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(The FASEB Journal. 2000;14:2097-2107.)
© 2000 FASEB

The loss of sympathetic nerve fibers in the synovial tissue of patients with rheumatoid arthritis is accompanied by increased norepinephrine release from synovial macrophages

LUITPOLD E. MILLER*, HANS-PETER JÜSTEN{dagger}, JÜRGEN SCHÖLMERICH* and RAINER H. STRAUB*1

* Laboratory of Neuroendocrinoimmunology, Department of Internal Medicine I, University Medical Center Regensburg, Germany; and
{dagger} Department of Orthopedic Surgery, Bavarian Red Cross Hospital, Bad Abbach, Germany

1Correspondence: Laboratory of Neuroendocrinoimmunology, Department of Internal Medicine I, University Medical Center Regensburg, Franz-Josef-Strauss-Allee 11, 93042 Regensburg, Germany. E-mail: Rainer.Straub{at}klinik.uni-regensburg.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Our objective was to investigate sympathetic and sensory nerve fibers in synovial tissue in rheumatoid arthritis (RA) and osteoarthritis (OA) in relation to histological inflammation and synovial cytokine and norepinephrine (NE) secretion. Immunohistochemistry was used to detect nerve fibers and inflammatory parameters. A superfusion technique of synovial tissue pieces was used to investigate cytokine and NE secretion. In RA, we detected 0.2 ± 0.04 tyrosine hydroxylase-positive (TH-positive=sympathetic) nerve fibers/mm2 as compared to 4.4 ± 0.8 nerve fibers/mm2 in OA (P<0.001). In RA, there was a negative correlation between the number of TH-positive nerve fibers and inflammation index (RRank=-0.705, P=0.002) and synovial IL-6 secretion (RRank=-0.630, P=0.009), which was not found in OA. Substance P-positive (=sensory) nerve fibers were increased in RA as compared to OA (3.5±0.2 vs. 2.3±0.3/mm2, P=0.009). Despite lower numbers of sympathetic nerve fibers in RA than in OA, NE release was similar at baseline (RA vs. OA: 152±36 vs. 106±21 pg/ml, n.s.). Basal synovial NE secretions correlate with the number of TH-positive CD 163+ synovial macrophages (RA: RRank=0.622, P=0.031; OA: RRank=0.299, n.s.), and synovial macrophages have been shown to produce NE in vitro. Whereas sympathetic innervation is reduced, sensory innervation is increased in the synovium from patients with longstanding RA when compared to the synovium from OA patients. The differential patterns of innervation are dependent on the severity of the inflammation. However, NE secretion from the synovial tissue is maintained by synovial macrophages. This demonstrates a loss of the influence of the sympathetic nervous system on the inflammation, accompanied by an up-regulation of the sensory inputs into the joint, which may contribute to the maintenance of the disease.—Miller, L. E., Jüsten, H.-P., Schölmerich, J., Straub, R. H. The loss of sympathetic nerve fibers in the synovial tissue of patients with rheumatoid arthritis is accompanied by increased norepinephrine release from synovial macrophages.


Key Words: osteoarthritis • synovium • norepinephrine • neuroimmunomodulators


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE ANALYSIS AND understanding of the intraarticular influence of nerve fibers on the inflammatory process in human arthritis is far from complete due to the complex nature of this matter. It is widely accepted that substance P, a neurotransmitter of the sensory afferents, is proinflammatory. For example, substance P stimulates interleukin 1 (IL-1) (1 2 3) , IL-2 (4 , 5) , IL-8 (6) , tumor necrosis factor (TNF; 1 , 7 ), prostaglandin E2 (8) , NF-{kappa}B (9) , and superoxide anion production (10) from various cell types. Local administration of substance P antagonists (11 , 12) or dorsal rhizotomy (13) markedly reduces the severity of inflammation in animal models. Furthermore, substance P is chemotactic for human monocytes (14) and it sensitizes articular afferent nerve fibers in normal and inflamed knee joints (15) , which leads to increased mechanosensitivity, pain, and continuous increased substance P release into the lumen of the joint. Aside from the proinflammatory mechanisms influenced by substance P, the effects of substance P also serve to continuously sense painful stimuli in the periphery.

