Aus der Abteilung für Rheumatologie im Zentrum Innere Medizin der Medizinischen Hochschule Hannover
Direktor: Prof. Dr. med. H. Zeidler seit April 2007 eingegliedert in die Klinik für Immunologie und Rheumatologie
Leiter: Prof. Dr. med. R. E. Schmidt
Expression von Neurotrophinen und ihren Rezeptoren bei Rheumatoider Arthritis und Spondyloarthritis
Dissertation zur Erlangung des Doktorgrades der Medizin
in der Medizinischen Hochschule Hannover
vorgelegt von
Christian Barthel aus Mühlhausen
Hannover 2011
Angenommen vom Senat der Medizinischen Hochschule Hannover am 24.11.2011
Gedruckt mit Genehmigung der Medizinischen Hochschule
Präsident: Prof. Dr. med. Dieter Bitter-Suermann Betreuer der Arbeit: Prof. Dr. med. Henning Zeidler,
Priv.-Doz. Dr. med. Markus Rihl Referent: Prof. Dr. med. Jürgen Wollenhaupt Korreferent: Prof. Dr. med. Klaus Hartung Tag der mündlichen Prüfung: 24.11.2011
Prüfungsausschussmitglieder: Prof. Dr. med. Anke Schwarz Prof. Dr. med. Gunnar Klein Prof. Dr. med. Bettina Wedi
1. Publikation mit Literaturverzeichnis
2. Zusammenfassung
2.1. Einleitung und Methoden 2.2. Ergebnisse und Diskussion 2.3. Zusammenfassung
2.4. Literaturverzeichnis
3. Lebenslauf
4. Erklärung nach §2 Abs. 2 Nr. 6 und 7 PromO
5. Danksagung
Open Access
Vol 11 No 3
Research article
Nerve growth factor and receptor expression in rheumatoid arthritis and spondyloarthritis
Christian Barthel1, Nataliya Yeremenko2, Roland Jacobs1, Reinhold E Schmidt1,
Michael Bernateck3, Henning Zeidler4, Paul-Peter Tak2, Dominique Baeten2 and Markus Rihl1
1Clinic for Immunology and Rheumatology, Hannover Medical School (MHH), Carl-Neuberg-Strasse 1, Hannover 30625, Germany
2Division of Clinical Immunology and Rheumatology, Academic Medical Center (AMC), University of Amsterdam, Meibergdreef 9, Amsterdam, 1105, The Netherlands
3Department of of Anesthesiology, Pain Clinic, Hannover Medical School (MHH), Carl-Neuberg-Strasse 1, Hannover, 30625, Germany
4Rheumatologikum Hannover, Rathenaustrasse 13/14, Hannover, 30159, Germany Corresponding author: Markus Rihl, rihl.markus@mh-hannover.de
Received: 12 Nov 2008 Revisions requested: 16 Dec 2008 Revisions received: 11 May 2009 Accepted: 2 Jun 2009 Published: 2 Jun 2009 Arthritis Research & Therapy 2009, 11:R82 (doi:10.1186/ar2716)
This article is online at: http://arthritis-research.com/content/11/3/R82
© 2009 Barthel et al.; licensee BioMed Central Ltd.
This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Introduction We previously described the presence of nerve growth factor receptors in the inflamed synovial compartment.
Here we investigated the presence of the corresponding nerve growth factors, with special focus on nerve growth factor (NGF).
Methods mRNA expression levels of four ligands (NGF, brain derived growth factor (BDNF), neurotrophin (NT)-3, NT-4) and their four corresponding receptors (tyrosine kinase (trk) A, trkB, trkC, NGFRp75) were determined in the synovial fluid (SF) cells of 9 patients with rheumatoid arthritis (RA) and 16 with spondyloarthritis (SpA) and compared with 7 osteoarthritis (OA) patients. NGF was also determined in synovial tissue (ST) biopsies of 10 RA and 10 SpA patients. The production of NGF by monocytes and lymphocytes was assessed by flow cytometry of SF cells, synovial tissue derived fibroblast-like synoviocytes (FLS) were assessed by ELISA on culture supernatant.
Results SF cell analysis revealed a clear BDNF and NGF mRNA expression, with significantly higher NGF expression in RA and SpA patients than in the OA group. NGF expression was higher in ST samples of RA as compared to SpA. Using intracellular FACS analysis, we could demonstrate the presence of the NGF protein in the two inflammatory arthritis groups on both CD3+ T lymphocytes and CD14+ cells, i.e. monocytes/macrophages, whereas cultured FLS did not produce NGF in vitro.
Conclusions Neurotrophins and especially NGF are expressed in the synovial fluid and tissue of patients with peripheral synovitis. The presence of neurotrophins as well as their receptors, in particular the NGF/trkA-p75 axis in peripheral synovitis warrants further functional investigation of their active involvement in chronic inflammatory arthritis.
Introduction
There is increasing evidence for the presence of neuronal growth factors in chronic inflammatory arthritis. Neurotrophins (nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and the neurotrophins NT-3 and NT-4) constitute a family of growth factors essential for the development, prolifer- ation, differentiation, and survival of neuronal as well as various non-neuronal cells. Neurotrophins bind to their specific high-
affinity receptors tyrosine kinase (trk) A (NGF), trkB (BDNF, NT-4), and trkC (NT-3), and to one low-affinity receptor p75 (or NGFRp75) that binds to all ligands. This p75 receptor is a member of the TNF receptor superfamily [1]. In particular, the NGF-trkA/p75 axis arouses increasing interest due to its role in chronic inflammatory arthritis in which a pathogenic function of this system has been postulated [2-6].
BDNF: brain-derived growth factor; BSA: bovine serum albumin; Ct: cycle threshold; DMEM: Dulbecco's modified eagle's medium; ELISA: enzyme- linked immunosorbent assay; FACS: fluorescence-activated cell sorter; FCS: fetal calf serum; FLS: fibroblast-like synoviocytes; G3PDH: glyceralde- hyde 3-phosphate dehydrogenase; mAb: monoclonal antibody; NGF: nerve growth factor; NSAID: non-steroidal anti-inflammatory drug; NT: neuro- trophin; OA: osteoarthritis; PBMC: peripheral blood mononuclear cells; PBS: phosphate-buffered saline; RA: rheumatoid arthritis; RT-PCR: reverse transcription polymerase chain reaction; SD: standard deviation; SpA: spondyloarthritis; SF: synovial fluid; SFMC: synovial fluid mononuclear cells;
ST: synovial tissue; TNF: tumor necrosis factor; trk: tyrosine kinase.
Using immunohistochemistry, we previously performed a detailed analysis on the synovial expression of neurotrophins showing convincingly high levels of both the trkA and the p75 NGF receptors in peripheral synovitis of patients with spondy- loarthritis (SpA). Their expression correlated with signs of inflammation and was modulated by effective treatment with anti-TNF [7]. However, apart from BDNF we were unable to demonstrate the presence of the ligands at the protein level.
