PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of annrheumdAnnals of the Rheumatic DiseasesVisit this articleSubmit a manuscriptReceive email alertsContact usBMJ
 
Ann Rheum Dis. 2007 July; 66(7): 871–879.
Published online 2007 February 23. doi:  10.1136/ard.2006.067066
PMCID: PMC1955126

β‐Endorphin, Met‐enkephalin and corresponding opioid receptors within synovium of patients with joint trauma, osteoarthritis and rheumatoid arthritis

Abstract

Objective

Intra‐articularly applied opioid agonists or antagonists modulate pain after knee surgery and in chronic arthritis. Therefore, the expression of β‐endorphin (END), Met‐enkephalin (ENK), and μ and δ opioid receptors (ORs) within synovium of patients with joint trauma (JT), osteoarthritis (OA) and rheumatoid arthritis (RA) were examined.

Methods

Synovial samples were subjected to double immunohistochemical analysis of opioid peptides with immune cell markers, and of ORs with the neuronal markers calcitonin gene‐related peptide (CGRP) and tyrosine hydroxylase (TH).

Results

END and ENK were expressed by macrophage‐like (CD68+) and fibroblast‐like (CD68) cells within synovial lining layers of all disorders. In the sublining layers, END and ENK were mostly expressed by granulocytes in patients with JT, and by macrophages/monocytes, lymphocytes and plasma cells in those with OA and RA. Overall, END‐ and ENK‐immunoreactive (IR) cells were more abundant in patients with RA than in those with OA and JT. ORs were found on nerve fibres and immune cells in all patients. OR‐IR nerve fibres were significantly more abundant in patients with RA than in those with OA and JT. μORs and δORs were coexpressed with CGRP but not with TH.

Conclusions

Parallel to the severity of inflammation, END and ENK in immune cells and their receptors on sensory nerve terminals are more abundant in patients with RA than in those with JT and OA. These findings are consistent with the notion that, with prolonged and enhanced inflammation, the immune and peripheral nervous systems upregulate sensory nerves expressing ORs and their ligands to counterbalance pain and inflammation.

Opioids elicit analgesic effects by activating both central and peripheral opioid receptors (ORs).1,2 Peripheral ORs have been demonstrated on sensory nerve terminals within subcutaneous tissue in animals.3,4,5 The activation of such peripheral ORs by exogenous and endogenous agonists can produce significant antinociceptive effects.5,6,7,8 Recently, ORs were identified by immunohistochemical staining and radioligand‐binding assay in synovial cells of patients with RA9,10 and in equine joints.11 Keates et al12 reported that the density of opioid‐binding sites was markedly enhanced in inflamed compared with control canine joint tissue. A growing body of clinical data documents the successful use of small doses of intra‐articular (IA) morphine for pain control in patients with osteoarthritis (OA), rheumatoid arthritis (RA) and joint trauma (JT; ie, after arthroscopic knee surgery) without central side effects.13,14,15,16,17,18 Opioid peptides are synthesised and processed within inflammatory cells1,19 and, on release, inhibit inflammatory pain by activating peripheral ORs.19,20,21,22 In humans, β‐endorphin (END) and Met‐enkephalin (ENK) have been found in synovial tissue.14,23,24 Such endogenous opioid peptides contribute to clinical pain inhibition as studies have shown that the IA administration of the opioid receptor antagonist naloxone can exacerbate postoperative pain.25 However, the exact cell types and disease‐specific differences in the occurrence of opioid peptides and receptors have not been examined so far. Therefore, we examined in detail the localisation of END and ENK and their corresponding ORs in synovium of patients with JT, OA and RA. We investigated the differences in the number and types of cells expressing END and ENK by use of different markers for macrophages/monocytes (CD68), granulocytes (CD15), T lymphocytes (CD3) and B lymphocytes (CD23). We also examined the expression and colocalisation of μORs and δORs with markers for primary afferent neurones (calcitonin gene‐related peptide (CGRP)) and for postganglionic sympathetic neurones (tyrosine hydroxylase (TH)).

Materials and methods

Patients

Synovial tissue was obtained from 28 patients (aged 23–81 years) during routine arthroscopy or open joint surgery for diagnostic and therapeutic procedures in patients with JT, or during elective knee joint replacement in those with OA and RA. Nine patients fulfilled the American College of Rheumatology criteria for RA,26 and nine patients met the criteria for OA.27 Ten patients with JT were operated following a 10–30‐day interval after knee injury (four patients with meniscal injury, three with patellar injury and three with cartilage damage). Histologically normal synovium (n = 2) was obtained during diagnostic arthroscopy from patients with minor knee JT. Before surgery, patients were informed about the purpose of the study and gave their written consent. The ethical committees of the Charité—Universitätsmedizin Berlin, Berlin, Germany, and the University Hospital Regensburg, Regensburg, Germany, approved the study. Table 11 gives the clinical and laboratory data of the patients. Parameters such as plasma C‐reactive protein and rheumatoid factor were measured by standard techniques, as described previously.28

Table thumbnail
Table 1 Clinical, laboratory and histological parameters of patients with joint trauma, osteoarthritis and rheumatoid arthritis

Reagents

The following antibodies were used: rabbit polyclonal antibodies against human END and ENK (Peninsula Laboratories, Merseyside, UK), μOR (Drs S Schulz and V Höllt, Magdeburg, Germany) and δOR (Dr R Elde, Minneapolis, Minnesota, USA); polyclonal guinea pig antibody against human CGRP (Peninsula Laboratories); monoclonal antibodies against human CD3 (clone PC3/188A; marker for T lymphocytes), CD15 (clone C3D‐1; granulocytes), CD23 (clone MHM6; B‐lymphocytes), CD68 (clone KP1; macrophages), CD163 (clone Ber‐MAC3; macrophages), type IV collagen (clone CIV 22, capillary vessels; all from Dako, Hamburg, Germany) and human TH (Incstar, Minneapolis, Minnesota, USA). Further, materials included the VectaStain Elite Kit, Vectashield, Texas red‐conjugated goat anti‐rabbit and fluorescein isothiocyanate‐conjugated donkey anti‐mouse secondary antibodies (Vector Laboratories, Burlingame, California, USA) and HistoGreen Peroxidase‐Substrate Kit (Linaris, Wertheim–Bettingen, Germany).