With respect to the sympathetic nervous system and its transmitters, the situation is not as uniform as with substance P. Since norepinephrine (NE) or adenosine, which are colocalized in vesicles of the sympathetic nerve terminal, are ligands of different receptor subtypes with opposing intracellular signal transduction pathways (summarized in ref 16 ), completely different effects may arise depending on the local concentration. At high concentrations of ~10-6 to 10-4 M (in the synaptic cleft), NE acts on {alpha}- and ß-adrenergic and adenosine on A1, A2, and A3 adenosine receptors, respectively. However, at low concentrations (<=10-7 M) effects are mediated mainly via {alpha}-receptors or A1 adenosine receptors, respectively. Stimulation of the ß-adrenoreceptor and the A2 receptor leads to an intracellular increase of cyclic AMP and thus marked down-regulation of arthritogenic TNF (17 18 19 20 21) , IL-12 (22 , 23) , or interferon-{gamma} (23 , 24) . In contrast, stimulation via the {alpha}2-adrenoreceptor (cAMP decrease) even stimulates TNF secretion (25) . Furthermore, ligation of ß-adrenoreceptors (26) and A2 receptors (27) has been shown to induce generally anti-inflammatory mechanisms in animal models of inflammation. Moreover, the anti-inflammatory mechanism of low-dose methotrexate is accomplished by extracellular adenosine that binds at A2 receptors (28) . Hence, early reports (29) on the modulation of arthritis by the sympathetic nervous system demonstrate proinflammatory effects (30) , whereas more recent studies (31) show anti-inflammatory effects of the sympathetic nervous system (13 , 32 , 33) . The differential effects of the sympathetic nervous system are dependent on either a {alpha}2- or ß-adrenergic stimulation, respectively (34) . Furthermore, opioids from the sympathetic nerve terminals were demonstrated to have anti-inflammatory properties (35 , 36) . These peptides exert a peripheral analgesic effect at sensory articular afferents (37) or a central analgesia on the spinal level (38) . Taken together, an increased sympathetic nervous system activity is accompanied by release of high amounts of norepinephrine, adenosine, and opioids, which induces an anti-inflammatory effect.

Comparative studies between patients with rheumatoid arthritis (RA) and osteoarthritis (OA) on sensory and sympathetic innervation of the synovial tissue using both morphometric and functional techniques are not available. Therefore, we investigated these two portions of the peripheral nervous system in the synovial tissue of patients with RA in comparison to OA through fluorescence immunohistochemistry. Histological markers of inflammation such as lining layer thickness, cellularity, macrophage density, T cell density, and vascularity were used to objectify the severity of local inflammation. In addition, superfusion of synovial tissue pieces with a superfusion technique (reviewed in ref 39 ) was used to investigate the spontaneous cytokine secreting capacity and the spontaneous and electrically releasable NE secretion.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients
Between July 1998 and July 1999, 17 patients with RA and 11 patients with OA who underwent knee joint replacement surgery were consecutively included without further selection. The patients were informed about the purpose of the study and gave written consent. The study was approved by the Ethical Committee of the University of Regensburg. Clinical and laboratory data were recorded immediately before surgery (Table 1 ). Erythrocyte sedimentation rate, C-reactive protein, blood leukocyte count, and rheumatoid factor (latex test) were measured by routine methods.


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  		Table 1. Basic characteristics of patients with osteoarthritis and rheumatoid arthritis who underwent knee joint replacement surgerya

Synovial tissue and preparation
Synovial tissue samples were obtained immediately after opening the knee joint capsule. The tissue samples were cleaned and washed in medium [RPMI 1640, 25 mM HEPES, 5% fetal calf serum (FCS), 1% penicillin/streptomycin, 30 µM mercaptoethanol, 0.57 mM ascorbic acid, 1.3 mM calcium, all additions from Sigma, Deisenhofen, Germany]. From the knee joint capsule, one piece of ~9 cm2 of the white and shining surface of the synovial tissue was dissected. Fat tissue and tissue with a large number of vessels were carefully removed. Thirty ~16 mm2 pieces were cut in order to be loaded into the superfusion chambers (see below) and 8 ~1 cm2 pieces of the same synovial area were used for histology. The samples intended for the hematoxylin-eosin (HE) and alkaline phosphatase anti-alkaline phosphatase (APAAP) staining were immediately placed in protective freezing medium (Tissue-Tek, Sakura Finetek Europe, Zoeterwoude, The Netherlands) and then quick-frozen floating on liquid nitrogen. The tissue samples used for the detection of nerve fibers were fixed for 12 to 24 h in phosphate-buffered saline (PBS) containing 4% formaldehyde and then incubated in PBS with 20% sucrose for 12 to 24 h The tissue was then bedded in Tissue-Tek and quick-frozen floating on liquid nitrogen. Each patient’s samples of synovial tissue were stored at -80°C until further use.