Accordingly, this study was designed to assess the expression of NGF and all other known neurotrophic ligands (BDNF, NT- 3, NT-4), as well as their receptors (trkA, NGFRp75, trkB, trkC), in order to provide evidence that all actors of this system are present and actively upregulated in the inflamed synovial compartment.
Materials and methods
Patients
Synovial fluid (SF) samples were collected from nine patients with rheumatoid arthritis (RA) fulfilling the American College of Rheumatology classification criteria and from 16 patients with SpA fulfilling the European Spondyloarthropathy Study Group classification criteria [8,9]. Also, seven patients with osteoar- thritis (OA) served as non-inflammatory controls. All patients had active synovitis of the knee. Patient characteristics and disease activity parameters are listed in Table 1. OA patients were graded according to the Kellgren and Lawrence classifi- cation [10]. The majority of patients had OA of the knee grade 2 (mean 2.3 ± standard deviation (SD) 1.0).
Synovial tissue (ST) biopsies were obtained from another panel of patients including 10 SpA and 10 RA patients with
early and active arthritis of the knee (disease duration <6 months, only NSAID treatment, but no steroids, no disease- modifying anti-rheumatic drugs, or biologics).
All subjects gave their written informed consent before inclu- sion in the study, which was approved by the local ethics com- mittee of the involved institutions.
Synovial fluid and synovial tissue biopsy samples and extraction of total RNA
SF was obtained by a conventional puncture of an actively inflamed knee joint. Samples were centrifuged for 15 minutes at 1000 g. Supernatants were removed, whole SF cell pellets were resuspended in RNAlater solution (Ambion, Austin, TX, USA), and frozen at -80°C until use. For intracellular fluores- cence-activated cell sorting (FACS) analysis, mononuclear cells derived from SF samples (SFMC) were obtained by standard Ficoll histopaque procedure and conserved in FCS and 10% dimethyl sulfoxide. They were kept in liquid nitrogen until use. ST biopsies were obtained by a standard procedure as previously described [11]. The Ficoll procedure was also used in order to obtain peripheral blood mononuclear cells (PBMC) from four healthy individuals used as controls for PCR. Total RNA was extracted from SF cell pellets, ST biop- sies, and PBMC using TRizol reagent (Invitrogen, Karlsruhe, Germany) and precipitation with isopropyl alcohol. All proce- dures were previously described in detail [12-14].
Quantitative real-time RT-PCR (TaqMan assay)
Generation of cDNA by reverse transcription and utilization of TaqMan® assay followed the protocol of the manufacturer Table 1
Clinical characteristics of neurotrophins and receptors in the synovial fluids of SpA, RA, and OA patients
Patient Groups Clinical characteristics
age gender DD (y) SJC TJC CRP (mg/l) ESR (mm) SFc/μl %PMN
SpA (n = 16) median 37 6 f/10 m 5 2* 1* 10.5* 20 7025* 72
SD 16.8 5.9 1.2 1.9 23 15 8825 26
(range) (16 to 65) (0.3 to 18) (1 to 4) (0 to 8) (1 to 99) (3 to 54) (2500 to 31100) (5 to 92)
RA (n = 9) median 49 8 f/1 m 5 5# 3# 11.7# 26 7950# 76
SD 15.3 9.2 3.2 1.8 29 8.8 3645 11
(range) (37 to 77) (1 to 26) (2 to 13) (2 to 7) (7.5 to -95) (12 to 67) (2600 to 1300) (50 to 90)
OA (n = 7) median 67 5 f/2 m 6 1 1 3 17 700 60
SD 5.8 5.7 0.5 0.4 1.1 5.6 1096 33
(range) (58 to 75) (2 to 18) (1 to 2) (1 to 2) (1.6 to 4.5) (12 to 25) (100 to 3250) (5 to 90) Table 1 shows the clinical data and parameters of disease activity of 16 spondyloarthritis (SpA), 9 rheumatoid arthritis (RA), and 7 osteoarthritis (OA) patients used for the RT-PCR measurements on the synovial fluid samples. Swollen joint count (SJC), tender joint count (TJC), C-reactive protein (CRP), and synovial fluid (SF) leukocyte counts were higher in both SpA (*) and RA (#) as compared with OA; Mann Whitney U test; P <
0.05).
DD = disease duration given in years; ESR = erythrocyte sedimentation rate; f = female; m = male; PMN = percentage of polymorphonuclear cells of SF leukocytes; OA = osteoarthritis; RA = rheumatoid arthritis; SD = standard deviation; SpA = spondyloarthritis.
(Applied Biosystems, Darmstadt, Germany). Briefly, 2 μg of total RNA were reverse transcribed using the MultiScribe® (Applied Biosystems, Darmstadt, Germany) reverse tran- scriptase. Duplicate PCR reactions were performed using the TaqMan® universal PCR master mix on ABI Prism® 7000 sequence detection system (Applied Biosystems, Darmstadt, Germany). After denaturation at 50°C for two minutes and 95°C for 10 minutes, 45 PCR reaction cycles were performed each at 95°C for 9 seconds and 60°C for one minute. The fol- lowing mRNA transcripts and assays were used (Applied Bio- systems, Warrington, UK): assays-on-demand for NGF-beta (assay no. Hs00171458), [GenBank:X52599], BDNF (assay no. Hs00156058), [GenBank:M61176], and NT-3 (assay no.
Hs00267375), [GenBank:M37763]; assay-by-design for NT- 4 (cat. no. 4332078; [GenBank:M86528]); assays-on demand for the high-affinity receptors trkA (assay no.
Hs00176787), [GenBank:X03541]; trkB (assay no.
Hs00178811), [GenBank:U12140]; trkC (assay no.
Hs00176797), [GenBank:U05012], and for the low-affinity receptor NGFRp75 (assay no. Hs00609976), [Gen- Bank:M14764].
Delta delta Ct method and statistical analysis
All PCR data were normalized to the expression of the glycer- aldehyde 3-phosphate dehydrogenase (G3PDH) housekeep- ing gene used as an internal control; SF data were also normalized with a set of four healthy PBMC used as an exter- nal control. The ST data were compared with each other. PCR data were obtained as cycle threshold (Ct) values. The Ct value is defined as the cycle with a fluorescence intensity sig- nificantly above the background fluorescence but within the exponential phase of the amplification [15,16]. The mean of two Ct measurements of one sample was calculated for both the given target gene and the G3PDH gene. Delta Ct was determined as the mean of the duplicate Ct values for the tar- get gene subtracted by the mean of the duplicate Ct values for the G3PDH gene. For each target gene, delta Ct measure- ments were performed separately for both the SF samples and the healthy PBMC. The delta delta Ct method represents the difference between the two cell types for a given target gene [17]. Expression levels are given as fold of expression and were compared between SpA, RA, and OA groups using the non-parametric Mann Whitney U test as appropriate.