Tissue preparation and histological evaluation

Immediately after surgery, synovial tissue was taken from all three groups, then fat tissue and tissue with a large number of vessels were removed. To obtain optimal tissue morphology, only intact synovial tissue was fixed in 4% (weight/volume) paraformaldehyde in 0.16 M phosphate buffered saline (PBS; pH 7.4) for 4 h and then cryoprotected overnight at 4°C in PBS containing 10% sucrose. The tissue was then embedded in tissue‐Tek compound (OCT, Miles, Elkhart, Indiana, USA) and frozen. These procedures are analogous to our previous studies that included positive controls—for example, in spinal cord29—thus precluding false‐negative results. All tissue samples from patients with JT, OA and RA were subjected to the same treatment protocol. For OR detection 40 μm thick sections were prepared on a cryostat and collected in PBS (floating sections). For histological examination, opioid peptide immunohistochemistry and cell identification, 7 μm thick sections from the same patient were mounted on gelatin‐coated slides. Histological evaluation was performed as described previously.28 For best results of OR demonstration, we used the most common method (floating thick tissue sections of 40 μm) to increase exposure and penetration of tissue by OR antibodies. In general, many antigens on nerve fibres can be more successfully demonstrated in floating thick tissue sections than in slide‐mounted tissue sections, which may, partly, be due to the three‐dimensional course of nerve fibres within tissue.3,30,31,29,4 However, the use of 7 μm thick sections mounted on slides is sufficient for demonstration of opioid peptide and identification of cell type. Cell density and lining layer thickness were determined in sections from at least three different tissue samples (H&E stained) per patient. The thickness of the lining layer was analysed by averaging the number of cells in a cross section at nine different locations (×400 magnification). The overall cell density was determined by counting all stained cell nuclei in 17 randomly selected high‐power fields of view (×400 magnification). The numbers of T cells, macrophages and vessels were evaluated in eight cryosections stained with antibodies against human CD3, CD163 and type IV collagen, respectively. The number of stained structures was averaged from 17 randomly selected high‐power fields (×400 magnification). The number of high‐power fields was derived from a histological study by Bresnihan et al.32

Single staining procedures

All sections were processed for immunohistochemistry with a vectastain avidin–biotin–peroxidase complex (ABC) kit (Vector Laboratories) as described previously.29 Unless otherwise stated, all incubations were performed at room temperature and PBS was used for washing (three times for 10 min) after each step. The floating (for ORs) and mounted (for opioid peptides) sections were incubated for 45 min in PBS with 0.6% H2O2 and 50% methanol to block endogenous peroxidase, and for 60 min in PBS containing 0.3% Triton X‐100, 1% bovine serum albumin, 5% goat serum and 5% donkey serum (blocking solution) to prevent non‐specific binding. The sections were then incubated overnight with antibodies against END, ENK, μOR or δOR (1:1000), and thereafter for 90 min with a goat anti‐rabbit biotinylated secondary antibody and for another 90 min with ABC. Finally, the sections were washed and stained with 3′,3′‐diaminobenzidine tetrahydrochloride (DAB; Sigma, Saint Louis, USA) containing 0.01% H2O2 in 0.05 M Tris‐buffered saline (pH 7.6) for 3–5 min. After the enzyme reaction, the floating sections (for OR detection) were mounted on gelatin‐coated slides. The slide‐mounted sections were washed in tap water, counterstained with thionin, dehydrated in alcohol, cleared in xylene and mounted in distrene, tricresyl phosphate and xylene (DPX; Merck, Darmstadt, Germany).

In addition to double staining, immunoreactive (IR) cells were identified by the following morphological criteria: (1) macrophages/monocytes by large cell bodies, vacuolated cytoplasm and irregular‐shaped nuclei; (2) lymphocytes by small cell bodies, large nuclei and small amounts of cytoplasm; (3) polymorphonuclear leucocytes by large cell bodies and multisegmented nuclei; (4) plasma cells by vacuolated cytoplasm and small dense eccentric nuclei; and (5) fibroblast‐like synoviocytes by dendritic‐shaped processes.

Double staining procedures

Double staining of opioid peptides with cell markers

Double staining was performed as described previously.19 Sections stained in the first sequence with an antibody against either END or ENK (as described above) were treated with 1% H2O2 for 30 min to inactivate peroxidase in ABC, washed in several changes of PBS and incubated with blocking solution. In a second step, sections mounted on gelatin‐coated slides were incubated with monoclonal mouse antibodies against human CD15, CD68, CD3 or CD23. All incubations were maintained overnight at 4°C, then slides were washed in PBS and exposed to the appropriate biotinylated secondary antibody for 1 h and to ABC for 45 min. Finally, these sections were washed and stained using Histogreen. The chromogen DAB used for the primary antiserum (END and ENK) appeared brown, whereas the Histogreen used for the second antiserum (cell markers) appeared green. After the enzymatic reaction, sections were mounted on gelatin‐coated slides. Without counterstaining, sections were washed in distilled water, dehydrated in alcohol, cleared in xylene and mounted in DPX.