Histological evaluation of inflammation
The frozen tissue samples were cut into 6–8 µm thick sections and placed on precoated slides (SuperFrost Plus, Menzel-Gläser, Braunschweig, Germany) and then fixed in ice-cold acetone for 10 min. The fixed sections used for the HE staining were stored in 0.1 M PBS, whereas the sections for the APAAP method were stored in 0.05 M Tris-buffered saline (TBS). To evaluate cell density and lining layer thickness, a standard HE staining was performed on ~ 45 sections for each individual patient (Mayer’s hematoxylin and eosin yellow from Sigma, Deisenhofen, Germany). At a magnification of 400x, the extent of the lining layer thickness was determined by averaging the number of cells in a lining layer cross section at nine different locations (Fig. 1A ). The cell density in the synovial tissue was determined by counting all stained cells in 17 randomly selected fields of view at a magnification of 400x and calculating the average number of cells per 1 mm2. To determine the number of T cells (Fig. 1B : CD3, Dako, Hamburg, Germany), macrophages (Fig. 1C : CD163, Dako), and capillary vessels (Fig. 1D : collagen IV, Dako) in the synovial tissue of each patient, eight cryosections were incubated with the above primary antibodies at a dilution of 1:100 in 0,05 M TBS containing 1% bovine serum albumin (BSA, Sigma, Deisenhofen, Germany). Specific immunohistological staining was achieved through a standard APAAP staining procedure (DAKO Universal APAAP Kit) and counterstained with Mayer’s hematoxylin. The number of T cells, macrophages, and vessels in the stained tissue was averaged from 17 randomly selected high-power fields at a magnification of 400x and expressed as the number of cells or vessels per 1 mm2. The microscopic evaluation of the inflammatory parameters was based on a recent work of Bresnihan et al. (40) .



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  		Figure 1. Histology of human synovial tissue. A) Cells in the lining layer of RA synovial tissue, 400x, hematoxylin/eosin stain. B) T cells (arrows) in the synovial tissue of an OA patient. Antibody directed against CD3, 400x, counterstained with Mayer’s hematoxylin. C) Macrophages (arrows) in RA synovial tissue. Antibody directed against CD163, 400x, counterstained with Mayer’s hematoxylin. D) Vessels (arrow) in synovial tissue of an RA patient. Antibody directed against collagen IV, 400x, counterstained with Mayer’s hematoxylin. E) Positive nerve fibers (arrows) in OA synovial tissue. Antibody directed against tyrosine hydroxylase, 400x. F) Substance P-positive nerve fibers (arrows) in RA synovial tissue. Antibody directed against substance P, 400x. G) Strongly positive stained nerve fibers for protein gene product 9.5 (arrows) in RA synovial tissue, 400x. H) Positive nerve fibers for tyrosine kinase B (arrows) in OA synovial tissue, 400x.

Determination of synovial tissue innervation
The formaldehyde-fixed tissue samples were cut into 7–9 µm thick sections. Six to eight cryosections for each nerve fiber-specific immunofluorescent staining were placed on precoated slides (SuperFrost Plus, Menzel-Gläser, Braunschweig, Germany). After air drying for 1 h at room temperature, the sections were rehydrated in 0.05 M TBS. The sections were blocked for 1 h with a solution of 10% whole serum (goat and donkey serum, Dako) from the host species of the respective secondary antibody in 0,05 M TBS containing 1% BSA and 0,01% NaH3 (Sigma, Deisenhofen, Germany). The slides were then washed 3 x 5 min in 0.05 M TBS. The tissue sections were incubated in a humid chamber for 12–18 h with primary antibodies against tyrosine hydroxylase (TH, the key enzyme for NE production in sympathetic nerve endings, Chemicon, Temecula, Calif.; Fig. 1E ), substance P (the key transmitter of the sensory nerve fibers, Chemicon; Fig. 1F ), tyrosine kinase B (Trk B, the intracellular tyrosine kinase coupled to the NGF receptor in sensory nerve fibers, Promega, Mannheim, Germany; Fig. 1H ), and protein gene product 9.5 (PGP 9.5, mixed marker of sympathetic and sensory nerve fibers, UltraClone, Wellow, U.K.; Fig. 1G ). The primary antibodies were diluted with 0.05 M TBS containing 1% BSA and 0.01% NaH3 to the following working dilutions: tyrosine hydroxylase 1:500, substance P 1:750, Trk B 1:100, and PGP 9.5 1:500. After another 3 x 5 min wash with 0.05 M TBS, a Cy3-labeled secondary antibody (Dianova, Hamburg, Germany) against the host species of the primary antibody was used to achieve an immunofluorescent staining (conc. 1:600, incubation for 1 h, followed by 4x5 min wash in the dark). The numbers of specifically stained nerve fibers and TH-positive cells per 1 mm2 were determined by averaging the number of stained nerve fibers with a minimum length of 50 µm (indicated by a small micrometer eyepiece) or TH-positive cells in 17 randomly selected fields of view at a magnification of 400x.

Cytokine analysis in the superfusate
In the superfusion experiments (see superfusion technique below), cytokine concentrations were determined at 120 and 240 min in a superfusate fraction of ~1 ml (collected over 15 min). Human IL-6, IL-8, and interferon-{gamma} in the superfusate fractions were determined by enzyme immunometric assay (detection limit in all assays <2 pg/ml, Endogen, Boston, Mass.). Human IL-2 (detection limit <7 pg/ml, R&D, Minneapolis, Minn.), IL-11 (detection limit <8 pg/ml, R&D), IL-15 (detection limit <1 pg/ml, R&D), IL-17 (detection limit <15 pg/ml, R&D), and TNF (detection limit <0.15 pg/ml, R&D) were determined by enzyme immunometric assay, too. Intra-assay and interassay coefficient of variation for all mentioned ELISAs were below 10%.

