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EXTENDED REPORT Ann Rheum Dis: first published as 10.1136/ard.61.6.492 on 1 June 2002. Downloaded from Identification of the advanced glycation end products Ne-carboxymethyllysine in the synovial tissue of patients with rheumatoid S Drinda, S Franke, C C Canet, P Petrow, R Bräuer, C Hüttich, G Stein, G Hein ......

Ann Rheum Dis 2002;61:488–492

Background: Generation of advanced glycation end products (AGEs) is an inevitable process in vivo and can be accelerated under pathological conditions such as oxidative stress. In serum and synovial fluid of patients with (RA) raised AGE levels have been found. Objective: To determine the presence of Nå-carboxymethyllysine (CML; marker of oxidative stress) in See end of article for RA synovial tissue by immunohistology. authors’ affiliations Methods: Frozen synovial tissue samples from 10 patients with RA and eight controls (four patients ...... without joint disease and four patients with osteoarthritis (OA)) were treated with rabbit-anti-CML-IgG Correspondence: to: Dr S and goat-antirabbit-IgG. Immunostaining was visualised by streptavidine-alkaline phosphatase Drinda, Friedrich Schiller (chromogen fuchsin). Cell differentiation was performed with against CD68, CD45RO, and Universität Jena, Klinik für CD20. Innere Medizin IV, Results: CML was detected in the synovial lining, sublining, and endothelium in 10/10 RA and 4/4 Rheumatologie/Osteologie, OA synovial specimens. In RA some (CD68+) and T cells (CD45RO+) showed positive Erlanger Allee 101, 07740 Jena, Germany; immunostaining for CML, whereas B cells were negative. Staining in OA synovial sublining was weak stefan.drinda@ compared with RA. med.uni-jena.de Conclusions: CML was detected for the first time in RA and OA synovial tissue. Different patterns of Accepted immunostaining in RA and OA and the presence of CML on macrophages and T cells, suggest a role 7 December 2001 for CML in the pathogenesis of RA. This might be due to presentation of new epitopes which can main- ...... tain or even trigger an autoimmune response.

dvanced glycation end products (AGEs) are formed dur- driven process in this disease, but a specific antigen has not yet ing the Maillard reaction by non-enzymatic glycation been found. Aand oxidation of proteins constituting a heterogeneous To obtain more detailed information on the accumulation of http://ard.bmj.com/ class of structures. The generation of AGEs is an inevitable AGE modified proteins in RA, this study aimed at investigat- process in vivo and their accumulation in different tissues has ing whether CML modifications are present in the synovial been implicated in the process of aging and also in the patho- tissues of patients with RA. genesis of several pathological conditions, including , , Alzheimer’s disease, renal failure, and renal MATERIALS AND METHODS replacement treatment with maintenance haemodialysis.1–5 Synovial tissue samples obtained by therapeutic synovectomia AGE modification can lead to tissue damage through from 10 patients with RA (mean age 57 years, range 33–83) on October 1, 2021 by guest. Protected copyright. alterations of tissue protein structure and function, and fulfilling the 1987 revised ACR criteria14 were examined in this stimulates cellular responses mediated by a specific receptor study. Synovial specimens obtained at post mortem from four (RAGE). patients who had no history of joint disease (mean age 52 Not only glucose but also reactive carbonyl intermediates years, range 21–86) and from four patients (mean age 74 derived from the Maillard reaction (3-deoxyglucosone), as years, range 66–80) with osteoarthritis (OA) served as well as the products of sugar oxidation (arabinose, glyoxal, controls. The OA samples were obtained during the surgical methylglyoxal) and lipid peroxidation (malondialdehyde) are implantation of a knee endoprosthesis. All patients were non- precursors causing chemical modification of proteins. Oxida- diabetic. Table 1 shows the patient characteristics. tive stress accelerates the formation of these products.67 The Sample preparation well characterised AGE Ne-carboxymethyllysine (CML) repre- Frozen synovial tissues (2 µm sections) were mounted on sents a chemically modified amino acid and originates in vivo polylysine coated slides (Menzel, Germany), fixed in acetone, from carbohydrate as well as from lipid derived precursors. It and blocked in 4% skimmed milk for 20 minutes. Slides were has been suggested that CML is a general marker of oxidative then incubated with rabbit-anti-CML-IgG (concentration 4 stress and tissue damage through protein alteration.8 Oxida- g/l, dilution 1:10 000; kindly provided by Roche Diagnostics, tive stress plays an important part in acute and chronic Germany) for one hour. Biotinylated goat-antirabbit-IgG con- inflammatory diseases, including rheumatoid arthritis (RA).9 jugated with streptavidine/alkaline phosphatase was used for Recently reported studies have provided evidence that the AGE pentosidine is raised in the articular cartilage as well as in the serum and synovial fluid of patients with RA.10–12 ...... Furthermore, the raised autoantibody level against CML in Abbreviations: AGEs, advanced glycation end products; CML, patients with diabetic renal failure indicates that CML Ne-carboxymethyllysine; IL, interleukin; OA, osteoarthritis; RA, rheumatoid 13 accumulated in vivo serves as an immunological epitope. On arthritis; RAGE, receptor for AGEs; TNFα, tumour necrosis factor α; TRX, the other hand, many findings in RA indicate an antigen thioredoxin

