What determines arthritogenicity of bacterial cell wall? A study on Eubacterium cell wall-induced arthritis
X. Zhang,
M. Rimpiläinen,
E.
imelyte and
P. Toivanen
Turku Immunology Centre, Department of Medical Microbiology, Turku University, Turku, Finland
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Abstract
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Objective. To study what determines the arthritogenicity of the bacterial cell wall (CW) using Eubacterium CW-induced arthritis in the rat.
Methods. Eubacterium aerofaciens, previously reported as arthritogenic, and E. limosum and E. alactolyticum, known as non-arthritogenic, were used. Gas chromatographymass spectrometry (GCMS) was applied to analyse the chemical composition of the bacterial cell wall. Cellular immune response was measured by concanavalin A (Con A) stimulation and FACScan analysis. Also, serum antibodies against the injected cell wall were determined.
Results. Unexpectedly, from the two strains of E. aerofaciens used only one proved to be arthritogenic (with a CW inducing chronic arthritis after a single intraperitoneal injection), even though these two strains were 100% identical by 16S rDNA analysis. CW of the other E. aerofaciens strain induced only transient acute arthritis; CW of E. limosum and E. alactolyticum induced weak signs of acute arthritis. Based on the GCMS analysis and on the results published previously, putative structures of peptidoglycan (PG) in the four CW preparations are presented. It is apparent that the presence of lysine in position 3 of the PG stem peptide contributes to arthritogenicity but is alone not decisive. Both strains of E. aerofaciens were immunosuppressive, when tested by Con A response at 2 weeks after CW injection. Such an immunosuppression was not observed after injection of CW from E. limosum or E. alactolyticum. FACScan analysis for six T cell markers and studies on serum antibody responses did not reveal any differences in the effect of the four bacterial strains used.
Conclusions. The results obtained suggest that the chemical structure of PG present in the bacterial CW is decisive in determining arthritogenicity/non-arthritogenicity. Therefore, from two bacterial strains belonging to normal human intestinal flora and 100% identical by 16S rDNA analysis, one proved to be arthritogenic and the other non-arthritogenic.
KEY WORDS: Intestinal flora, Peptidoglycan, Gas chromatography, Mass spectrometry, Lysine.
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Introduction
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From the animal models of rheumatoid arthritis, those based on the use of a single i.p. injection of Gram-positive cell walls (CW) into rats, are among the most popular. The bacteria tried in these experiments include Streptococcus, Lactobacillus, Bifidobacterium, Clostridium, Peptostreptococcus, Coprococcus, Propionibacterium, and Eubacterium [15]. In Eubacterium CW-induced arthritis, histopathological findings of the joint inflammation closely resemble those observed in human rheumatoid arthritis, including synovial infiltration by T lymphocytes, synovial lining cell hyperplasia, pannus formation, tendonitis, and cartilage and bone erosions [4, 6]. Likewise, the presence of T helper cells and macrophages in the synovial tissue has been reported [7], and even autoreactive T cell lines which can behave as arthritogenic have been described [8].
A major constituent of the Gram-positive CW is the peptidoglycanpolysaccharide (PGPS) complex, which is known to initiate and sustain a chronic destructive inflammation. This capacity is dependent upon resistance of PGPS to biodegradation and on the consequent persistence in the tissues [4, 912]. The PG moiety of the PGPS complex possesses multiple immunological activities [13, 14]. The inflammatory properties of PG are decreased after degradation by lysozyme, N-acetylmuramyl-L-alanine amidase [15, 16], or mutanolysin [17], implying that PG has an important role in the pathogenesis of the chronic inflammation. The PS moiety, covalently bound to the PG, seems also to possess relevant biological activity; streptococcal PGPS could not induce chronic arthritis if the complex was separated into PG and PS [18, 19], indicating that the inflammatory PG might be easily degraded in the absence of PS.