Inflammation index
The morphological (lining layer thickness, T cell density, macrophage density, vascularity, and cellularity; Fig. 1A , B , C , D ) and functional (spontaneous IL-6 and IL-8 secretion, see superfusion technique below) inflammatory parameters were combined in order to establish an inflammation index. In each patient group (RA/OA), the mean of each individual marker was calculated and set to 100 percent (e.g., mean cellularity in RA = 1226 cells/ mm2 set to 100%, mean cellularity in OA = 857.1 cells/ mm2 set to 100%). The value of each patient was expressed in percent of this individual mean. If a patient had a percent value below (above) 100%, the respective value was below (above) the mean of all patients. For each patient, the geometric mean of all parameters of inflammation (n=7) was that individual’s inflammation index in percent. This technique allows the combination of all parameters even in case of very different levels and units. The inflammation index gives an idea whether a patient has more or less inflammation in relation to the other patients of the same disease group.

Superfusion technique of synovial tissue
As described previously in detail for spleen slices (41 42 43) , we used a microsuperfusion chamber apparatus to superfuse pieces of synovial tissue with culture medium (RPMI 1640, 25 mM HEPES, 5% FCS, 1% Pen/Strep, 30 µM mercaptoethanol, 0.57 mM ascorbic acid, 1.3 mM calcium, all additions from Sigma, Deisenhofen, Germany). These superfusion chambers had a volume of 80 µl and were equipped with two perforated gold disc electrodes forming the bottom and the top of each chamber, respectively. By means of these gold electrodes, computer-assisted electrical stimulation of the synovial tissue was possible. Superfusion was performed for 4 h at a temperature of 37°C and a flow rate of 66 µl/min (one piece per chamber, 24 chambers in parallel). To study spontaneous basal norepinephrine release, all pieces were superfused during the first 150 min with culture medium without electrical stimulation. During the second part of the superfusion period (150th–240th min), electrical stimulation was applied to induce secretion of neurotransmitters. Pieces were electrically stimulated using two trains of monophasic rectangular pulses (2 ms, 1 Hz, 43 mA, 1500 pulses; ref 41 ) at 150 and 195 min.

Measurement of released NE in the superfusate
To determine the amount of endogenously released NE from each patient’s 24 tissue samples used in the superfusion experiments, 1 ml of superfusate from five electrically stimulated and four unstimulated chambers was collected at 60, 120, 180, and 240 min of the superfusion experiment. Since electrical stimulation occurred between 150 and 240 min, we were able to investigate NE concentration at two time points with either baseline conditions or electrical stimulation. Electrically released NE (mean of the 180th min and 240th min) was expressed in percent of baseline NE (mean of the 60th min and 120th min). The superfusate was immediately frozen and stored for short periods at -20°C (max. 3 days). The amount of NE in the superfusate was measured by radioimmunometric assay (IBL, Hamburg, Germany). The highly sensitive protocol used with this kit has a detection limit of 10 pg/ml. Test samples analyzed with high-performance liquid chromatography showed that the radioimmunoassay (RIA) produced comparable results.

Measurement of secreted NE in the supernatant of synovial macrophages
A piece of RA synovial tissue (~5 cm2) was placed in a petri dish containing 7 ml of PBS (mentioned above) and homogenized with surgical scissors under sterile conditions. The tissue was then transferred to a 50 ml test tube (Falcon, Heidelberg, Germany) and digestion enzymes were added at the following concentrations: collagenase I 1 mg/ml, DNase 0,3 mg/ml, Hyaluronidase 2 mg/ml (all Sigma, Deisenhofen, Germany). After a digestion period of 1 h at 37°C with constant agitation, the reaction was stopped through the addition of RPMI tissue culture medium containing 10% FCS (mentioned above). The digested tissue was filtered through a 40 µm cell strainer and the isolated cells were then centrifuged at 1200 rpm for 5 min. The cells were washed with PBS and centrifuged again. The cells were then incubated with a mouse anti-human CD 163 antibody (mentioned above) for 15 min at 4°C, followed by another wash with PBS. To isolate the CD 163-positive macrophages, the cells were incubated with a rat anti-mouse magnetic microbead antibody (Miltenyi, Auburn, Calif.) for 15 min at 4°C and washed again. CD 163-positive cells were separated from the total cell suspension through a standard magnetic cell sorting protocol (Miltenyi). Approximately 5 x 104 cells from each, the CD 163-positive selection and the negative selection were placed in 24-well cell culture plates containing 1.5 ml RPMI medium with 10% FCS and 1% penicillin/streptomycin. The cell culture supernatant was collected after 12 h and the NE content was determined through NE RIA (as described above).