Fluorescence-activated cell sorting for detection of NGF SF mononuclear cells (SFMC) of three RA and three SpA patients from the panel described in Table 1, as well as PBMC from two healthy controls were prepared by density gradient centrifugation using biocoll (1.077 g/ml; Biochrom, Berlin, Germany). SFMC were harvested from the interphase and washed twice at 1000 g and 300 g, respectively. The cells were finally resuspended in PBS/BSA and stained for surface markers (CD3 PerCP, clone SK7; CD14 FITC, Leu-M3; CD56 APC, NCAM 16.2; all from Becton Dickinson, Heidelberg, Germany) for 20 minutes. After two washes with PBS/BSA
(300 g/three minutes) the cells were fixed for 10 minutes at room temperature in PBS containing 4% paraformaldehyde.
Cells were then washed once and resuspended in saponin buffer (PBS supplemented with 5 mM HEPES and 0.1%
saponin) in order to perforate the cell membranes. Subse- quently, aliquots were stained with monoclonal antibodies (mAb) against NGF (biotinylated anti-human β-NGF, cata- logue number BAF256; R&D systems, Minneapolis, MN, USA). Unspecific binding of the mAb via Fc-receptors was dis- criminated by adding human IgG solution (Octagam; Octap- harma, Langenfeld, Germany). After 30 minutes of incubation at 4°C, cells were washed three times with PBS/BSA and resuspended again in saponin buffer. PE-labeled streptavidin (SA-PE, Becton Dickinson, Heidelberg, Germany) for second- ary staining of biotinylated NGF was added and cells were incubated for 30 minutes at 4°C. After three washes (300 g/
three minutes) with PBS/BSA, cells were ready for FACS analysis.
Phenotypic analyses were performed as multicolor immunoflu- orescences. At least 104 cells per appropriate lymphocyte or monocyte gate, respectively, according to forward scatter vs side scatter properties were analyzed using a dual-laser cytometer with Cell Quest Pro (FACSCalibur, Becton Dickin- son, Heidelberg, Germany) and Summit 4.3 (Beckman Coul- ter, Krefeld, Germany) software.
Culture of fibroblast-like synoviocytes to determine NGF production
Fibroblast-like synoviocytes (FLS) were isolated from synovial biopsies of one RA and one SpA patient as described previ- ously [18]. After three passages, cells were resuspended at 10,000 cells/ml Dulbecco's modified eagle's medium (DMEM) with 10% FCS and plated at 2 ml/well in 24 well plates. Cells were grown for three days until confluence, then the normal medium (DMEM + 10% FCS) was replaced by starvation medium (DMEM + 1% FCS). After 24 hours, cells were stim- ulated with either medium alone (DMEM 1% FCS); TNF-alpha at 10 ng/ml; or IL-1 beta at 10 ng/ml, or lipopolysaccharide at 1 ug/ml. After 72 hours of culture with stimulation, superna- tants were collected and used undiluted for measuring NGF by ELISA (R&D Systems, Minneapolis, MN, USA; lowest detection level: 30 pg/ml).
Results
The mRNA expression levels of the four neurotrophic ligands and the four receptors as determined in the SF cells are depicted in Table 2 and outlined in detail by scatter plots in Figure 1 (ligands) and Figure 2 (receptors).
As for the transcripts encoding the ligands, BDNF revealed high expression levels in all three groups (RA median: 163, SpA median: 92 with a high range from 71 to 444, OA median:
137). The highest mRNA expression in SF samples was found for NGF revealing significantly higher levels in RA and SpA
(median 418 and 323, respectively) as compared with OA (median 49; P = 0.001 for both comparisons). Transcripts encoding NT-3 and NT-4 were expressed on lower levels and slightly higher by trend in RA as compared with SpA and OA.
The NGF mRNA expression levels as determined in ST biop- sies were found to be significantly higher in RA (mean expres- sion level 1.6 ± 1.2 SD) as compared with SpA (mean expression level 0.7 ± 0.3 SD) patients (P = 0.02) indicating that NGF is produced locally, at least in the arthritic synovium of RA patients (Figure 3).
In agreement with our previous immunohistochemistry data on ST samples [7], the mRNA transcripts encoding both the high- affinity NGF receptor trkA and the common low-affinity recep- tor p75 revealed the highest expression levels in SF of the SpA and the RA group being significantly higher expressed as compared with the OA group. The highest values were found
for trkA in the SpA group (trkA median in SpA 20 vs OA 4.7;
P = 0.003; p75 median in SpA 13.4 vs OA 4.8; P = 0.03) and for p75 in the RA group (trkA median values: RA 16 vs OA 4.7;
P = 0.004; p75 median values: RA25 vs OA 4.8; P = 0.01 as determined by the Mann Whitney U test). Expression levels of trkB and trkC receptors were clearly lower than the ones of trkA and p75. Expression of trkB and trkC in SpA and OA was similar. However, trkB expression in the RA group was signifi- cantly higher as compared with the OA group (trkB median in RA 7.7 vs OA 4.1; P = 0.04).
We also measured NGF expression by staining on a single cell level using flow cytometry. Concomitant staining of cell sur- face markers and intracellular NGF revealed the presence of NGF in T lymphocytes (CD3+) and monocytes (CD14+). In contrast, B lymphocytes (CD19+) and nearly all natural killer cells (CD16+) were NGF negative in patients as well as in Figure 1
Scatter plots showing mRNA expression of the neurotrophic ligands
Scatter plots showing mRNA expression of the neurotrophic ligands. The scatter plots a to d depict the expression levels of the four neurotrophic lig- ands. (a) nerve growth factor (NGF). (b) Brain-derived growth factor (BDNF). (c) Neurotrophin (NT)-3. (d) NT-4). Bold horizontal lines represent the median. The highest levels were found for BDNF and NGF. Significantly higher expression was revealed for NGF in both spondyloarthritis (SpA) and rheumatoid arthritis (RA) as compared with osteoarthritis (OA; P = 0.001 for both comparisons).
healthy controls. In both the SpA and the RA group, percent- ages of NGF expressing T lymphocytes and monocytes were considerably higher as compared with healthy controls (Figure 4). As we did notice clear mRNA expression for NGF not only in SF but also in ST, we additionally investigated the produc- tion of NGF by FLS. In vitro cultured FLS from RA as well as SpA did not secrete detectable levels of NGF, even upon stim- ulation with various proinflammatory cytokines (data not shown). Taken together, these data suggest that infiltrating T lymphocytes and myeloid cells are the main source of NGF in the inflamed peripheral joint.
Discussion
The present study is a descriptive comprehensive quantitative expression analysis of mRNA transcripts encoding the four
known human neurotrophins and their four corresponding receptors in the synovial compartment of arthritis patients.
The presence of neurotrophic factors in the inflamed joint has been described earlier [2,3]. Focusing on the NGF/trkA-p75 axis in our own and other work, we could previously demon- strate high trkA and p75 NGF receptor expression at the pro- tein level in the inflamed ST in peripheral SpA synovitis. This expression was correlated with inflammatory disease activity and was downregulated by TNF-blocking treatment indicating that their expression is not constitutive but actively modulated in inflammation [7]. However, the high-affinity receptors trkB and trkC as well as the ligands NGF, NT-3, and NT-4 were expressed in the minority of patients or not detectable by immunohistochemistry.