The composition of PG varies from one bacterial species to another and even between strains within a single species. PG consists of several layers (up to 70) of adjacent N-acetylglucosamine and N-acetylmuramic acid molecules; the layers are bound by peptides to each other in a regular fashion. Peptides comprising four to five amino acids (called stem peptides) are attached to N-acetylmuramic acid to form the glycan backbone of PG. Backbones are covalently bound to each other by interpeptide bridges which may or may not contain one or more additional amino acid [20]. The structure of PG is schematically depicted in Fig. 1
. The greatest variation of amino acids in the stem peptide occurs in position 3. Variation also occurs in the mode of cross-linkage and in the interpeptide bridge. According to the mode of cross-linkage, PG is divided into group A (cross-linkage between positions 3 and 4 of the stem peptide) and the less frequent group B (cross-linkage between positions 2 and 4). Further division into subgroups is made according to variation of the interpeptide bridge. In group A, the amino acid linked to muramic acid is always L-alanine, followed by D-glutamic acid in position 2, to form the minimal immunoadjuvant structure muramyl dipeptide (MDP). In group B, the amino acid linked to muramic acid is usually glycine or serine, followed by D-glutamic acid in position 2.

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FIG. 1. Schematic picture of PG. The figures indicate the position of amino acids in the stem peptide. M, N-acetylmuramic acid; N, N-acetylglucosamine; Ala, alanine; Glu, glutamic acid; Orn, ornithine; Dap, diaminopimelic acid; Ser, serine; Lys, lysine.
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Eubacterium CW-induced arthritis is of special interest, since E. aerofaciens of human intestine was among the top 10 bacterial species (out of 54) which distinguished patients with early rheumatoid arthritis from controls [21]. In addition, it is the only one of those 10 in which the CW has been used for arthritis induction. However, CWs of all Eubacterium species are not arthritogenic. For instance, E. limosum CW is not arthritogenic [22]. Even though resistance to biodegradation and persistence in the tissues seem to be important for arthritogenicity, it has remained unknown what characteristics of the CW itself determine these properties, as well as the capacity for arthritis induction. Burroughs et al. [23], working with Haemophilus influenzae, demonstrated a structureactivity relationship for the inflammatory properties of PG degradation products. According to their findings, the pro-inflammatory activity is highly dependent on structural variation, both in the stem peptide and the cross-linkage. Therefore, it is possible that, within Eubacterium PG, variation of the stem peptides and cross-linkage leads to different responses by the host immune system. In the present work, we aimed to study these questions by using the CW of four Eubacterium strains. For this purpose, we chose a strain of E. aerofaciens proven to be arthritogenic (causing chronic arthritis) and a strain of E. limosum known as non-arthritogenic [22]. Both of these species belong to the normal intestinal flora in man. Additionally, we used another strain of E. aerofaciens and a strain of E. alactolyticum, presuming they were arthritogenic and non-arthritogenic, respectively; E. alactolyticum is found in human dental plaque.
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Materials and methods
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Bacteria
Eubacterium aerofaciens ATCC 25986 was obtained from the Culture Collection, University of Gothenburg, Sweden, with a nomination CCUG 28087. Eubacterium aerofaciens ATCC 35085, E. limosum ATCC 8486 and E. alactolyticum ATCC 17927 (now recognized as Pseudoramibacter alactolyticus [24]) were purchased from the American Type Culture Collection, Rockville, MD, USA. All strains were grown overnight under strictly anaerobic conditions at 37°C in BBL Schaedler Broth (Becton Dickinson, MD, USA) to the late logarithmic phase. The strains were characterized by 16S rDNA sequence analysis as described by Jalava et al. [25], by biochemical reactions [26] and by gas chromatographic (GC) analysis of cellular fatty acids (CFA) as described by Eerola and Lehtonen [21, 27]. CW of E. aerofaciens ATCC 25986 and E. limosum ATCC 8486 have been proven to be arthritogenic and non-arthritogenic, respectively [22]. Arthritis induction by the other two strains has not been previously studied.