Presentation of the data and statistical analysis
All data are given as mean ± SE. Correlations were calculated by Spearman Rank Correlation analysis (SPSS/PC, Advanced Statistics, V9.0.0, SPSS Inc., Chicago, Ill.) and graphically demonstrated by linear regression analysis. Group means were compared by the nonparametric Mann-Whitney test (SPSS). Group means over time in two different groups were compared by the general linear model (SPSS) and P<0.05 was the significance level.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Histologically determined inflammation in patients with RA and OA
The lining layer was significantly thinner in patients with OA (mean: 5.7±2.3 cells) as compared to patients with RA (mean: 12.5±2.8 cells, P<0.01 for the difference). Cellularity in the synovial tissue was significantly higher in RA as compared to OA (Fig. 2 ). However, there were no significant differences in T cell density, macrophage density, and vascularity between the two groups (Fig. 2) . In both disease groups, RA and OA, macrophages were significantly more prevalent than T cells (Fig. 2) . The correlation matrix in Table 2 demonstrates that lining layer thickness was independent of the other histological markers of inflammation. T cell density and macrophage density correlated significantly in patients with RA and OA (Table 2) . However, vascularity correlated with cellularity and T cells only in patients with RA (Table 2) . In patients with RA, macrophage density significantly correlated with erythrocyte sedimentation rate (RRank=0.533, P<0.05). The three patients with methotrexate therapy had significantly lower T cell density, macrophage density, and vascularity (with MTX vs. without MTX: T cells 52.3±23.6 vs. 153.2±27.7; macrophages 248.3±26.1 vs. 449.2±80.1; vascularity 84.9±13.1 vs. 215.6±23.7; p at least < 0.05). Other effects of the prescribed medication on the investigated parameters were not evident.



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  		Figure 2. Histological markers of inflammation in the synovial tissue of patients with RA (black circles) and OA (open circles). The number of all cells, T cells, macrophages, and vessels in the stained tissue was averaged from 17 randomly selected high-power fields at a magnification of 400x and expressed as the number of cells or vessels per 1 mm2.


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  		Table 2. Matrix of correlation coefficients for the relation between various histological markers of inflammation in patients with RA and OAa

Cytokine secretion from synovial tissue
IL-6 concentration in patients with OA was significantly higher as compared to RA (Fig. 3 ). In contrast, with respect to IL-8 and TNF, secretion of these two cytokines was significantly higher in RA as compared to OA (Fig. 3) . Due to the large sample size used to calculate the levels of cytokine secretion, differences in the size of the investigated tissue samples are negligible. This is further supported by extensive superfusion experiments with murine spleen slices (41 42 43 44) . In patients with RA, prior administration of drugs (prednisolone, MTX, sulfasalazine, azathioprine, NSAID) did not influence the cytokine secretion during superfusion. Typical T cell cytokines such as interferon-{gamma}, IL-2, IL-11, IL-15, and IL-17 were below the detection limit in the collected superfusate at both investigated time points (120 and 240 min).



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  		Figure 3. Superfusate concentrations of IL-6, IL-8, and TNF from RA (black bars) and OA (open bars) synovial tissue at 120 min. Pieces of synovial tissue (n for each condition is given in the bars) were transferred to minisuperfusion chambers and superfused for 2 h at a temperature of 37°C and a flow rate of 66 µl/min. Superfusate was collected at 120 min for 15 min to measure the cytokine superfusate concentration by ELISA.

Sensory and sympathetic innervation of synovial tissue in relation to inflammation
Examples for tissue innervation are given in Fig. 1 . RA patients had significantly lower numbers of TH-positive nerve fibers in comparison to patients with OA (Fig. 4 ). In contrast, the number of substance P-positive nerve fibers were significantly higher in patients with RA as compared to patients with OA (Fig. 4) . There was a direct correlation between the density of TH-positive nerve fibers and the density of substance P-positive nerve fibers in patients with RA (RRank=0.599, P=0.018) but not in OA (RRank=0.535, P=0.090; data not shown). This indicates a similar behavior of TH-positive and substance P-positive nerve fibers in RA and probably in OA, but on a completely different level. Trk B and PGP 9.5-positive nerve fibers were also found in higher densities in RA patients than in OA patients, but the differences did not reach significant levels (Fig. 4) .



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  		Figure 4. Number of nerve fibers in synovial tissue of patients with RA (black bars) and OA (open bars). Abbreviations: TH, tyrosine hydroxylase (sympathetic); SP, substance P (sensory); TrkB, tyrosine kinase B (mixed: detects the intracellular tyrosine kinase at the nerve growth factor receptor); PGP 9.5, protein gene product 9.5 (mixed sympathetic and sensory). The total number of examined high-power fields for each type of nerve fiber and disease group is given on or in the bars.