Figure 2
Scatter plots showing mRNA expression of neurotrophin receptors
Scatter plots showing mRNA expression of neurotrophin receptors. The scatter plots a to d depict the expression levels of the four neurotrophin receptors. (a) Tyrosine kinase (trk)A. (b) p75. (c) trkB. (d) trkC. Bold horizontal lines represent the median. The highest levels were found for trkA and p75, revealing significantly higher expression levels in spondyloarthritis (SpA; P = 0.0003, P = 0.003 respectively) and rheumatoid arthritis (RA;
P = 0.004 and P = 0.001, respectively) vs osteoarthritis (OA).
In order to investigate the NGF/trkA-p75 axis as well as all other neurotrophic ligands and receptors at the transcript level, we used quantitative real-time RT-PCR to determine their expression in a larger panel of SpA and RA patients with active peripheral synovitis of the knee. Our data confirm the high expression of both the trkA and p75 NGF receptors at the
transcript level in the synovial compartment of SpA and RA patients. Of note, we now provide evidence that the NGF lig- and is also expressed in the SF and tissue biopsy samples of peripheral synovitis indicating that this system is active in chronic inflammatory arthritis. However, we need to state, that the high NGF transcript expression is in contrast to our previ- ous ELISA data in SF samples [7]. This discrepancy between mRNA and protein expression has been reported earlier in studies on brain tissue [19]. Reasons for this phenomenon might involve post-transcriptional modifications of NGF [1,20].
We also can not exclude technical reasons such as the NGF antibody used for the previous quantitative immunoassay.
The cellular source of NGF in humans has been investigated in several studies. Under unstimulated conditions, NGF is pro- duced mainly by CD4+ T and B lymphocytes [1,21]. Under inflammatory conditions such as allergy and arthritis, NGF can be produced, stored, and released by eosinophils, mast cells, lymphocytes, and synovial fibroblasts, as well as monocytes and macrophages [22]. Using intracellular FACS analysis, we could demonstrate the presence of the NGF protein in the two inflammatory arthritis groups on both CD3+ and CD14+ cells, that is, T lymphocytes and monocytes/macrophages, which are known to be involved in the major pathways of both SpA and RA. However, ST-derived FLS do not seem to produce NGF as measured by ELISA. This finding might indicate the mere pro-inflammatory potential of NGF as opposed to factors released by fibroblasts, which are predominantly involved in structural damage. On the other hand, we can not definitely rule out the production of NGF by FLS. One explanation would be, that FLS loose their ability to produce NGF when cultured Table 2
RT-PCR expression levels of neurotrophins and receptors in the synovial fluids of SpA, RA, and OA patients
Patient Groups RT-PCR results
TrkA p75 trkB trkC NGF BDNF NT-3 NT-4
SpA (n = 16) median 20 13.4 6.7 7.1 323 92 7.0 7.3
SD 12 7.7 3.9 6.0 296 113 2.9 5.0
(range) (3.6 to 45) (3.5 to 28) (1.1 to 17) (2.4 to 25) (74 to 1225) (71 to 444) (1.3 to 9.6) (1 to 15)
RA (n = 9) median 16 25 7.7 11 418 163 7.5 10
SD 10 12 4.3 3.4 275 101 3.5 7 to 6
(range) (8.9 to 40) (2.3 to 41) (4.1 to 18) (4.3 to 14) (267 to 1104) (71 to 374) (0.5 to 11) (4.9 to 22)
OA (n = 7) median 4.7 4.8 4.1 6.7 49 137 3.4 8.6
SD 4.2 3.7 2.8 3.8 37.7 98.5 2.2 3.1
(range) (1.7 to 15) (3.2 to 15) (1.3 to 9.8) (3.2 to 15) (18 to 129) (25 to 238) (1.7 to 7.8) (4.3 to 13) Table 2 shows the RT-PCR expression levels of mRNA transcripts of four neurotrophins (nerve growth factor (NGF), brain-derived growth factor (BDNF), and neurotrophin (NT)-3, NT-4) and their corresponding receptors (high-affinity receptors tyrosine kinase (trk)A, trkB, trkC and the low- affinity NGF receptor p75) of all 32 patients are shown (see also Figures 1 and 2 for detailed data and statistics).
DD = disease duration given in years; ESR = erythrocyte sedimentation rate; f = female; m = male; PMN = percentage of polymorphonuclear cells of SF leukocytes; OA = osteoarthritis; RA = rheumatoid arthritis; SD = standard deviation; SpA = spondyloarthritis.
Figure 3
mRNA expression of NGF in synovial tissue samples
mRNA expression of NGF in synovial tissue samples. mRNA expression of nerve growth factor (NGF) as the prototype of neurotrophins was measured in the synovium of both 10 spondyloarthritis (SpA) and 10 rheumatoid arthritis (RA) patients; the expression levels were compared with each other (relative expression) showing a twice as high and thus significantly higher NGF expression in RA as compared with SpA (P = 0.02).
over three passages in vitro. Another explanation would be that NGF is produced but not secreted, at least not in large amounts. Nevertheless, FLS are most likely one of the targets of NGF.
To date, the functional role of neurotrophins in inflammatory joint disorders is unclear. A pathogenic role for the NGF/trkA- p75 axis and other neurotrophins has been postulated for air- way inflammation [23], atopic dermatitis [24], psoriasis [25], inflammatory bowel disease [26], and arthritis [2-7]. In inflam- matory syndromes, NGF has been attributed to upregulating TNF-alpha, promoting the differentiation of B cells to plasma cells, enhancing chemotaxis and production of superoxide by
neutrophils [22]. NGF is also involved in humoral immune responses by acting as an autocrine survival factor maintaining the viability of memory B-cells and macrophages [27]. NGF and its receptors have also tissue remodeling capacities exert- ing a strong fibrotic stimulus on skin and lung fibroblasts [28].
Upon binding to trkA, NGF induces its auto-phosphorylation and subsequently the activation of both phospholipase PLCγ and protein kinase C, which in turn activates the mitogen-acti- vated protein kinase pathway involving the c-jun N-terminal, the p38, and the extracellular-regulated protein kinases (ERK1/2) all of which have been identified in arthritis as well.
Interestingly, the wnt proteins, which have been described as regulating neurotrophin expression [29], have recently also Figure 4
NGF staining by flow cytometry
NGF staining by flow cytometry. PBMC of (a) healthy controls (HC) and (b) synovial fluid mononuclear cells (SFMC) from spondyloarthritis (SpA), and (c) rheumatoid arthritis (RA) patients were first stained with surface markers (CD3 and CD14) and permeabilized in order to enable intracellular detection of nerve growth factor (NGF). The cells were analysed by flow cytometry after setting lymphocyte (left column) and monocyte (right col- umn) gates according to forward scatter vs side scatter properties of the cells. Dot plots of one representative individual of each group are shown.
been found to play a pathogenic role in spondyloarthritis [30].