CW preparation
The bacterial CW was isolated as described previously [18]. Briefly, the cells were harvested by centrifugation, washed twice with phosphate-buffered saline (PBS) at pH 7.2, heated at 80°C for 30 min to inactivate autolytic enzymes [5], and disrupted with glass beads (Ø 0.450.50 mm) in a MSK Cell Homogenizer (B. Braun Melsungen AG, Melsungen, Germany). The CWs were collected by centrifugation (Sorvall RC5C, Du Pont, Wilmington, USA) at 10 000 g, +4°C, for 30 min, treated with deoxyribonuclease I (1 mg/g wet weight), ribonuclease (10 mg/g wet weight) (both enzymes from Sigma Chemical Co., MO, USA) and trypsin (20 mg/g wet weight; Fluka Chemika AG, Buchs SG, Switzerland), washed twice with PBS and once with distilled water, and sonicated in an ice bath for 75 min (Branson Sonifier, Smith Kline Co., Danbury, CT, USA). The sonicated CW suspension was centrifuged at 10 000 g, +4°C, for 20 min. The supernatant was centrifuged by ultracentrifugation (Sorvall Ultracentrifuge OTD65B, rotor 60 Ti, Du Pont) at 100 000 g, +4°C, for 60 min. The pellet containing the CW called 100p, for a precipitate obtained by centrifugation at 100 000 g was suspended in water, dialysed (MWCO 3500, Spectrum, CA, USA) against distilled water at +4°C for 4 days by changing the water several times and lyophilized for the chemical analysis. For i.p. injection into rats, the CW preparation was suspended in PBS and heated at 90°C for 30 min [28]. The sterility of the preparation was checked by plating on agar plates at 37°C and room temperature under aerobic and anaerobic conditions; no bacterial growth was detected after 2 days of culture. The endotoxin tests by E-TOXATE (Sigma) were also found to be negative.
GCmass spectrometry (GCMS)
GCMS was used to analyse the chemical composition of the CW preparations [29]. The derivatized molecules were ionized by the electron impact method and analysed in the selected ion monitoring (SIM) mode using single positive ions at a mass-to-charge ratio (m/z). Sugars were analysed as alditol acetate derivatives with fucose, allose, and N-methyl-glucamine (Sigma) as internal standards. The ions monitored were the same as those described by Gilbart et al. [29], the only exception was our use of m/z 289 for rhamnose. Amino acids were analysed as butyl heptafluorobutyl derivatives using L-norleucine, L-methionine and L-tryptophan (Sigma) as internal standards. The ions were selected as described by Gilbart et al. [29]. One microlitre of the derivative was injected in the splitless mode and analysed by GC (model HP 5890A; Hewlett-Packard, Wilmington, DE, USA) equipped with a fused silica capillary column (SE-54; Nordian Instruments, Helsinki, Finland) and coupled directly with a TRIO-1 mass spectrometer (VG Instruments, Manchester, UK). For sugars, the column oven temperature, started at 50°C, was programmed to 270°C at the rate of 10°C/min and held for 1 min. Finally, the column was heated to 290°C and held for 5 min. For amino acids, the column oven temperature, started at 85°C, was programmed to 280°C at the rate of 10°C/min, held for 1 min, and finally heated to 290°C and held for 5 min.
Animals and induction of arthritis
Inbred female Lewis rats weighing approximately 150 g were purchased from Harlan SpragueDawley, IN, USA. Arthritis was induced by i.p. injection of CW (200 µg dry weight/g rat body weight) suspended in sterile PBS. Control rats were injected with an equal volume of sterile PBS. To monitor the development of arthritis, each limb was assigned a score of 04, based on the degree of erythema, oedema, painfulness and functional disorder of the ankle and metatarsal joints (wrist and metacarpal joints), by two independent observers, as described previously [30]. Such an evaluation has been widely used by us; the results are parallel to those by histological grading [4, 3032].