Double staining of ORs and neuronal markers

Floating sections stained in the first sequence with an antibody against either μOR or δOR (as described above) were treated with 1% H2O2 for 30 min to inactivate peroxidase in ABC, washed in several changes of PBS and incubated with blocking solution. Then, sections were incubated with a guinea pig antibody against human CGRP overnight at 4°C. The slides were then washed in PBS, exposed to the appropriate biotinylated secondary antibody (Vector Laboratories) for 1 h and to ABC for 45 min. Finally, the sections were washed and stained using DAB. During staining with DAB, nickel solution was used to differentiate the double immunostaining. The chromogen DAB/nickel used for the primary antisera (μOR and δOR) appeared grey/black, whereas the one (DAB) used for the second antiserum (CGRP) appeared brown. After the enzymatic reaction, the floating sections were mounted on gelatin‐coated slides, washed in distilled water, dehydrated in alcohol, cleared in xylene and mounted in DPX. For double immunofluorescence staining for TH with μOR or δOR, floating sections were incubated with rabbit polyclonal antibodies against μOR or δOR in combination with a mouse monoclonal antibody against TH (1:1000), washed with PBS and then incubated with secondary Texas red‐conjugated goat anti‐rabbit antibody in combination with fluorescein isothiocyanate‐conjugated donkey anti‐mouse antibody. Thereafter, the floating sections were washed with PBS, mounted on gelatin‐coated slides and Vectashield, and viewed under a Zeiss 510 laser‐scanning microscope.

Specificity controls

To demonstrate specificity of staining, the following controls were included (see also Mousa et al19): (1) preabsorption of diluted antibody against END, ENK, μOR or δOR with 5 μg/ml of purified END, ENK (Peninsula Laboratories) or synthetic peptide antigen for μOR and δOR (Gramsch Laboratories‐Biotechnology, Schwabhausen, Germany), respectively, for 24 h at 4°C; (2) preincubation of END antiserum with 7 μg/ml purified ENK, and preincubation of ENK antiserum with 7 μg/ml purified END to exclude cross reactivity; and (3) omission of either the primary antisera, the secondary antibodies or ABC.

Quantification of immunostaining

END‐ or ENK‐IR cells as well as OR‐IR nerve fibres or cells were counted using only intact tissue exhibiting optimal morphology to avoid misleading results. END‐ or ENK‐IR cells were counted by a blinded experimenter in four sections per patient. Fifteen squares (384 μm2 each) per section were analysed using a Zeiss microscope (objective ×20×10) as described previously.29,19 The percentage of stained (for END or ENK) polymorphonuclear or mononuclear cells was determined by the formula: stained polymorphonuclear or mononuclear cells/total number of stained cells ×100. OR‐IR nerve fibres were counted by a blinded experimenter in five sections per patient and 10 squares (38.4 mm2 each) per section using a Zeiss microscope (objective ×20×10). Seven patients per group were used for quantitative analysis.

Statistical analysis

Data are represented as means (SEM). Sample comparisons were made using one‐way analysis of variance (ANOVA) followed by Student–Newman–Keuls or Dunnett's post hoc test in the case of normally distributed data, and Kruskal–Wallis one‐way ANOVA on ranks followed by Dunn's test in the case of data not distributed normally. Differences were considered significant if p<0.05. All tests were performed using Sigma Stat V.2.03 statistical software.

Results

Parameters of inflammation in serum and synovial tissue

To compare the severity of inflammation among patients with JT, OA and RA, we investigated laboratory serum data, the thickness of the lining layer, overall cellularity, density of CD3 T cells and CD163 macrophages, and vascularity. Parameters of inflammation were more overt in patients with RA than in those with JT and OA (table 11).). C‐reactive protein, cellularity and thickness of the synovial lining increased from patients with JT to OA to RA and were significantly higher in patients with RA than in those with JT and OA (p<0.05; table 11).). Synovial vascularity was similar in patients with OA and RA and significantly higher than in those with JT (table 11).). The overall density of CD3 T cells and CD163 macrophages increased from patients with JT to OA to RA. This increase was significant for CD163 macrophages in patients with RA (p<0.05, ANOVA on ranks followed by Dunn's test). Erythrocyte sedimentation rate (ESR) was significantly higher in patients with RA than in those with OA (p<0.05; table 11).

Localisation of END and ENK

Lining layers

Staining of tissue of patients with JT demonstrated END‐ and ENK‐IR cells scattered within and directly under the lining layer of inflamed, but rare in non‐inflamed, synovial tissue of patients with minor trauma ((figsfigs 1A, 2C2C).). The number of END‐ and ENK‐IR cells was similar, and both cell types were significantly more abundant in patients with OA and RA than in those with JT (table 22).). The morphological appearances (dendritic‐shaped processes) of some END‐ and ENK‐IR cells were consistent with fibroblast‐like (type B) synoviocytes (fig 1A,C,E1A,C,E).). In all diseases, double immunohistochemistry demonstrated that most of the cells containing END or ENK were also positive for CD68 (macrophages; data not shown).

figure ar67066.f1
Figure 1 Localisation of endorphin (END) (A, B) in patients with joint trauma (JT), (C, D) in those with osteoarthritis (OA) and (E, F) in those with rheumatoid arthritis (RA). (A) Most macrophages (M), fibroblast‐like cells (arrows) in ...
figure ar67066.f2
Figure 2 Localisation of enkephalin (ENK; A–D) and μ opioid receptors (μORs; E) in synovium of patients with non‐inflamed (C) rheumatoid arthritis (RA; A,B,E) and osteoarthritis (OA; D). (A) ENK‐immunoreactive ...
Table thumbnail
Table 2 Number of endorphin‐ and enkephalin‐immunoreactive (IR) immune cells (per 384 μm2) and of μ opioid receptor (μOR)‐ and δOR‐IR nerve fibres (per 38.4 mm2) in ...