www.annrheumdis.com Carboxymethyllysine (CML-AGE) in RA synovial tissue 489

Table 1 Clinical data on investigated patients Ann Rheum Dis: first published as 10.1136/ard.61.6.492 on 1 June 2002. Downloaded from Duration/ exacerbation of Radiological WBC BSG Patient Diagnosis disease (years) Age (years) Sex signs* Treatment† (×109/l) CRP (mg/l) (mm)

1RA3064FL41,2,3105238 2 RA 5 54 F L5 2 10 55.9 68 3 RA 1 75 F L4 4 10.2 21.5 19 4 RA 10 52 F L5 1, 2 5.6 34.7 52 5 RA 10 44 F L4 2 9.8 41.4 38 6 RA 2 63 F NA 1,2,3,5 6.8 <5 12 7 RA 2 37 F L3 1, 2, 3, 6 12.1 63.2 27 8 RA 2 62 F L4 1, 2, 3 10.1 7.7 18 9 RA 1.5 83 F L4 No 8.4 46.8 50 10 RA 1.5 33 M NA 2 8.7 7,. 24 11 OA 0.17 (2 m) 77 F K4 2 4.9 <5 10 12 OA 0.75 66 F K4 2 7.7 8,5 13 13 OA 0.5 74 F K4 No 5.2 <5 17 14 OA 1 80 F K4 2 4.9 <5 24 15 Liver cirrhosis NA 28 M – – – – – 16 Cardiac failure NA 74 F – – – – – 17 Gastric sarcoma NA 21 F – – – – – 18 Peritonitis NA 86 M – – – – –

*Radiological signs: grading after Larsen (L) and Kellgren (K)28; †treatment: 1, MTX; 2, NSAID; 3, cortisone; 4, etanercept; 5, azathioprine; 6, leflunomide. NA, not applicable; F, female; M, male. detection (concentration 1 mg/ml, dilution 1:400, incubation DAKO Denmark), and CD45RO (activated T lymphocytes; time 40 minutes; DAKO Denmark). Immunostaining was DAKO Denmark). Tissue samples were examined in two visualised using the streptavidine/alkaline phosphatase tech- staining steps: in the first step slides were incubated with nique with the chromogen fuchsin. Negative controls included rabbit-anti-CML-IgG (see above), staining was visualised by (a) omission of the primary ; (b) treatment of the alkaline phosphatase anti-alkaline phosphatase technique sections with rabbit immunoglobulin of irrelevant specificities (chromogen neufuchsin). In the second staining step, slides at the same concentration as the primary antibody; (c) were treated with antibodies against CD20 and CD68 by non-arthritic synovial tissue (table 1). applying the peroxidase technique (chromogen diaminoben- For cell differentiation we used antibodies against CD68 zidine) or CD45RO using the streptavidine-biotin- (macrophages; DAKO Denmark), CD20 (B lymphocytes; immunogold-silver technique. For monostaining the semiquantitative measurement was assigned to the following ranges: no positive cells, <5% positive http://ard.bmj.com/ on October 1, 2021 by guest. Protected copyright.