Lymphocyte stimulation assay
To determine the cellular reactivity to concanavalin A (Con A), rat splenocytes were isolated by using Lympholyte-Rat (Cedarlane Laboratories, Ontario, Canada) at 2 weeks after the CW injection. The splenocytes were incubated with Con A (1.25 µg/ml) in round-bottom microtitre wells (Nunclon, Roskilde, Denmark) containing 1 x 105 cells/0.2 ml culture medium. The cells were pulsed with 1 µCi [3H] thymidine 18 h before harvesting on day 3 with an automatic cell harvester (Harvest 96, Tomtec, Germany); the incorporated radioactivity was counted in a beta counter (Wallac, Turku, Finland). All samples were assayed in quadruplicate.
FACScan analysis
Fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated monoclonal antibodies OX-52, OX-35, OX-40, OX-39, R73, V65 and isotype-matched mouse IgG (PharMingen, San Diego, CA, USA) were used. For double-colour staining, splenocytes isolated by Lympholyte-Rat 2 weeks after CW injection were incubated with FITC-conjugated monoclonal antibodies followed by PE-conjugated monoclonal antibodies at +4°C for 30 min in the dark. After washing twice with PBS supplemented with 2% fetal calf serum containing 0.01% sodium azide, labelled lymphocytes were analysed using FACScan (Becton Dickinson, CA, USA) flow cytometer and CELLquest software. To quantify the expression of the surface antigens, the mean fluorescence intensity for each staining was measured.
Antibody assays
Sera for antibody assays were collected by cardiac puncture 2 weeks after the CW injection. Serum IgM, IgG, and IgA class antibodies specific for CW were quantified by a modification of a previously published enzyme-linked immunosorbent assay (ELISA) [32]. The water extract of sterile rat food pellets was used as a control antigen. The food pellets were composed of wheat, barley, soy, wheat, fish powder, minerals and vitamins with a minimal contamination of bacterial structures (Lactamin AB, Stockholm, Sweden). The pellet was smashed and dissolved in sterile water; after low-speed centrifugation, the supernatant was collected and the protein concentration was measured by the Lowry protein assay. The supernatant was used as the antigen. Dynatech 96 microtitre plates (Nunc) were coated with CW (equivalent 5 µg rhamnose CW/well) or with rat food antigens (5 µg protein/well) in PBS. Rat sera diluted 1:200 were incubated with alkaline phosphatase-conjugated sheep anti-rat IgG 1:500 (The Binding Site Limited, Birmingham, UK), or with unconjugated mouse anti-rat IgA 1:20 000, or with unconjugated mouse anti-rat IgM 1:20 000 (Zymed Laboratories, CA, USA). For IgA and IgM detection, alkaline phosphatase-conjugated goat anti-mouse IgG + IgM 1:2000 (Caltag Laboratories, CA, USA) was used as a second antibody. The absorbances were measured at a wavelength of 405 nm using a Titertek Multiscan plus spectrophotometer (Labsystems, Helsinki, Finland).
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Results
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Induction of arthritis
As expected, i.p. injection of CW from E. limosum or E. alactolyticum caused extremely slight signs of acute arthritis and no chronic arthritis (Fig. 2
). However, from the two strains of E. aerofaciens, only CW of ATCC 25986 induced chronic arthritis as expected, whereas i.p. injection of CW from the strain ATCC 35085 resulted only in acute arthritis, which subsided in about 10 days without any evidence for chronicity. On this basis and for the sake of simplicity, E. aerofaciens ATCC 25986 is called the arthritogenic strain and the strain ATCC 35085 as well as the strains of E. limosum and E. alactolyticum are designated non-arthritogenic.

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FIG. 2. Arthritis development in rats injected i.p. with CWs from four different strains of Eubacterium. Each symbol represents the mean ± standard error of the mean (S.E.M.) of five to 12 rats.