To investigate the relationship between the severity of inflammation and presence of nerve fibers, correlations between the inflammation index and the number of specific nerve fibers were calculated. Figure 5 demonstrates a negative correlation between the inflammation index and the number of TH-positive nerve fibers in RA but not in OA patients. A similar result was obtained for the correlation of spontaneous IL-6 release at 120 min and number of TH-positive nerve fibers in RA but not in OA patients (Fig. 6 ). With respect to substance P, number of nerve fibers tended to decrease with a higher inflammation index in RA (RRank=-0.525, P=0.054) but not in OA (RRank=-0.023, n.s.) (data not shown).



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  		Figure 5. Relationship between degree of synovial inflammation and number of tyrosine hydroxylase-positive nerve fibers (TH, sympathetic) in patients with RA (A) and OA (B). The linear regression line, the Spearman rank correlation coefficient, and its P value are shown.



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  		Figure 6. Relationship between IL-6 superfusate concentration at 120 min and number of tyrosine hydroxylase-positive nerve fibers (TH, sympathetic) in patients with RA (A) and OA (B). The linear regression line, the Spearman rank correlation coefficient, and its P value are shown.

Spontaneous and electrically releasable NE secretion
As demonstrated above, the number of sympathetic nerve fibers was significantly lower in RA as compared to OA. Therefore, we investigated spontaneous NE release from superfused synovial tissue (measured in 14 RA and 10 OA patients without any selection). Average spontaneous NE concentration in baseline samples (mean of the 60th and 120th min of superfusion) was higher in patients with RA as compared to OA but this did not reach a significant level (152.0±36.0 vs. 106.0±21.0 pg/ml, P=0.36). A ratio between the number of tyrosine hydroxylase-positive nerve fibers and the baseline NE secretion was established for both disease groups. A Spearman rank correlation showed an inverse correlation of the above described ratio to the IL-6 production before electrical stimulation in both disease groups (RA: Rrank=-0.598; P=0.031, OA: Rrank=-0.850; P=0.004). Since numbers of TH-positive nerve fibers were much lower in RA as compared to OA, another source of the NE production was likely.

Immunofluorescent histochemistry with the antibody against TH (mentioned above) also demonstrated a marked positive staining of synovial cells (Fig. 7A ). A salient number of TH-positive cells were stained in both RA and OA patients (29.1±8.9 vs. 25.7±10.8 TH-positive cells/ mm2, n.s. difference). A double labeling protocol consisting of an APPAP-based immunohistological staining against CD 163 (1:75, not counterstained with HE; mentioned above, Fig. 7B ), followed by an immunofluorescent staining of TH (1:500, mentioned above, Fig. 7A ) was used to determine the cell type of the TH-positive cells. Figure 7 clearly shows that the CD 163-positive macrophages are also TH positive. An exemplary experiment with freshly isolated CD 163-positive macrophages further substantiated the hypothesis that synovial macrophages are able to secrete NE. In the supernatant of CD 163-positive synovial macrophages, a mean NE concentration of 73 pg/ml was determined while the mean concentration of NE in the supernatant of the negative selection was found to be 43 pg/ml. The number of TH-positive cells significantly correlated with baseline NE release in RA but not in OA patients (Fig. 8A , B ). Therefore, it is likely that NE is produced by TH-positive macrophages in the synovial tissue, particularly in RA patients.



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  		Figure 7. A micrograph of identical fields of view (400x) in a section of double-stained OA synovial tissue with antibodies directed against tyrosine hydroxylase (A), photographed at 400x magnification under fluorescent light, and CD 163 (B), photographed at 400x under bright light conditions clearly identified synovial macrophages as being tyrosine hydroxylase positive.



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  		Figure 8. Baseline norepinephrine release in relation to tyrosine hydroxylase-positive cells (RA in panel A, OA in panel B) and electrically released norepinephrine (RA in panel C, OA in panel D). The linear regression line, the Spearman rank correlation coefficient, and its P value are shown. Baseline norepinephrine was the average superfusate concentrations of samples taken at 60.and 120 min of superfusion. In experiments with electrical stimulation (C, D), electrical stimulation occurred between 150 and 240 min. Electrically released norepinephrine (mean of the 180th min and 240th min) was expressed in percent of baseline NE (mean of the 60th min and 120th min) (C, D). Abbreviations: TH, tyrosine hydroxylase; NE, norepinephrine.

If NE is stored in nerve terminals, electrical stimulation should lead to an increase of NE release as demonstrated in spleen slices (41 42 43) . Only 8 out of 14 patients with RA and 8 out of 10 patients with OA demonstrated an increase of NE above baseline during electrical stimulation (above the 100% level in Fig. 8C , 8D ). Baseline NE secretion correlated inversely with the electrically induced change of NE in both RA and OA patients (Fig. 8C , D ). This indicates that NE release can be electrically induced only from synovial tissue pieces with a low spontaneous baseline NE secretion (lower than ~ 200 pg/ml, Fig. 8C , D ).