Sublining layers

In JT, 69.9% (1.7%) of END‐IR and 66.4% (2.7%) of ENK‐IR cells were granulocytes (fig 1B1B).). Consistently, the majority of END‐IR and ENK‐IR cells were positive for CD15 (granulocytes) and only a few cells were positive for CD3 (fig 3A,B3A,B).

figure ar67066.f3
Figure 3 Synovial tissue from patients with joint trauma (JT; A, B), osteoarthritis (OA; C, D) and rheumatoid arthritis (RA; E–H) double immunostained for endorphin (END)/CD15, enkephalin (ENK)/CD15 (A, B), END/CD68 (C, E), ENK/CD68 (D, ...

In contrast, in OA, 94.6% (2.4%) of END‐IR and 90.4% (1.5%) of ENK‐IR cells were mononuclear cells. Similarly, in RA, 91.1% (0.6%) of END‐IR cells and 93.0% (1.2%) of ENK‐IR cells were mononuclear cells ((figsfigs 1D,F, 2A,C2A,C).). Accordingly, a high number of END‐IR and ENK‐IR cells were positive for CD68 (macrophages) and CD3 (T lymphocytes) in OA and RA (fig 3C–H). Some END‐IR and ENK‐IR cells were also positive for CD23 (B lymphocytes) in RA and OA (data not shown). In some samples of synovium from patients with OA or RA, fibroblasts and plasma cells, respectively, were the main population of cells expressing END and ENK (fig 2B,C2B,C).). Overall, END‐ and ENK‐IR immune cells were more abundant in RA than in JT and OA (p<0.05; table 22).). END‐ and ENK‐IR immune cells were absent in non‐inflamed tissue (fig 2C2C).). In both inflamed and non‐inflamed tissue, ENK (but not END) was occasionally detected singly or in bundles of nerve fibres (fig 2A2A).

Preabsorption of END or ENK antibodies with their respective antigens or omission of primary or secondary antibodies completely abolished immunostaining (data not shown). Preincubation of END antiserum with purified ENK or ENK antiserum with purified END had no effect on END or ENK immunoreactivity, respectively, excluding cross reactivity between END or ENK antisera (data not shown). Omission of either the primary or the secondary antibody did not produce the first or second (double) colour, respectively (data not shown).

Localisation of ORs

μOR‐ or δOR‐IR nerve fibres were identified within the sublining tissue of all patients. These consisted partly of rather thick bundles of fibres and partly of thin varicose structures that may correspond to single nerve fibres or endings (fig 4A–F). The density of μOR‐ and δOR‐IR nerve fibres was significantly greater in RA than in OA and JT (fig 4A–C; p<0.05; table 22).). μORs and δORs were also expressed on immune cells (fig 2D2D)) and on fibroblasts infiltrating the lining and sublining layers in all diseases. The number of OR‐IR immune cells was greater in RA (μOR: 5.1 (1.6); δOR: 5.9 (2.0)/384 μm2) than in OA (μOR: 3.2 (1.0); δOR: 3.1 (1.1)/384 μm2) or in JT (μOR: 2.8 (1.1); δOR: 3.0 (1.4)/384 μm2; p<0.05; Kruskal–Wallis test followed by Dunn's test). All μOR‐ and δOR‐IR nerve fibres coexpressed CGRP. Some fibres expressed CGRP alone (fig 4I4I D–F). In contrast, close proximity of OR‐ and TH‐IR fibres, but no colocalisation, was observed in all diesases (fig 4II4II A,B). Preabsorption of OR antibodies with their respective antigens or omission of primary or secondary antibodies completely abolished immunostaining (data not shown).

figure ar67066.f4
Figure 4 (I) Localisation of opioid receptors (μOR; D, E) and δOR (A–C, F) in joint trauma (JT; A), osteoarthritis (OA; B, D) and rheumatoid arthritis (RA; C, E, F). δORs are localised on nerve fibres in JT (A), ...

Discussion

We found the highest amount of synovial lining cells, vascularity and infiltrating immune cells in patients with RA compared with those with JT and OA. END and ENK were expressed predominantly in granulocytes in patients with JT, and in macrophages, lymphocytes and plasma cells in those with OA and RA. Overall, END‐ and ENK‐IR cells were significantly more abundant in patients with RA than in those with JT and OA. μORs and δORs were localised on immune cells and on sensory nerve fibres but not on sympathetic nerve fibres, and were significantly more abundant in patients with RA than in those with JT and OA.

The lining layer of the synovial membrane contains macrophage‐like (type A cells) and fibroblast‐like cells (type B cells).33 Our double staining demonstrated that most of the lining layer cells containing END or ENK were positive for CD68 (macrophages). Relatively few END‐ and ENK‐IR cells had dendritic‐shaped processes, consistent with fibroblast‐like synoviocytes (type B).34 In the sublining layers, we found different patterns of opioid peptide‐containing leucocyte subsets in patients with JT, OA and RA. END and ENK were expressed predominantly in granulocytes in patients with JT, and in macrophages, lymphocytes and plasma cells in those with OA and RA. These results extend previous reports of END and ENK expression in humans14,25 and in dogs35 that did not differentiate the synovial cell types. The pattern of opioid‐expressing cells described here is similar to the known distribution of cell types in patients with JT, OA and RA.36,37,38,39 Occasionally, ENK immunoreactivity was also found in nerve fibres, consistent with previous studies on animals and humans.40,41,42,43