In addition, NGF has been identified as a proangiogenic factor, another significant pathogenic pathway in chronic inflamma- tory arthritis such as SpA and RA [31,32].
Conclusions
Taken together, this comprehensive analysis demonstrates the expression of the pleiotropic NGF and its two receptors in peripheral synovitis of SpA and RA. The knowledge of neuro- trophin expression on cells from the inflamed synovial com- partment in arthritis patients adds to the potential evaluation of pathogenic mechanisms and the development of new thera- peutic strategies (e.g. the pharmacological blockade of the NGF receptor and their signaling pathway by using receptor antagonists). Our findings prompt further functional as well as clinical studies on the role of neurotrophins and their therapeu- tic potential in arthritis.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
CB performed the RT-PCR experiments, the analysis of the data, and drafted the manuscript. NY did the FLS isolation and the ELISA. MB provided technical assistance in collecting the samples. RJ performed the FACS analysis. RES, HZ, PPT, and DB provided assistance in interpretation of the data and draft- ing the manuscript. MR designed the study and provided assistance in analysis and interpretation of the data and draft- ing the manuscript.
Acknowledgements
This work was supported by the Competence Network Rheumatology (KNR, BMBF, Berlin).
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11. Baeten D, Bosch F Van den, Elewaut D, Stuer A, Veys EM, De Key- ser F: Needle arthroscopy of the knee with synovial biopsy sampling: technical experience in 150 patients. Clin Rheuma- tol 1999, 18:434-441.
12. Gu J, Rihl M, Märker-Hermann E, Baeten D, Kuipers JG, Song YW, Maksymowych WP, Burgos-Vargas R, Veys EM, De Keyser F, Deister H, Xiong M, Huang F, Tsai WC, Yu DT: Clues to patho- genesis of spondyloarthropathy derived from synovial fluid mononuclear cell gene expression profiles. J Rheumatol 2002, 29:2159-2164.
13. Rihl M, Baeten D, Seta N, Gu J, De Keyser F, Veys EM, Kuipers JG, Zeidler H, Yu DT: Technical validation of cDNA based microar- ray as screening technique to identify candidate genes in syn- ovial tissue biopsy specimens from patients with spondyloarthropathy. Ann Rheum Dis 2004, 63:498-507.
14. Wendt K, Wilk E, Buyny S, Buer J, Schmidt RE, Jacobs R: Gene and protein characteristics reflect functional diversity of CD56dim and CD56bright NK cells. J Leukoc Biol 2006, 80:1529-1541.
15. Bustin SA: Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 2000, 25:169-193.
16. Pfaffl MW: A new mathematical model for relative quantifica- tion in realtime RT-PCR. Nucleic Acids Res 2001, 29:e45.
17. Fleige S, Walf V, Huch S, Prgomet C, Sehm J, Pfaffl MW: Com- parison of relative mRNA quantification models and the impact of RNA integrity in quantitative real-time RT-PCR. Biotechnol Lett 2006, 28:1601-1613.
18. Vandooren B, Cantaert T, ter Borg M, Noordenbos T, Kuhlman R, Gerlag D, Bongartz T, Reedquist K, Tak PP, Baeten D: Tumor necrosis factor alpha drives cadherin 11 expression in rheu- matoid inflammation. Arthritis Rheum 2008, 58:3051-3062.
19. Zhang HT, Li LY, Zou XL, Song XB, Hu YL, Feng ZT, Wang TT:
Immunohistochemical distribution of NGF, BDNF, NT-3, and NT-4 in adult rhesus monkey brains. J Histochem Cytochem 2007, 55:1-19.
20. Freund-Michel V, Frossard N: The nerve growth factor and its receptors in airway inflammatory diseases. Pharmacol Ther 2008, 117:52-76.
21. Lambiase A, Bracci-Laudiero L, Bonini S, Bonini S, Starace G, D'Elios MM, De Carli M, Aloe L: Human CD4+ T cell clones pro- duce and release nerve growth factor and express high-affin- ity nerve growth factor receptors. J Allergy Clin Immunol 1997, 100:408-414.
22. Bonini S, Rasi G, Bracci-Laudiero ML, Procoli A, Aloe L: Nerve growth factor: neurotrophin or cytokine? Int Arch Allergy Immunol 2003, 131:80-84.
23. Rochlitzer S, Nassenstein C, Braun A: The contribution of neuro- trophins to the pathogenesis of allergic asthma. Biochem Soc Trans 2006, 34:594-599.
24. Dou YC, Hagstromer L, Emtestam L, Johansson O: Increased nerve growth factor and its receptors in atopic dermatitis: an immunohistochemical study. Arch Dermatol Res 2006, 298:31-37.
25. Raychaudhuri SP, Raychaudhuri SK: Role of NGF and neuro- genic inflammation in the pathogenesis of psoriasis. Prog Brain Res 2004, 146:433-437.
26. Reinshagen M, von Boyen G, Adler G, Steinkamp M: Role of neu- rotrophins in inflammation of the gut. Curr Opin Investig Drugs 2002, 3:565-568.
27. Manni L, Lundeberg T, Fiorito S, Bonini S, Vigneti E, Aloe L: Nerve growth factor release by human synovial fibroblasts prior to and following exposure to tumor necrosis factor-alpha, inter- leukin-1 beta and cholecystokinin-8: the possible role of NGF
in the inflammatory response. Clin Exp Rheumatol 2003, 21:617-624.
28. Micera A, Vigneti E, Pickholtz D, Reich R, Pappo O, Bonini S, Maquart FX, Aloe L, Levi-Schaffer F: Nerve growth factor dis- plays stimulatory effects on human skin and lung fibroblasts, demonstrating a direct role for this factor in tissue repair. Proc Natl Acad Sci USA 2001, 98:6162-6167.
29. Patapoutian A, Backus C, Kispert A, Reichardt LF: Regulation of neurotrophin-3 expression by epithelial-mesenchymal inter- actions: the role of Wnt factors. Science 1999, 283:1180-1183.
30. Diarra D, Stolina M, Polzer K, Zwerina J, Ominsky MS, Dwyer D, Korb A, Smolen J, Hoffmann M, Scheinecker C, Heide D van der, Landewe R, Lacey D, Richards WG, Schett G: Dickkopf-1 is a master regulator of joint remodeling. Nat Med 2007, 13:156-163.
31. Lazarovici P, Marcinkiewicz C, Lelkes PI: Cross talk between the cardiovascular and nervous systems: neurotrophic effects of vascular endothelial growth factor (VEGF) and angiogenic effects of nerve growth factor (NGF)-implications in drug development. Curr Pharm Des 2006, 12:2609-2622.
32. Szekanecz Z, Koch AE: Mechanisms of disease: angiogenesis in inflammatory diseases. Nat Clin Pract Rheumatol 2007, 3:635-643.