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Identification of bacteria
Due to the unexpected findings with the other strain (ATCC 35085) of E. aerofaciens, the identity of all four strains at the DNA level was studied. For this purpose, genes coding for 16S ribosomal RNA were sequenced (320452 base pairs). The results obtained indicated 100.0% identity between the two strains of E. aerofaciens, 78.6% identity between E. aerofaciens and E. limosum or between E. aerofaciens and E. alactolyticum. Similarity between E. limosum and E. alactolyticum was 92.6%. Despite the complete identity in this DNA analysis, the two strains of E. aerofaciens were slightly different both in their biochemical and CFA profiles. The main differences in the biochemical reactions were in the acid production from glucose, lactose, mannitol, rhamnose and salicin. In the CFA profiles, the main differences were in the relative amounts of the fatty acid methyl esters 12:0, 16:0, 18:1-cis-9, 18:0 and in dimethyl acetates 11:0 and 14:0. These results indicate that the two strains of E. aerofaciens do not represent the same bacterial clone.
Chemical analysis of CW
The results of the chemical analysis of the four CW preparations are shown in Table 1
. All four preparations had a significant amount (7.220.4% of dry weight) of rhamnose which has been claimed to be important for arthritogenicity [33]. Of interest was the finding that CW of E. limosum and E. alactolyticum, which were extremely mild in the acute arthritogenicity, had a small amount (2.03.9%) of N-acetylgalactosamine, whereas CWs of both strains of E. aerofaciens were completely devoid of this compound. The total amount of carbohydrates in the four CWs varied from 39.9 to 58.9% and that of amino acids from 27.7 to 48.7%. Regarding the quantitative content of PG components N-acetylglucosamine and N-acetylmuramic acid, no such differences between the four preparations were observed that could be considered significant for arthritogenicity/non-arthritogenicity.
The amino acids detected (Table 1
) can be divided into those known to be part of PG and those belonging to the proteins outside PG. The latter ones are intimately attached to PG, and their complete separation from the CW preparations would not have been possible without breaking the PG structure. Therefore, amino acids belonging to proteins outside PG are found in minor quantities in CW preparations. The results obtained indicate the occurrence of PG amino acids as follows: E. aerofaciens ATCC 25986: alanine, glutamic acid, lysine, aspartic acid; E. aerofaciens ATCC 35085: alanine, glutamic acid, ornithine, aspartic acid; E. limosum ATCC 8486: alanine, glutamic acid, lysine, ornithine, serine; E. alactolyticum ATCC 17927: alanine, glutamic acid, diaminopimelic acid. For PG amino acids the results are also calculated in micromoles and as molar ratios, revealing tentative PG types (Table 2
). The total amount of PG amino acids was 25.5% of dry weight in E. aerofaciens ATCC 25986, 30.1% in E. aerofaciens ATCC 35085, 36.1% in E. limosum, and 28.9% in E. alactolyticum. The amounts of protein amino acids were <2.2, <4.9, 12.6 and 9.0%, respectively.
Host's immune status
To study the T cell function of the rats injected with different preparations of Eubacterium CW, Con A response of spleen lymphocytes taken at 2 weeks after the CW injection was determined. Cells from the rats injected with CW of either strain of E. aerofaciens did not at all respond to Con A, whereas spleen cells from the rats injected with CW of E. limosum or E. alactolyticum or with PBS responded in a normal way (Table 3
).
The lack of Con A responses cannot be explained by changes in the T cell populations, since FACScan analysis using six different markers did not reveal any differences in the splenocytes between the four groups of rats (Table 4
). The markers analysed for this purpose were Pan-T,
ßTCR, 
TCR, CD4 and T cell activation markers CD25 and OX-40. The monoclonal antibody used for staining CD4 (OX-35) also reacts with monocytes and macrophages, resulting in values of CD4+ cells exceeding the Pan-T numbers.