Both END‐ and ENK‐IR cells were significantly more abundant in patients with RA than in those with JT and OA, in parallel with the highest degree of inflammatory parameters in patients with RA. These findings suggest that increases of opioid peptide‐containing cells are correlated with the inflammatory activity in the joint. Consistently, we have shown that ongoing Freund's adjuvant‐induced inflammation stimulates a progressively increasing recruitment of opioid peptide‐containing immune cells to the site of injury in the rat paw.44 In this model, opioid‐containing granulocytes predominate at early stages, whereas opioid‐producing monocytes/macrophages, T lymphocytes and B lymphocytes follow at later stages.8,45,46,47 In addition to their main role as producers of antibodies and cytokines, Blymphocytes also play a role in the synthesis and release of opioid peptides.8,45,48,49 The recruitment of opioid‐containing leucocytes from the circulation to the site of inflammation is orchestrated by a successive expression of chemokines and adhesion molecules, primarily CXCR2 ligands, L and P selectins, α42 integrins and intercellular adhesion molecule 1.21,50,51,52

We next examined the localisation of ORs. Previous studies have shown such receptors in human,14 canine12 and equine11 synovium based on radioligand‐binding assays and autoradiography. We have now extended these studies by use of specific antibodies against the human μOR and δOR to identify the exact anatomical structures and possible differences in the expression of ORs between the three diseases. Our results show that μORs and δORs are expressed on nerve fibres and on various types of immune cells in all disorders. The latter findings are in agreement with previous studies that detected ORs in human immune and synovial cells.9,10,53,54,55 Such ORs have been reported to modulate various functions such as proliferation, antibody production, chemotaxis, phagocytosis, tumoricidal activity and gene expression in vitro, but their roles in vivo remain to be investigated in more detail.10,56

Since a large number of animal studies have shown that inflammatory pain can be inhibited by activation of ORs on peripheral sensory neurones (reviewed in Stein et al1), we investigated these ORs in more detail. Using immunohistochemical analysis on floating sections, we were able clearly to define the anatomical localisation and appearance of nerve fibres expressing OR in the sublining synovial layers. The number of μOR‐ and δOR‐IR fibres was significantly higher in patients with RA than in those with JT and OA. Again, this suggests a correlation with inflammatory activity in the joint and is in agreement with animal studies that have revealed an upregulation of μORs and δORs in sensory neurones in models of Freund's adjuvant‐induced inflammation,31,57,58,59 and with a higher number of sensory nerve fibres in synovial tissue of patients with RA than in those with OA.28 Double staining for μORs and δORs with CGRP disclosed coexpression in both cases. In contrast, neither μORs nor δORs colocalised with TH. Since CGRP and TH are specific markers of primary afferent C fibres and sympathetic efferent fibres, respectively,30 our findings agree with animal studies suggesting that μORs and δORs are localised on nociceptive C fibres but not on sympathetic fibres.30,60,61,62 However, since there is a loss of sympathetic neurones in synovial tissue in patients with RA, a definitive conclusion could not be drawn from our experiments at this time.28 Together, these findings suggest that neuronal ORs most probably modulate sensory afferent functions rather than sympathetic efferent functions within inflamed joints and provide an anatomical correlate to clinical studies demonstrating that IA opioid agonists (eg, morphine) inhibit postsurgical and chronic arthritic pain.14,15,17,63,64,65,66 Moreover, the greater increase of sensory nerve endings expressing ORs in RA than in JT and OA is consistent with reports that IA morphine produces a more pronounced analgesic effect in patients with RA than in those with OA.18

In summary, our findings extend recent concepts regarding the interaction of ORs and opioid peptides in inflamed tissues. Animal studies have shown that cytokines, hormones and chemokines can stimulate the release of immune cell‐derived opioid peptides, and that the activation of neuronal ORs by these peptides results in inhibition of inflammatory pain.19,21,22,45,58,67,68 Importantly, clinical studies have shown that the IA administration of an OR antagonist exacerbates pain in JT,25 indicating that opioid peptides are not only secreted on artificial stimulation but are tonically released from inflammatory cells and from sensory nerves and, thus, maintain a certain level of intrinsic pain control. We have now provided the anatomical correlates in human tissue by demonstrating that two different opioid peptides are expressed in granulocytes, monocytes/macrophages and lymphocytes in synovium and that the corresponding ORs are expressed on sensory neurones and immune cells. Both peptides were significantly more abundant in patients with RA than in those with JT and OA, and μOR‐ and δOR‐expressing sensory nerve endings were increased more in patients with RA than in those with JT and OA. Although the functional role of opioids and neuronal ORs in the control of pain has been established, it remains to be clarified which clinical outcome parameters are modulated by ORs on inflammatory cells.

Acknowledgements

We thank Drs Robert Elde, Stefan Schulz and Volker Höllt for donating μOR and δOR antibodies used in this study. We also thank Professor Wolfgang Ertel and Dr Christoph E Heyde (Department of Trauma and Reconstructive Surgery, Charité Campus Benjamin Franklin, Berlin, Germany) for their help. This study was supported by the Deutsche Forschungsgemeinschaft (KFO 100/1).

Abbreviations

ABC - avidin–biotin–peroxidase complex

ANOVA - analysis of variance

CGRP - calcitonin gene‐related peptide

DAB - 3′,3′‐diaminobenzidine tetrahydrochloride

DPX - distrene, tricresyl phosphate and xylene

END - endorphin

ENK - enkephalin

IA - intra‐articular

IR - immunoreactive

JT - joint trauma, OA, osteoarthritis

OR - opioid receptor

PBS - phosphate buffered saline

RA - rheumatoid arthritis

TH - tyrosine hydroxylase

Footnotes

Competing interests: None declared.