2.1. Einleitung und Methoden
Die Neurotrophine „nerve growth factor“ (NGF), „brain-derived neurotrophic factor“
(BDNF), „neurotrophin 3” (NT-3) und „neurotrophin 4” (NT-4) bilden eine Gruppe von Wachstumsfaktoren, die für die Entwicklung, Proliferation, Differenzierung und das Überleben sowohl von neuronalen als auch nicht-neuronalen Zellen wichtig sind.
Neurotrophine binden an ihre spezifischen hochaffinen Rezeptoren Thyrosinkinase A (TrkA: NGF), Thyrosinkinase B (TrkB: BDNF und NT-4) sowie Thyrosinkinase C (TrkC: NT-3). Zusätzlich ist ein weiterer, niedrigaffiner Rezeptor bekannt (Rezeptor p75 bzw. „Nerve growth factor receptor p75“: NGFRp75 oder auch p75), der alle Liganden bindet. Dieser p75-Rezeptor gehört zur Tumornekrosefaktor-Rezeptor- Superfamilie [1].
Eine pathogenetische Rolle des NGF/TrkA-p75-Systems und weiterer Neurotrophine wurde für entzündliche Atemwegserkrankungen [2], Psoriasis [3], entzündliche Darmerkrankungen [4] und Arthritiden [5-10] beschrieben. Bei entzündlichen Syndromen wird NGF eine hochregulierende Wirkung für TNF-alpha zugeschrieben, ebenso eine förderliche Rolle in der Differenzierung von B-Zellen zu Plasmazellen, Förderung der Chemotaxis und Produktion von Superoxiden durch Neutrophile [11].
NGF ist außerdem an der humoralen Immunantwort beteiligt, indem es als autokriner antiapoptotischer Faktor die Lebensfähigkeit von Gedächtnis-B-Zellen und Makrophagen erhält [12]. NGF und seine Rezeptoren haben außerdem eine gewebsumbauende Wirkung, indem sie einen starken fibrotischen Stimulus auf Haut- und Lungenfibroblasten bewirken [13]. Durch die Bindung an TrkA induziert NGF seine Autophosphorylierung und anschließend die Aktivierung der Phospholipase PLCγ und der Proteinkinase C. Diese beiden Enzyme wiederum aktivieren den mitogen-aktivierenden Proteinkinase-Pfad (MAPK) unter Einbeziehung des c-jun N- Endes, der p38 und der extrazellulär-regulierten Proteinkinasen (ERK1/2), die jeweils in der Pathogenese der rheumatoiden Arthritis identifiziert wurden. Wie kürzlich beschrieben, scheinen interessanterweise wnt-Proteine, die auf die Expression der Neurotrophine eine regulatorische Funktion ausüben [14], eine Rolle in der Pathogenese der Spondyloarthritis zu spielen [15]. Darüber hinaus wurde NGF als proangiogenetischer Faktor identifiziert, ein wichtiger Pathogeneseweg chronisch entzündlicher Erkrankungen wie RA und SpA [16,17]. Besonders den Bindungen von
In jüngeren Arbeiten anderer Gruppen wurden Neurotrophine bei entzündlichen Atemwegserkrankungen und der Psoriasis untersucht. Hier konnte gezeigt werden, dass Neurotrophine als Mediatoren aktiv an einer Immunantwort mitwirken [21].
Die zelluläre Herkunft von humanem NGF wurde bereits in verschiedenen Studien beschrieben. Unter normalen Bedingungen ohne Zellstimulation wird NGF hauptsächlich durch CD4-positive T- und B-Zellen produziert [1,22]. Unter entzündlichen Bedingungen können jedoch auch Eosinophile, Mastzellen, Lymphozyten, synoviale Fibroblasten sowie Monozyten und Makrophagen NGF produzieren, speichern und abgeben.
In einer vorangegangenen Untersuchung konnten wir mit immunhistochemischen Färbungen zeigen, dass in der Synovialis von Patienten mit Spondyloarthritis (SpA) sowohl TrkA als auch p75 höher exprimiert werden als in der gesunden Kontrollgruppe [10]. Die Höhe der Expression korrelierte mit dem Grad der Entzündungsaktivität und wurde effektiv durch die medikamentöse Behandlung mit dem TNF-Blocker Infliximab moduliert. Der Nachweis der einzelnen Neurotrophine in Verbindung mit einer systematischen Untersuchung der Expressionshöhe auf Transkriptebene wurde in dieser Arbeit nicht geführt. In der hier zusammengefassten und dieser Promotion zugrunde liegenden experimentellen Arbeit wurde daher die Genexpression aller bekannten menschlichen Neurotrophine (NGF, BDNF, NT-3, NT-4) und ihren Rezeptoren (TrkA, TrkB, TrkC, p75) der Hypothese folgend, dass neben NGF auch weitere Neurotrophine bei verschiedenen Arthritiden im entzündeten Gelenk nachweisbar sind, untersucht.
Da das technische Vorgehen und die jeweiligen Methoden detailliert in der o.g.
Originalpublikation beschrieben sind, sollen diese hier nur kurz erwähnt werden.
Es wurde die Synovialflüssigkeit von 9 Patienten mit Rheumatoider Arthritis (RA) und von 16 Patienten mit Spondyloarthritis (SpA) untersucht. Die RA-Patienten erfüllten die ACR-Kriterien (American College of Rheumatology) für die RA, die SpA-Patienten die ESSG-Kriterien (European Spondyloarthropathy Study Group) [23-24]. Bei allen Patienten bestand eine aktive Synovialitis des Kniegelenks. Als nicht- inflammatorische Kontrollgruppe wurden 7 Patienten mit Arthrose (Osteoarthritis, OA)
Arthrose 2. Grades (Mittelwert 2,3 ± Standardabweichung 1,0).
Biopsien von Synovialgewebe wurden von einem anderen Patientenkollektiv, bestehend aus 10 Patienten mit SpA und 10 Patienten mit RA, mittels minimal invasiver Nadelarthroskopie gewonnen [26]. Diese hatten eine frühe Arthritis des Kniegelenks, d.h. die Dauer der Erkrankung betrug maximal 6 Monate und sie wurden nur mit einem nichtsteroidalen Antirheumatikum (NSAR) aber nicht mit einer immunmodulatorischen, langwirksamen Basistherapie, Biologika oder Glukokortikoiden behandelt.
Alle Patienten gaben ihr schriftliches Einverständnis, bevor sie in die Studie aufgenommen wurden. Diese wurde durch die Ethikkommissionen der beteiligten Institutionen (Medizinische Hochschule Hannover und AMC, Universität Amsterdam) befürwortet.
Synovia (Gelenkflüssigkeit) wurde durch eine Punktion des Kniegelenks gewonnen.
Hieraus wurden mononukleäre Zellen (SFMC, synovial fluid mononuclear cells) mittels Standard-Ficoll-Histopaque-Verfahren extrahiert. Diese Methode wurde ebenfalls genutzt, um mononukleäre Zellen des peripheren Blutes (PBMC, peripheral blood mononuclear cells) von vier gesunden Probanden zu gewinnen. Diese wurden als Kontrollen für die Messungen mittels PCR (polymerase chain reaction) genutzt.