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TABLE 4. Ex vivo phenotype analysis of splenocytes at 2 weeks after injection of different bacterial CW preparations
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To determine whether an effect on B cell functions could explain the different arthritogenicities of different CWs, we studied the serum antibody response against the homologous (injected) CW. It is apparent that the results obtained do not reveal any explanation for the varying arthritogenicity (Table 5
). The most vigorous antibody response against the injected CW was observed in the rats injected with the non-arthritogenic E. alactolyticum CW, particularly in IgG class antibodies which were quite low in the other groups. It is also noteworthy that the other non-arthritogenic CW (E. limosum) did not induce an antibody response to the same magnitude as did CWs of E. alactolyticum or of the two E. aerofaciens strains. As a control, we also determined antibody responses against an irrelevant antigen to find out whether the experimental groups would behave differently in this respect. As such an antigen we used an extract of food pellets, since all animals were exposed to this in exactly the same way. Responses against the food antigen and the bacterial CW preparations cannot be quantitatively compared, but the results (Table 6
) clearly reveal that the four experimental groups did not differ in their response against the unrelated antigens. It is evident, however, that the CW injection stimulated antibody responses against food antigens over the level observed in the rats injected with PBS alone (Table 6
). On the basis of these findings, including responses against the CW, we concluded that antibody responses do not provide any explanation for the difference in arthritogenicity between the eubacterial strains.
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TABLE 5. Serum antibodies at 2 weeks after injection of different bacterial CW preparations; homologous CW was used as antigen
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TABLE 6. Serum antibodies 2 weeks after injection of different bacterial CW preparations; extract of rat food pellets was used as antigen
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Discussion
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Several authors have suggested that bacterial CW fragments derived from the intestinal flora may pass the bowel wall, distribute within the body [9, 10, 34, 35] and cause a local or systemic reaction leading to arthritis [3638]. Studies by Severijnen et al. [22] have shown that i.p. injection of CW of E. aerofaciens, a main resident in the human intestinal flora, induces a chronic arthritis in the rat. In the present study, two strains of E. aerofaciens were chosen to represent arthritogenic strains, and E. limosum and E. alactolyticum to represent non-arthritogenic bacterial strains. To our surprise, the CW isolated from E. aerofaciens ATCC 35085 induced only acute arthritis, whereas the other E. aerofaciens strain (ATCC 25986) induced both acute and chronic arthritis, as expected. These two strains are 100% identical by the 16S rDNA analysis, but they have slightly different biochemical and CFA profiles, indicating that they are different clones of the same species. Therefore, they provide a challenging opportunity to study what determines the arthritogenicity of the bacterial CW.
The chemical composition of complex biological materials, including bacterial CWs, can be analysed qualitatively and quantitatively by GCMS. Relying on this unique methodology and on the results published previously, we here present putative structures of PGs in the four CW preparations used (Fig. 3
). PG of the arthritogenic E. aerofaciens (ATCC 25986) contains alanine, glutamine/glutamic acid, lysine and aspartic acid; most probably it represents PG subgroup A4
having D-aspartic acid in the interpeptide bridge and L-lysine in position 3 of the stem peptide. The non-arthritogenic E. aerofaciens (ATCC 35085) has the same amino acids except that lysine is replaced by ornithine. Therefore, its structure most likely represents subgroup A4ß having D-aspartic acid in the interpeptide bridge and L-ornithine in position 3 of the stem peptide. One must admit that the molar ratios observed (Table 2
) do not exactly support the quantitative ratios required for the subgroup A4ß. However, the molar ratios obtained can be regarded only as rough guidelines, particularly since the CW and not the purified PG was used in the analyses. PG of E. limosum contains alanine, glutamic acid, lysine, ornithine and serine. The composition agrees with the E. limosum PG structure of subgroup B2
suggested by Guinand et al. [39]. This subgroup contains D-lysine in the interpeptide bridge and L-ornithine in position 3 of the stem peptide. Finally, PG of E. alactolyticum contains only alanine, glutamic acid and diaminopimelic acid. Its structure has previously been described by Severin et al. [40] to belong to subgroup A1
, without an interpeptide bridge and with diaminopimelic acid in position 3 of the stem peptide; our findings are in concert with this (Fig. 3
).