References

1. Stein C, Schäfer M, Machelska H. Attacking pain at its source: new perspectives on opioids [review]. Nat Med 2003. 91003–1008.1008 [PubMed]
2. Oeltjenbruns J, Schäfer M. Peripheral opioid analgesia: clinical applications [review]. Curr Pain Headache Rep 2005. 936–44.44 [PubMed]
3. Coggeshall R E, Zhou S, Carlton S M. Opioid receptors on peripheral sensory axons. Brain Res 1997. 764126–132.132 [PubMed]
4. Brack A, Rittner H L, Machelska H, Shaqura M, Mousa S A, Labuz D. et al Endogenous peripheral antinociception in early inflammation is not limited by the number of opioid‐containing leukocytes but by opioid receptor expression. Pain 2004. 10867–75.75 [PubMed]
5. Barber A, Gottschlich R. Opioid agonists and antagonists: an evaluation of their peripheral actions in inflammation. Med Res Rev 1992. 12525–562.562 [PubMed]
6. Stein C, Millan M J, Shippenberg T S, Peter K, Herz A. Peripheral opioid receptors mediating antinociception in inflammation. Evidence for involvement of mu, delta and kappa receptors. J Pharmacol Exp Ther 1989. 2481269–1275.1275 [PubMed]
7. Stein C, Hassan A H S, Przewlocki R, Gramsch C, Peter K, Herz A. Opioids from immunocytes interact with receptors on sensory nerves to inhibit nociception in inflammation. Proc Natl Acad Sci USA 1990. 875935–5939.5939 [PubMed]
8. Przewlocki R, Hassan A H S, Lason W, Epplen C, Herz A, Stein C. Gene expression and localization of opioid peptides in immune cells of inflamed tissue: functional role in antinociception. Neuroscience 1992. 48491–500.500 [PubMed]
9. Takeba Y, Suzuki N, Kaneko A, Asai T, Sakane T. Endorphin and enkephalin ameliorate excessive synovial cell functions in patients with rheumatoid arthritis. J Rheumatol 2001. 282176–2183.2183 [PubMed]
10. Shen H, Aeschlimann A, Reisch N, Gay R E, Simmen B R, Michel B A. et al Kappa and delta opioid receptors are expressed but down‐regulated in fibroblast‐like synoviocytes of patients with rheumatoid arthritis and osteoarthritis. Arthritis Rheum 2005. 521402–1410.1410 [PubMed]
11. Sheehy J G, Hellyer P W, Sammonds G E, Mama K R, Powers B E, Hendrickson D A. et al Evaluation of opioid receptors in synovial membranes of horses. Am J Vet Res 2001. 621408–1412.1412 [PubMed]
12. Keates H L, Cramond T, Smith M T. Intraarticular and periarticular opioid binding in inflamed tissue in experimental canine arthritis. Anesth Analg 1999. 89409–415.415 [PubMed]
13. Stein C, Comisel K, Haimerl E, Yassouridis A, Lehrberger K, Herz A. et al Analgesic effect of intraarticular morphine after arthroscopic knee surgery. N Engl J Med 1991. 3251123–1126.1126 [PubMed]
14. Stein C, Pflüger M, Yassouridis A, Hoelzl J, Lehrberger K, Welte C. et al No tolerance to peripheral morphine analgesia in presence of opioid expression in inflamed synovia. J Clin Invest 1996. 98793–799.799 [PMC free article] [PubMed]
15. Kalso E, Smith L, McQuay H J, Moore R A. No pain, no gain: clinical excellence and scientific rigour—lessons learned from IA morphine. Pain 2002. 98269–275.275 [PubMed]
16. Likar R, Schafer M, Paulak F, Sittl R, Pipam W, Schalk H. et al Intraarticular morphine analgesia in chronic pain patients with osteoarthritis. Anesth Analg 1997. 841313–1317.1317 [PubMed]
17. Stein A, Yassouridis A, Szopko C, Helmke K, Stein C. Intraarticular morphine versus dexamethasone in chronic arthritis. Pain 1999. 83525–532.532 [PubMed]
18. Tanaka N, Sakahashi H, Sato E, Hirose K, Ishii S. The efficacy of intra‐articular analgesia after total knee arthroplasty in patients with rheumatoid arthritis and in patients with osteoarthritis. J Arthroplasty 2001. 16306–311.311 [PubMed]
19. Mousa S A, Shakibaei M, Sitte N, Schäfer M, Stein C. Subcellular pathways of beta‐endorphin synthesis, processing, and release from immunocytes in inflammatory pain. Endocrinology 2004. 1451331–1341.1341 [PubMed]
20. Schäfer M, Carter L, Stein C. Interleukin 1 beta and corticotropin‐releasing factor inhibit pain by releasing opioids from immune cells in inflamed tissue. Proc Natl Acad Sci USA 1994. 914219–4223.4223 [PubMed]
21. Machelska H, Cabot P J, Mousa S A, Zhang Q, Stein C. Pain control in inflammation governed by selectins. Nat Med 1998. 41425–1428.1428 [PubMed]
22. Rittner H L, Mousa S A, Labuz D, Beschmann K, Schafer M, Stein C. et al Selective local PMN recruitment by CXCL1 or CXCL2/3 injection does not cause inflammatory pain. J Leukoc Biol 2006. 791022–1032.1032 [PubMed]
23. Suzuki N, Yoshino S, Nakamura H. A study of opioid peptides in synovial fluid and synovial tissue in patients with rheumatoid arthritis. Arerugi 1992. 41615–620.620 [PubMed]
24. Shiga H, Yoshino S, Nakamura H, Koiwa M. Role of opioid peptide in rheumatoid arthritis—detection of methionine‐enkephalin and leucine‐enkephalin in synovial tissue. Arerugi 1993. 42243–249.249 [PubMed]