Building subgroups of patients with low (<200 pg/ml) and high (>= 200 pg/ml) baseline NE release demonstrates that NE is only electrically released from synovial tissue pieces with low baseline NE concentration in both RA (Fig. 9A, B) and OA patients (Fig. 9C , D ).



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  		Figure 9. Electrically released norepinephrine in relation to baseline release in RA (A, B) and OA (C, D) patients. Pieces of synovial tissue were transferred to minisuperfusion chambers and superfused for 4 h at a temperature of 37°C and a flow rate of 66 µl/min. Superfusate was collected for 15 min to measure norepinephrine superfusate concentration at 60, 120, 180, and 240 min. Electrical stimulation occurred between 150 and 240 min (black horizontal bar). Open circles represent electrically stimulated pieces of synovial tissue; black circles represent unstimulated pieces of synovial tissue. Electrically released norepinephrine (mean of the 180th min and 240th min) was expressed in percent of baseline NE (mean of the 60th min and 120th min). The left panels demonstrate patients (A: 9 RA, C: 8 OA) with a baseline release below 200 pg norepinephrine/ml superfusate; the right panels demonstrate patients (B: 5 RA, D: 2 OA) with a baseline release equal or above 200 pg norepinephrine/ml superfusate. The P value for the comparison of the two curves is given in each figure. NE, norepinephrine.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The study demonstrates that sympathetic nerve fibers are significantly reduced and substance P-positive nerve fibers were significantly increased in chronic RA compared to OA. The loss of sympathetic nerve fibers in patients with RA was correlated to an increase in the degree of inflammation. Despite the loss of sympathetic nerve fibers in patients with RA, a high amount of NE was still secreted from synovial tissue. Further studies demonstrated that TH-positive synovial macrophages probably were the source of the secreted NE. In 43% of the patients with RA and 20% of patients with OA, NE release from the synovial tissue is not typical neuronal transmitter secretion, but is most likely produced by TH-positive synovial macrophages.

Transmitters of the sensory nervous system such as substance P are proarthritogenic since they induce proinflammatory cytokines such as IL-1, IL-8, and TNF (1 2 3 4 5 6 7) via stimulation of NF-{kappa}B (9) . High concentrations of sympathetic neurotransmitters such as NE and adenosine are anti-inflammatory since they inhibit arthritogenic cytokines such as TNF, IL-12, and interferon-{gamma} (17 18 19 20 21 22 23 24) . Moreover, NE at high concentrations induces the anti-inflammatory cytokine IL-10 (22 , 45) and adenosine at high concentrations inhibits collagenase (46) . Thus, these two portions of the peripheral nervous system seem to have completely different effects on immune competent cells.

The differential role of sympathetic and sensory nerves on immune competent cells inspired us to study both types of nerve fibers in the same patients with RA and OA using the same extensive histological and functional techniques. For the first time, we found a differential loss of sympathetic nerve fibers in longstanding RA as compared to OA patients. This differential loss was not found in one earlier study in patients with RA as compared to normal subjects (47) . The different results between the earlier investigation (47) as compared to the present study may be due to 1) different source of tissue (arthroscopy vs. surgery), 2) different mean age of patients (48 vs. 68 year), 3) a lower number of investigated tissue sections per patient (3 vs. 6–8), and 4) blocking the tissue with serum from the host species of the respective secondary antibody (without vs. with). Furthermore, Pereira et al. (47) did not indicate the number of randomly selected high-power fields for each specific stain (17 in the present study) and nor did they define the significant length of a nerve fiber (minimal length was 50 µm in the present study). From our point of view, these are marked differences that can lead to a strong bias, which has been demonstrated in a quantitative analysis of synovial tissue (40) . To our knowledge, there is no other study that investigated both types of nerve fibers in a parallel fashion. Generally, a varying degree of depletion of nerve fibers was found in the synovial tissue of patients with RA and in animals with experimental arthritis (47 48 49 50 51) . However, some studies also demonstrated an increase with respect to substance P nerve fibers (52 , 53) . Our study demonstrated a significantly higher level of substance P nerve fibers in RA as compared to OA patients. A comparative study of the overall depletion or increase of sympathetic and sensory nerve fibers between RA patients and normal subjects was not possible due to the difficulty of obtaining sufficient tissue samples from healthy subjects. Summarizing, the anti-inflammatory nerve fibers were significantly decreased whereas the proinflammatory nerve fibers were significantly increased in RA when compared to OA. The differential pattern of innervation (ratio of sensory to sympathetic nerve fibers) could be a contributing factor in the perpetuation of the inflammation seen in RA patients. The loss of the sympathetic nerve fibers in RA patients is most likely a consequence of the initial synovial inflammation. The reduction or lack of sympathetic innervation may be a contributing factor to the development of chronic RA.