Figure 1 RA synovial tissue showing CML-expression (red) in the lining layer, the subsynovial interstitium, and in endothelial cells Figure 2 RA synovial tissue with , which shows double (×20). staining: CML (red) and CD68+ (brown) (×40).

www.annrheumdis.com 490 Drinda, Franke, Canet, et al

Table 2 Immunohistological staining of structures in synovial tissue Ann Rheum Dis: first published as 10.1136/ard.61.6.492 on 1 June 2002. Downloaded from

Immunohistology

Staining in Staining in Patient Staining in synovial vascular No/disease synovial lining sublining endothelium

1/RA NA +++ ++ 2/RA +++ +++ +++ 3/RA ++ ++ ++ 4/RA +++ ++ +++ 5/RA ++ +/− +++ 6/RA +++ +++ +++ 7/RA +++ ++ +++ 8/RA NA + +/− 9/RA NA +/− ++ 10/RA ++ +++ +++ 11/OA ++ +/− +++ 12/OA ++ +/− +++ 13/OA ++ +/− ++ 14/OA +++ + + 15/Control NA +/− +/− 16/Control −− + 17/Control −− ++ 18/Control −− +

Semiquantitative; −, no positive cells; +/−, <5% positive cells; ++, 30–60% positive cells; +++, >60% positive cells; NA not applicable.

Figure 3 RA synovial tissue with activated T cells, which shows double staining: CML (red) and CD45RO+ (black) (×40). http://ard.bmj.com/ on October 1, 2021 by guest. Protected copyright.

Figure 5 OA synovial tissue showing CML expression (red) in the lining layer, the subsynovial interstitium, and in endothelial cells (×20). Overall, fewer cells are present compared with RA tissue.

numerous sublining and stroma cells as well as in the Figure 4 RA synovial tissue with follicle of B cells (brown staining). endothelium of synovial vessels (fig 1). Some macrophages CML positive cells are organised outside the follicle (×20). (CD68+) and activated T cells (CD45RO+) showed positive immunostaining for CML (figs 2 and 3), whereas B cells were cells, 30–60% positive cells, >60% positive cells, and not negative (fig 4). About 10% of CML positive cells were found in applicable. For double staining no measurement was made. macrophages and about 30% in activated T cells. In contrast, in control tissues from patients without joint disease no signal RESULTS was detected in synovial lining and sublining, and only weak CML was detected in 10/10 RA synovial specimens. CML- to moderate immunostaining was seen in vascular endothe- specific staining was localised in the synovial lining and in lium cells (table 2). No staining was seen in tissue sections

www.annrheumdis.com Carboxymethyllysine (CML-AGE) in RA synovial tissue 491 after omission of the primary antibody or treatment with rab- and synovial tissue of patients with RA in comparison with 29

bit immunoglobulin, demonstrating the specificity of the anti- levels in patients with other joint diseases. Ann Rheum Dis: first published as 10.1136/ard.61.6.492 on 1 June 2002. Downloaded from CML-antibody used. Synovial tissues from patients with OA In contrast, no immunostaining was found in the synovial showed, in the synovial lining and vascular endothelium, a lining or sublining of the control patients without a history of staining pattern and intensity comparable with that of RA joint disease. samples, but the number of lymphocytes and macrophages Detection of CML in control tissue samples from patients was lower than in RA tissue. Staining in OA synovial sublining with OA indicates that the presence of AGEs in higher was weak compared with RA synovial tissue (fig 5). concentration is not a specific phenomenon of RA and is not restricted to those diseases with autoimmune pathways. OA tissue samples showed in synovial lining and vascular DISCUSSION endothelium a staining pattern and intensity comparable with Besides the physiological occurrence of AGEs, these changed that of RA samples, but staining in the synovial sublining was protein structures seem to play an important part in different weak. This might be due to the lower number of lymphocytes diseases.1–5 As far as we know, this study is the first to show and macrophages followed by a lower presence of AGE CML that CML accumulates in RA synovial tissue. CML was than in the RA tissue. To understand the role of AGEs in auto- detected in all samples of synovial tissue from patients with immune pathways further studies should examine whether RA. the presence of AGEs is followed by an invasion of immune CML is an AGE which can be used as a marker of oxidative competent cells, like macrophages or lymphocytes, or if AGEs stress.67 In RA, inflammatory changes and destruction of are only a “by product” in the autoimmune response. joints are seen.9 The accumulation of CML in RA synovial tis- sue might be the result of oxidative stress during local and ACKNOWLEDGEMENTS systemic inflammation. The generation of AGEs is an inevita- We thank the German Ministry for Education and Research (grant ble process in vivo; yet, on the other hand, effects of FKZ 01 ZZ 9602) and Roche Diagnostics for their generosity in inflammation, such as oxidative stress, can cause different providing the rabbit-anti-CML-antibody. “metabolic changes”, leading to the mutation of key regulatory . This may help to transform inflammation ...... into chronic disease.15 In RA, a disease with a probable autoimmune pathogenesis, the triggering factors of the Authors’ affiliations S Drinda, S Franke, G Stein, G Hein, Department of Internal Medicine immunological process are still unknown and up to now a IV, Friedrich Schiller University Jena, Germany specific antigen is still missing. Yang et al showed that in older C C Canet, P Petrow, R Bräuer, C Hüttich, Institute of Pathology, patients AGEs represent new epitopes and contain new Friedrich Schiller University Jena, Germany antigenic structures,16 thereby possibly contributing to the generation of autoimmune responses.17 This theory is sup- REFERENCES ported by the findings of Miyata et al and Takahashi et al,who 1 Vlassara H. 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Arthritis Rheum 1994;37:724–8. 12 Chen JR, Takahashi M, Suzuki M, Kushida S, Miyamoto S, Inoue T. process by changing the antigen structure of different tissue Comparison of the concentrations of pentosidine in the synovial fluid, compartments, or by activating the secretion of inflammation serum and urine of patients with rheumatoid arthritis and osteoarthritis. mediators. The fact that a possibly consequence of inflamma- Br J Rheumatol 1999;38:1275–8. 13 Shibayama R, Araki N, Nagai R, Horiuchi S. Autoantibody against tion can maintain the inflammation itself can also be seen in N-carboxymethyllysine: an advanced glycation end product of the the recent findings on the relation between thioredoxin (TRX) Maillard reaction. Diabetes 1999;48:1842–9. and oxidative stress. TRX is a cellular reducing catalyst 14 Arnett FC, Edworthy SM, Bloch DA, Mc Shane DJ, Fries JF, Cooper NS, induced by oxidative stress. The effect of TRX on TNFα- et al. 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COVER STORY......