25. Stein C, Hassan A H S, Lehrberger K, Giefing J, Yassouridis A. Local analgesic effect of endogenous opioid peptides. Lancet 1993. 342321–324.324 [PubMed]
26. Arnett F C, Edworthy S M, Bloch D A, McShane D J, Fries J F, Cooper N S. et al The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988. 31315–324.324 [PubMed]
27. Altman R D, Asch E, Block D, Bole G, Borenstein D, Brandt K. et al Development of criteria for the classification and reporting of osteoarthritis: classification of osteoarthritis of the knee. Arthritis Rheum 1986. 291039–1049.1049 [PubMed]
28. Miller L E, Justen H P, Scholmerich 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. FASEB J 2000. 142097–2107.2107 [PubMed]
29. Mousa S A, Machelska H, Schäfer M, Stein C. Immunohistochemical localization of endomorphin‐1 and endomorphin‐2 in immune cells and spinal cord in a model of inflammatory pain. J Neuroimmunol 2002. 1265–15.15 [PubMed]
30. Wenk H N, Honda C N. Immunohistochemical localization of delta opioid receptors in peripheral tissues. J Comp Neurol 1999. 408567–579.579 [PubMed]
31. Mousa S A, Zhang Q, Sitte N, Ji R, Stein C. Beta‐endorphin‐containing memory‐cells and mu‐opioid receptors undergo transport to peripheral inflamed tissue. J Neuroimmunol 2001. 11571–78.78 [PubMed]
32. Bresnihan B, Cunnane G, Youssef P, Yanni G, Fitzgerald O, Mulherin D. Microscopic measurement of synovial membrane inflammation in rheumatoid arthritis: proposals for the evaluation of tissue samples by quantitative analysis. Br J Rheumatol 1998. 37636–642.642 [PubMed]
33. Graabaek P M. Characteristics of the two types of synoviocytes in rat synovial membrane. An ultrastructural study. Lab Invest 1984. 50690–702.702 [PubMed]
34. Iwanaga T, Shikichi M, Kitamura H, Yanase H, Nozawa‐Inoue K. Morphology and functional roles of synoviocytes in the joint. Arch Histol Cytol 2000. 6317–31.31 [PubMed]
35. Karahan S, Kincaid S A, Baird A N, Kammermann J R. Distribution of beta‐endorphin and substance P in the shoulder joint of the dog before and after a low impact exercise programme. Anat Histol Embryol 2002. 3172–77.77 [PubMed]
36. Koch A E, Kunkel S L, Shah M R, Hosaka S, Halloran M M, Haines G K. et al Growth‐related gene product alpha. A chemotactic cytokine for neutrophils in rheumatoid arthritis. J Immunol 1995. 1553660–3666.3666 [PubMed]
37. Hansch A, Stiehl P, Geiler G. Quantification of macrophages and granulocytes at the joint cartilage—pannus junction in rheumatoid arthritis. Z Rheumatol 1996. 55401–409.409 [PubMed]
38. Konig A, Krenn V, Gillitzer R, Glockner J, Janssen E, Gohlke F. et al Inflammatory infiltrate and interleukin‐8 expression in the synovium of psoriatic arthritis–an immunohistochemical and mRNA analysis. Rheumatol Int 1997. 17159–168.168 [PubMed]
39. Pallister I, Bhatia R, Katpalli G, Allison D, Parker C, Topley N. Alteration of polymorphonuclear neutrophil surface receptor expression and migratory activity after isolation: comparison of whole blood and isolated PMN preparations from normal and postfracture trauma patients. J Trauma 2006. 60844–850.850 [PubMed]
40. Bergstrom J, Ahmed M, Li J, Ahmad T, Kreicbergs A, Spetea M. Opioid peptides and receptors in joint tissues: study in the rat. J Orthop Res 2006. 241193–1199.1199 [PubMed]
41. Weihe E, Nohr D, Millan M J, Stein C, Muller S, Gramsch C. et al Peptide neuroanatomy of adjuvant‐induced arthritic inflammation in rat. Agents Actions 1988. 25255–259.259 [PubMed]
42. Gronblad M, Liesi P, Korkala O, Karaharju E, Polak J M. Innervation of human bone periosteum by peptidergic nerves. Anat Rec 1984. 209297–299.299 [PubMed]
43. Gronblad M, Korkala O, Liesi P, Karaharju E. Innervation of synovial membrane and meniscus. Acta Orthop Scand 1985. 56484–486.486 [PubMed]
44. Rittner H L, Brack A, Machelska H, Mousa S A, Bauer M, Schäfer M. et al Opioid peptide‐expressing leukocytes: identification, recruitment, and simultaneously increasing inhibition of inflammatory pain. Anesthesiology 2001. 95500–508.508 [PubMed]
45. Cabot P J, Carter L, Gaiddon C, Zhang Q, Schäfer M, Loeffler J P. et al Immune cell‐derived β‐endorphin: production, release and control of inflammatory pain in rats.J Clin Invest 1997. 100142–148.148 [PMC free article] [PubMed]
46. Brack A, Labuz D, Schiltz A, Rittner H L, Machelska H, Schafer M. et al Tissue monocytes/macrophages in inflammation: hyperalgesia versus opioid‐mediated peripheral antinociception. Anesthesiology 2004. 101204–211.211 [PubMed]
47. Brack A, Rittner H L, Machelska H, Shaqura M, Mousa S A, Labuz D. et al Endogenous peripheral antinociception in early inflammation is not limited by the number of opioid‐containing leukocytes but by opioid receptor expression. Pain 2004. 10867–75.75 [PubMed]