There are quite a few possible explanations for the loss of sympathetic nerve fibers in the synovial tissue of chronic RA patients. One mechanism possibly leading to this loss could be connected to the regulatory levels of semaphorins in the synovial tissue. Semaphorins are nerve-repellent factors that are known to act as mediators of neuronal apoptosis (54) and to repulse and inhibit neuronal development (55) . Semaphorin III specifically influences the sensory nervous system (55) , whereas semaphorin IV specifically interacts with the sympathetic nervous system (56) . Another factor that could influence the differential pattern of synovial innervation is the level of nerve growth factor (NGF) in the tissue. Although it is unlikely that the lack of NGF in the tissue is responsible for the loss of sympathetic nerve fibers, there are indications that very high concentrations of NGF (often found in the synovial fluid from RA patients) can cause neuronal apoptosis (57) .

We used an extensive protocol to verify inflammation in the synovial tissue in order to evaluate the relationship between inflammation and nerve fiber density. The quantitative analysis of synovial inflammatory parameters (lining layer thickness, cellularity, T cell infiltration, macrophage infiltration, and vascularity) was based on an earlier study (40) . To add functional parameters to the evaluation of inflammation, we used a superfusion system to investigate spontaneous IL-6 and IL-8 secretion from each patient’s tissue samples. TNF secretion was not a useful marker because it was below the detection limit in 80% of all patients. The results indicate that anti-inflammatory sympathetic nerve fibers are strongly reduced when the tissue is inflamed. It is interesting that substance P-positive nerve fibers also tended to decrease with increasing inflammation. However, overall number of substance P-positive nerve fibers remained high when compared to TH-positive nerve fibers in patients with RA.

To receive a functional marker of sympathetic innervation of the tissue, we studied spontaneous NE release into the superfusate from synovial tissue in the same RA and OA patients. Normally, the spontaneous secretion of NE in tissue is due to continuous release of low amounts of this transmitter from nerve endings (41) . Even though the density of TH-positive nerve fibers was significantly lower in RA than in OA, NE release tended to be higher in patients with RA as compared to OA. This was indicative of another source of NE in the tissue. During a second staining of synovial tissue from all patients, we were able to identify TH-positive synovial cells within the tissue. These cells were identified as CD 163-positive macrophages. The basal NE release correlated with the number of TH-positive synovial cells in RA patients, which demonstrates that NE is most likely secreted by synovial macrophages. The importance of the NE released from synovial macrophages on the local inflammatory process is unclear at the moment, since NE effects are concentration dependent. At low concentrations, NE has a proinflammatory effect whereas at high concentrations it has an anti-inflammatory effect. If macrophages are found in high densities in the areas of high inflammation within the synovial tissue, it is likely that an anti-inflammatory effect through the inhibition of tumor necrosis factor via ß-adrenergic receptors could occur in the direct surroundings of these macrophages (58) .

In earlier studies of murine spleen slices, we demonstrated that electrical stimulation of the tissue induces NE release (41 42 43) . With respect to the synovial tissue, we wanted to verify whether or not NE release is electrically inducible from the tissue. In 57% of patients with RA and 80% of patients with OA, electrical stimulation leads to an increase of NE when compared to basal levels. In those patients with high basal NE levels, electrical stimulation did not induce NE release. In a subanalysis of patients with high or low basal NE levels, it was verified that NE is electrically released only in samples with low basal NE secretion below ~200 pg/ml at baseline. This indicates that in samples with high baseline NE secretion, the transmitter was not released from nerve terminals in a typical fashion, but must originate from another source. Control experiments with the sodium channel blocker tetrodotoxin (inhibitor of neuronal activity) were not possible because this agent by itself changes the cytokine secretion pattern of isolated cells in vitro.

In conclusion, this study found low numbers of sympathetic nerve fibers in synovial tissue of RA as compared to OA patients, which was dependent on the degree of inflammation in RA patients. The baseline NE secretion was similar in both patient groups (RA and OA). The baseline NE secretion from the tissue of RA patients with a reduced density of sympathetic nerve fibers is most likely produced by NE-secreting synovial macrophages. With respect to spontaneous NE secretion, this study indicates that ‘sympathetic’ cells seem to replace sympathetic nerve fibers in the synovial tissue. We do not know at this time whether these cells are also able to secrete neuropeptide Y, ATP, or endogenous opioids and therefore are comparable to sympathetic nerve endings. The loss of sympathetic nerve fibers will lead to the uncoupling of the synovial tissue from the hypothalamus-autonomic nervous system axis. Both loss of endogenous sympathetic anti-inflammatory neurotransmitters and uncoupling of the synovial tissue may lead to an unfavorable situation supporting the disease process, particularly in view of high numbers of sensory proinflammatory nerve fibers.


   ACKNOWLEDGMENTS
 
We thank Angelika Gräber for excellent technical assistance and Prof. Dr. Neuhuber, Institute of Anatomy, University of Erlangen, for his technical advice with respect to immunohistochemistry of nerve fibers. The study was supported by the Deutsche Forschungsgemeinschaft (STR 511/5–1).

Received for publication January 5, 2000. Revision received March 29, 2000.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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