Colchicum autumnale from Het Cruydtboeck by Rembertus Dodonaeus

he cover illustration (fig 1) shows a remained a standard not only as a botanical woodcut representing colchicum autum- work but also as a pharmacopoeia, and was Tnale from the famous Cruydtboeck improved and reprinted several times. (Herbal) by Dodonaeus. Nowadays, Rember- Colchicum autumnale (meadow saffron) tus Dodonaeus is considered to be the was already known to the ancient Greeks for founder of modern botany. its beneficial effect on podagra, but also for its He was born in 1517 in Mechelen, at that toxicity. The active components, the alka- time an important Flemish town and judicial loids, are extracted from the seed as well as capital of the Netherlands. After graduating in from the tuber. Strikingly, the plant flowers in medicine at the University of Louvain at the autumn, hence the specification “autum- age of 18, Dodonaeus returned to his native nale” in its name. Colchicum is said to refer to town to work there as a general physician. He the Greek region Colchis, near the Black Sea http://ard.bmj.com/ had a special interest in botany and began from where the flower originates. Some even describing in detail the external appearance, speculate that the sorceress Medea, daughter inflorescence, flowering time, and medical of the King of Colchis, used colchicum properties of herbs and plants. This meticu- autumnale as a poison to kill her children as lous work resulted in 1554 in the publication a revenge for the adultery of her husband of the famous Flemish herbal Het Cruydtboeck. Jason. The book contains descriptions of more than 1000 herbs and plants and is illustrated with Acknowledgement 715 woodcuts based on drawings by Peter van We thank Mr Bekkers and Mr van Druenen from the on October 1, 2021 by guest. Protected copyright. der Borcht. Later, the book was translated into “Maaslands Antiquariaat” in the Stokstraat, Maas- Latin in order to reach a larger public. tricht, The Netherlands, who kindly allowed us to take the photographs from their version of Het The work of Dodonaeus was an inspiration Cruydtboek. for two other famous physician-herbalists in the early 16th century, Clusius (1526–1609) A Boonen and Lobelius (1538–1616), who expanded Department of Rheumatology, University Hospital and improved his work. From 1574 until 1577 Maastricht, The Netherlands Dodonaeus was the personal physician of L van de Putte Emperor Maximilian at the imperial court of Vienna. In 1582 he was nominated professor Department of Rheumatology, University Medical Centre Nijmegen, The Netherlands of medicine at the Leiden University in Holland where he died in 1585. Until Figure 1 Colchicum autumnale. Correspondence to: Dr A Boonen, University Linneaus (1707–1778) introduced the new Hospital Maastricht, PO Box 5800, 6202 AZ classification of plants in 1735, Het Cruydtboeck Maastricht, The Netherlands; [email protected]

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