48. Weyand C M, Seyler T M, Goronzy J J. B cells in rheumatoid synovitis [review]. Arthritis Res Ther 2005. 7S9–12.12 [PMC free article] [PubMed]
49. Kavelaars A, Ballieux R E, Heijnen C J. The role of IL‐1 in the corticotropin‐releasing factor and arginine‐vasopressin‐induced secretion of immunoreactive beta‐endorphin by human peripheral blood mononuclear cells. J Immunol 1989. 1422338–2342.2342 [PubMed]
50. Brack A, Rittner H L, Machelska H, Leder K, Mousa S A, Schäfer M. et al Control of inflammatory pain by chemokine‐mediated recruitment of opioid‐containing polymorphonuclear cells. Pain 2004. 112229–238.238 [PubMed]
51. Machelska H, Mousa S A, Brack A, Schopohl J K, Rittner H L, Schafer M. et al Opioid control of inflammatory pain regulated by intercellular adhesion molecule‐1. J Neurosci 2002. 225588–5596.5596 [PubMed]
52. Machelska H, Brack A, Mousa S A, Schopohl J K, Rittner H L, Schafer M. et al Selectins and integrins but not platelet‐endothelial cell adhesion molecule‐1 regulate opioid inhibition of inflammatory pain. Br J Pharmacol 2004. 142772–780.780 [PMC free article] [PubMed]
53. Sharp B M, McAllen K, Gekker G, Shahabi N A, Peterson P K. Immunofluorescence detection of delta opioid receptors (DOR) on human peripheral blood CD4+ T cells and DOR‐dependent suppression of HIV‐1 expression. J Immunol 2001. 1671097–1102.1102 [PubMed]
54. Tomassini N, Renaud F L, Roy S, Loh H H. Mu and delta receptors mediate morphine effects on phagocytosis by murine peritoneal macrophages. J Neuroimmunol 2003. 1369–16.16 [PubMed]
55. Karaji A G, Khansari N, Ansary B, Dehpour A. Detection of opioid receptors on murine lymphocytes by indirect immunofluorescence: mature normal and tumor bearing mice lymphocytes. Int Immunopharmacol 2005. 51019–1027.1027 [PubMed]
56. Tegeder I, Geisslinger G. Opioids as modulators of cell death and survival—unraveling mechanisms and revealing new indications. Pharmacol Rev 2004. 56351–369.369 [PubMed]
57. Calza L, Pozza M, Arletti R, Manzini E, Hokfelt T. Long‐lasting regulation of galanin, opioid, and other peptides in dorsal root ganglia and spinal cord during experimental polyarthritis. Exp Neurol 2000. 164333–343.343 [PubMed]
58. Ballet S, Conrath M, Fischer J, Kaneko T, Hamon M, Cesselin F. Expression and G‐protein coupling of mu‐opioid receptors in the spinal cord and dorsal root ganglia of polyarthritic rats. Neuropeptides 2003. 37211–219.219 [PubMed]
59. Zöllner C, Shaqura M A, Bopaiah C P, Mousa S, Stein C, Schäfer M. Painful inflammation‐induced increase in mu‐opioid receptor binding and G‐protein coupling in primary afferent neurons. Mol Pharmacol 2003. 64202–210.210 [PubMed]
60. Bartho L, Stein C, Herz A. Involvement of capsaicin‐sensitive neurones in hyperalgesia and enhanced opioid antinociception in inflammation. Naunyn Schmiedebergs Arch Pharmacol 1990. 342666–670.670 [PubMed]
61. Zhang Q, Schäfer M, Elde R, Stein C. Effects of neurotoxins and hindpaw inflammation on opioid receptor immunoreactivities in dorsal root ganglia. Neuroscience 1998. 85281–291.291 [PubMed]
62. Zhou L, Zhang Q, Stein C, Schäfer M. Contribution of opioid receptors on primary afferent versus sympathetic neurons to peripheral opioid analgesia. J Pharmacol Exp Ther 1998. 2861–7.7 [PubMed]
63. Joshi G P, McCarroll S M, Brady O H, Hurson B J, Walsh G. Intra‐articular morphine for pain relief after anterior cruciate ligament repair. Br J Anaesth 1993. 7087–88.88 [PubMed]
64. McSwiney M M, Joshi G P, Kenny P, McCarroll S M. Analgesia following arthroscopic knee surgery. A controlled study of intra‐articular morphine, bupivacaine or both combined. Anaesth Intens Care 1993. 21201–203.203
65. Sammarco J L, Conzemius M G, Perkowski S Z, Weinstein M J, Gregor T P, Smith G K. Postoperative analgesia for stifle surgery: a comparison of intra‐articular bupivacaine, morphine, or saline. Vet Surg 1996. 2559–69.69 [PubMed]
66. Le Loet X, Pavelka K, Richarz U. Transdermal fentanyl for the treatment of pain caused by osteoarthritis of the knee or hip: an open, multicentre study. BMC Musculoskelet Disord 2005. 631
67. Cabot P J, Carter L, Schäfer M, Stein C. Methionine‐enkephalin‐ and dynorphin A‐release from immune cells and control of inflammatory pain. Pain 2001. 93207–211.211 [PubMed]
68. Labuz D, Berger S, Mousa S A, Zöllner C, Rittner H L, Shaqura M A. et al Peripheral antinociceptive effects of exogenous and immune cell‐derived endomorphins in prolonged inflammatory pain. J Neurosci 2006. 264350–4358.4358 [PubMed]

Articles from Annals of the Rheumatic Diseases are provided here courtesy of BMJ Group