Hier soll einschränkend angemerkt werden, dass der Vergleich von PCR-Daten aus Synovia mit PBMC von Gesunden (in unserem Fall wurden drei männliche und eine weibliche Mitarbeiter/in der Klinik im Alter von 28 ± 5,3 Jahren herangezogen) nicht etabliert ist und daher nur eingeschränkte Aussagen zulässt. Dieses Verfahren wurde aufgrund der Ermangelung an Synovia von Gesunden gewählt. Im Rahmen einer früheren Arbeit aus unserer Arbeitsgruppe wurden aber Genexpressionsprofile aus der nicht entzündlichen Synovia von OA-Patienten und gesunden PBMC verglichen und zeigten keine signifikanten Unterschiede [28].
Proben aus Synovialis (Gewebe) wurden nach einer beschriebenen Methode aufgearbeitet [26]. Aus allen Zellen wurde die Gesamt-RNA zur weiteren Analyse extrahiert [27-29]. Durch reverse Transkription wurde cDNA generiert und mit quantitativer Real-Time-PCR analysiert. Die Auswertung erfolgte mit der Delta-Delta-
Whitney-U-Test herangezogen, das Signifikanzniveau wurde mit p<0,05 definiert.
SFMC wurden aus der Synovia von drei Patienten mit RA und drei Patienten mit SpA sowie PBMC aus dem Vollblut von zwei gesunden Personen mittels Dichtegradientenzentrifugation gewonnen. Anschließend wurden Oberflächenmoleküle für T-Lymphozyten (CD3), Monozyten/Makrophagen (CD14), B-Lymphozyten (CD19) und Natürliche Killerzellen (CD16) sowie intrazelluläres NGF mit monoklonalen Antikörpern markiert und durchflusszytometrisch gemessen, um die NGF-produzierende Zellart zu identifizieren.
Fibroblasten-ähnliche Synoviozyten (FLS, fibroblast-like synoviocytes) wurden von Synovialisbiopsien eines Patienten mit RA und eines Patienten mit SpA isoliert [33].
Die Zellen wurden zunächst in einem normalen Nährmedium (DMEM+10% FCS) kultiviert, anschließend wurde dieses durch ein nährstoffreduziertes Medium (DMEM+1% FCS) ersetzt. Nach 24 Stunden wurden die Zellen mit TNF-alpha, IL-1 beta und Lipopolysaccharid (LPS) stimuliert. Ein Teil der Zellen wurde nicht stimuliert und im nährstoffreduzierten Medium belassen. Nach 72 Stunden Stimulation wurde der Überstand entnommen und es wurde aus der unverdünnten Lösung NGF mittels ELISA bestimmt.
Bei der vorliegenden in 2009 in Arthritis, Research and Therapy publizierten Arbeit handelt es sich um eine deskriptive quantitative Auswertung der mRNA-Transkription der vier bekannten Neurotrophine und ihrer Rezeptoren im Gelenk von Patienten mit aktiver rheumatoider Arthritis und Spondyloarthritis mit peripherer Gelenkbeteiligung.
In der damaligen Ausgabe der o.g. Zeitschrift wurde ein Editorial publiziert, dass die Daten zusammenfasst und deren Bedeutung erläutert [34].
Die Ergebnisse der Real-Time-PCR zeigten, dass BDNF in allen Patientenkollektiven (RA, SpA und OA) im Vergleich zur Kontrollgruppe (PBMC von Gesunden) hochreguliert war (angegeben sind jeweils Mittelwerte mit Standardabweichung; RA:
163 ± 101, SpA: 92 ± 113, OA: 137 ± 98,5). Die höchste mRNA-Expression in der Synovia mit einer signifikant höheren Expression bei RA und SpA fand sich für NGF (418 ± 275 und 323 ± 296) im Vergleich zur OA-Gruppe (49 ± 37,7; p=0,001 für beide Vergleiche). Die Expression von NT-3 und NT-4 war vergleichsweise deutlich geringer und etwas höher in der RA-Gruppe, verglichen mit SpA und OA. Die Expression der für NGF kodierenden Transkripte aus der Synovialis war signifikant höher bei der RA (durchschnittlich 1,6-fach) im Vergleich zur SpA (0,7-fach; p=0,02).
Dies könnte darauf hinweisen, dass NGF lokal produziert wird, zumindest bei der Synovialitis von Patienten mit RA.
In Übereinstimmung mit früheren immunhistochemischen Daten [10] war sowohl der hochaffine NGF-Rezeptor TrkA als auch der niedrigaffine p75-Rezeptor in Zellen in der Synovia von RA und SpA hochreguliert. Die höchsten Werte für TrkA fanden sich in der SpA-Gruppe (TrkA in SpA 20 ± 12, in OA 4,7 ± 4,2; p=0,003; p75 in SpA 13,4 ± 7,7, in OA 4,8 ± 3,7; p= 0,03; jeweils bezogen auf gesunde PBMC) und für p75 in der RA-Gruppe (TrkA in RA 16 ± 10; p=0,004; p75 in RA 25 ± 12; p=0,01). Die Expression von TrkB und TrkC in Proben von Patienten mit RA und SpA war deutlich geringer nachweisbar als die Expression von TrkA und p75, wobei die Expression von TrkB in der RA-Gruppe signifikant höher im Vergleich zur OA-Gruppe war (TrkB in RA 7,7 ± 4,3, in OA 4,1 ± 2,8; p=0,04).
In einem weiteren Schritt wurde die aktive Synthese von NGF auf Zellebene durchflusszytometrisch untersucht. Begleitende Färbungen von Oberflächenmarkern und intrazellulärem NGF zeigten das Vorhandensein von NGF in T-Lymphozyten
Lymphozyten (CD19+) und fast alle natürlichen Killerzellen (CD16+) sowohl in Patientenproben als auch in den gesunden Kontrollen negativ für NGF. In der SpA- und der RA-Gruppe war die NGF-Expression deutlich höher im Vergleich zu den gesunden Kontrollen. Dieser Befund veranlasste uns zu der Hypothese, dass NGF- mRNA in der Synovialis exprimiert sein könnte. Die Produktion von NGF wurde daher an in vitro-kultivierte FLS von RA- und SpA-Patienten untersucht. Es fanden sich jedoch keine nachweisbaren Mengen an sezerniertem NGF. Dieser Befund legt nahe, dass infiltrierende T-Lymphozyten und Zellen der myeloischen Reihe die Hauptquelle für NGF im entzündeten peripheren Gelenk sind. Dieser Befund deutet auf das pro-inflammatorische Potential von NGF hin. Somit könnte NGF im Gegensatz zu Faktoren stehen, die von Fibroblasten sezerniert werden und überwiegend an strukturellen Schäden beteiligt sind, wie dies z.B. für Matrixmetalloproteinasen der Fall ist. Andererseits können wir nicht mit Sicherheit ausschließen, dass NGF durch FLS produziert wird. Möglicherweise verlieren FLS die Fähigkeit, NGF zu produzieren, wenn sie über mehrere Generationen in vitro kultiviert werden. Eine andere Erklärung wäre, dass NGF produziert, jedoch nicht aus der Zelle sezerniert wird, zumindest nicht in größeren Mengen. Jedenfalls konnten Raychaudhuri et al. an einem größeren Patientenkollektiv zeigen, dass FLS NGF produzieren [35]. Umgekehrt stellen FLS höchstwahrscheinlich ein Zielgewebe für NGF dar.
