70 positive cell surface expression on synovial cells derived from RA-affected joints. However, the difference in Hsp70 gene expression between RA-derived synovial cells and healthy control peripheral blood leukocytes was not observed. We also found significantly lower relative gene expression in peripheral blood leukocytes of RA patients in comparison with healthy controls. We speculate that this observation might be explained by the administration of immunosupressive therapy in a cohort of RA patients. However, Schett et al. [18] described that non-steroidal anti-inflammatory drugs triggered only an incomplete heat shock response, with HSF1 activation but not Hsp70 induction, whereas steroids and immunosupressive drugs did not affect the heat shock response at all.
Interestingly, the highest Hsp70 gene expression was observed in RA non-affected skin dermis gained from the operation wound when compared to autologous RA-affected synovial tissue, autologous and/or control peripheral blood leukocytes.Skin as a barrier to the environment is much more exposed to various external factors, such as heat, UV light, microorganism, chemicals, etc., causing mainly apoptosis of epidermal keratinocytes. Some of them may affect even the deeper layers containing dermal fibroblasts. Hsp70 expression is important as a molecular repair factor that maintains the protective function of the normal skin. As a physiological response to potentially harmful enviromental stress factors, these cells produce higher levels of Hsp70 comparing to the cells of internal organs and tissues [24].
We speculate that the Hsp70 expression in skin dermis might be intensified also by the shock caused by the desinfection and sterile folio cover of the field of the operation, however, dermal fibroblasts were growing from the primary tissue very well in most patients.We summarise that an increased Hsp70 gene expression in RA-affected synovial tissue is followed by Hsp70 cell surface expression on fibroblast-like synovial cells growing from RA synovial tissue. Hsp70 may be translocated to the cell surface from the cytosol and/or Hsp70 released from inflamed synovial tissue may be captured onto the membrane of synovial cells from the extracellular space via the CD91 receptor as we have reported recently [14]. The significance of the Hsp70 presence on the cell surface of RA-affected synovial cells remains undefined but may take part in the development of a higher resistance to stress- induced LF3 apoptosis.