Das Vorhandensein neurotropher Faktoren in der Synovialitis wurde bereits früher beschrieben [5,6]. Daten aus funktionellen Studien sind bislang nicht verfügbar.
Unsere Untersuchungen zeigen, dass das Neurotrophin-Rezeptor-System und insbesondere die NGF-TrkA-p75-Achse in Synovia und Synovialis von Arthritis- Patienten und damit bei chronisch-entzündlichen Gelenkerkrankungen aktiviert ist.
Nach unserer Hypothese spielen Neurotrophine bei der Arthritis eine pro- inflammatorische Rolle (im Gegensatz zu anderen Untersuchungen, wo sie eine eher anti-inflammatorische oder pro-fibrotische Rolle spielen) und sind als auto- oder parakrine Faktoren an der Perpetuierung einer chronischen Entzündung beteiligt.
Andere Hypothesen zielen darauf ab, dass insbesondere NGF autoreaktive T-Zellen aktiviert.
Ziel dieser Arbeit war der Nachweis von Neurotrophinen und ihren Rezeptoren auf Transkriptebene im Gelenk von Patienten mit rheumatoider Arthritis (RA) und Spondyloarthritis (SpA). Als Kontrollen dienten Patienten mit Arthrose (OA) und mononukleäre Zellen aus dem Blut von Gesunden (PBMC). Ein zentraler Befund unserer Arbeit ist der Nachweis aller hochregulierten Neurotrophine und ihrer Rezeptoren in Synovia und Synovialis von Patienten mit RA und SpA, signifikant im Vergleich zu OA und Gesunden sind NGF und seine beiden Rezeptoren TrkA und p75 exprimiert.
Mittels intrazellulärer Durchflusszytometrie konnten wir zeigen, dass NGF überwiegend von T-Lymphozyten und Monozyten/Makrophagen synthetisiert wird, nicht jedoch von B-Lymphozyten und natürlichen Killerzellen. Aus der Synovialis stammende Fibroblasten (FLS) produzieren kein NGF.
Zusammenfassend weist diese Arbeit die Expression von pleiotropem NGF und seiner beiden Rezeptoren im Gelenk von RA und SpA nach. Eine pro- inflammatorische Rolle von Neurotrophinen in der Pathogenese entzündlicher Gelenkerkrankungen liegt nahe; dies konnte auch für andere entzündliche Erkrankungen nachgewiesen werden [2-4,5-10,36]. Neben der pathogenetischen Forschung, die in Zukunft funktionelle Untersuchungen zur Rolle von Neurotrophinen bei der Arthritis mit einbeziehen sollte, könnten künftige Therapiestrategien in der Behandlung von SpA und RA die pharmakologische Blockade von NGF und/oder seinen Rezeptoren und damit die Hemmung der durch die Ligand-Rezeptorbindung ausgelösten Signalkaskade nutzen.
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Nat Med 2007, 13:156-163.
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Curr Pharm Des 2006, 12:2609-2622.
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Nat Clin Pract Rheumatol 2007,3:635-643.
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Arthritis Rheum 1988, 31:315-324.
25. Kellgren JH, Lawrence JS: Radiological assessment of osteoarthritis.
Ann Rheum Dis 1957, 16:494-501.
26. Baeten D, Bosch F Van den, Elewaut D, Stuer A, Veys EM, De Keyser F: Needle arthroscopy of the knee with synovial biopsy sampling: technical
experience in 150 patients.
Clin Rheumatol 1999, 18:434-441.
27. Gu J, Rihl M, Märker-Hermann E, Baeten D, Kuipers JG, Song YW,
Maksymowych WP, Burgos-Vargas R, Veys EM, De Keyser F, Deister H, Xiong M, Huang F, Tsai WC, Yu DT: Clues to pathogenesis of spondyloarthropathy derived from synovial fluid mononuclear cell gene expression profiles.
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Ich erkläre, dass ich die der Medizinischen Hochschule Hannover zur Promotion eingereichte Dissertation mit dem Titel
Expression von Neurotrophinen und ihren Rezeptoren bei Rheumatoider Arthritis und Spondyloarthritis
in der Abteilung Rheumatologie der Medizinischen Hochschule Hannover unter Betreuung von Herrn Priv.-Doz. Dr. med. Markus Rihl (Betreuer) und Herrn Prof. Dr.
med. Henning Zeidler (Doktorvater) ohne sonstige Hilfe durchgeführt und bei der Abfassung der Dissertation keine anderen als die dort aufgeführten Hilfsmittel benutzt habe.
Die Gelegenheit zum vorliegenden Promotionsverfahren ist mir nicht kommerziell vermittelt worden. Insbesondere habe ich keine Organisation eingeschaltet, die gegen Entgelt Betreuerinnen und Betreuer für die Anfertigung von Dissertationen sucht oder die mir obliegenden Pflichten hinsichtlich der Prüfungsleistungen für mich ganz oder teilweise erledigt.
Ich habe diese Dissertation bisher an keiner in- oder ausländischen Hochschule zur Promotion eingereicht. Weiterhin versichere ich, dass ich den beantragten Titel bisher noch nicht erworben habe.
Ergebnisse der Dissertation wurden in folgendem Publikationsorgan veröffentlicht:
Arthritis Research & Therapie 2009, 11:R82
Hannover, den
(Christian Barthel)
Ich möchte mich herzlich bei Herrn Prof. Dr. H. Zeidler bedanken, der mich als Doktorand in die Abteilung Rheumatologie der MHH aufgenommen und mir die Durchführung der experimentellen Arbeit in seiner Abteilung ermöglicht hat.
Mein spezieller Dank gilt Herrn Priv.-Doz. Dr. M. Rihl für die freundliche Überlassung des Themas, die umfassende und kompetente Betreuung im Labor sowie die Hilfestellung bei der Publikation der Originalarbeit. Seine kontinuierliche Unterstützung und Motivation haben mir immer sehr geholfen.
Ich danke Herrn Prof. Dr. R. Jacobs und Frau Dipl.-Biol. K. Wendt für die Hilfe bei der intrazellulären Durchflusszytometrie und die Unterstützung bei der Interpretation der Daten.
Herrn Prof. Dr. D. Baeten und Frau Dr. N. Yeremenko danke ich für die Isolation der FLS und die Durchführung der ELISA zur Vervollständigung unserer Daten.
Allen ehemaligen und derzeitigen Mitarbeiterinnen und Mitarbeitern der Abteilung Rheumatologie an der Medizinischen Hochschule Hannover danke ich für die angenehme und freundliche Arbeitsatmosphäre.