Does the chemical structure of PG explain why only one of the four CW preparations used induces chronic arthritis in the rat? Two types of observation might help here. First, PGs of group A have been reported to be significantly more powerful immunostimulants than those of group B [20, 41]. Second, lysine in position 3 of the stem peptide is known to contribute to the phlogistic capacity of Gram-positive CWs [23]. On this basis, it would be understandable that PG of group B from E. limosum as well as group A PGs from E. alactolyticum and E. aerofaciens ATCC 35085, all lacking lysine in position 3, do not induce chronic arthritis. It will be of interest to see, by studying other bacterial species and strains, whether such a presence of lysine is decisive for arthritogenicity. It remains possible that other factors are also required. For example, a further difference between the two strains of E. aerofaciens is the higher PS content (49.4%) in the arthritogenic strain when compared with that in the non-arthritogenic strain (27.5%); the PGPS complex is known to be resistant to mammalian enzymes, and the high carbohydrate content may contribute to prolonged tissue persistence [11].
If the chemical differences explain how only PG of E. aerofaciens ATCC 25986 caused chronic arthritis, a question remains why a significant development of acute arthritis was seen after injection of PG from E. aerofaciens ATCC 35085 but not after injection of PG from E. limosum or E. alactolyticum. Three different observations may be important in this regard. First, the total amount of protein amino acids was lower (<2.2% and <4.9% of dry weight) in CWs of E. aerofaciens strains than in those of E. limosum (12.6%) or E. alactolyticum (9.0%). Second, CWs of both E. aerofaciens strains lack N-acetylgalactosamine (Table 1
). Third, these two strains were immunosuppressive, when tested by Con A response at 2 weeks after the CW injection, at a stage when signs of the acute arthritis had already subsided and only the chronic arthritis was present (Fig. 2
). Such an immunosuppression was not observed after injection of CW from E. limosum or E. alactolyticum. It is apparent that the same cellular mechanisms are not involved in the arthritis induction and Con A response. The importance of these three types of observation regarding the capacity of CWs to induce acute arthritis remains at the present unanswered. It is of interest that the presence of MDP within the PG structure is not sufficient for induction of acute arthritis, since our strain of E. alactolyticum has MDP and yet no acute arthritis was observed.
We also studied proliferative lymphocyte responses against the CW preparations used. However, they proved to have extremely weak if any stimulatory capacity (data not shown), without any difference between the four bacterial strains. Likewise, no significant, meaningful differences between the four groups of rats were observed in the FACScan analysis with six T cell markers or in antibody responses.
Taken together, the present results suggest that the chemical structure of the bacterial CW is decisive in determining arthritogenicity/non-arthritogenicity. Our results agree with the view that the presence of lysine in position 3 of the PG stem peptide contributes to the phlogistic capacity [23]. How this contribution is mediated and what else is required cannot be answered on the basis of the present experiments. It remains to be seen, for example, how the presence of lysine in the critical position affects resistance to enzymatic degradation and tissue persistence known to be important for the development of chronic arthritis [, 912]. Likewise, the questions of how and why an i.p. injection of a phlogistic compound leads predominantly to arthritis remain open.
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Acknowledgments
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We gratefully thank Leena Kivistö, Marja-Riitta Teräsjärvi, and Heli Niittymäki for excellent technical assistance, Jari Jalava for 16S rDNA sequence analysis, and Hannele Jousimies-Somer for biochemical reactions and CFA profiles of the four Eubacterium strains. We also thank Yong Zhang for help in evaluating the arthritis, and Erkki Eerola, Janne Komi and Jaakko Uksila for their kind advice and discussions. This work was supported by EVO of Turku University Central Hospital.
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Notes
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Correspondence to: X. Zhang, Department of Medical Microbiology, Turku University, Kiinamyllynkatu 13, FIN-20520 Turku, Finland. 
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Submitted 2 June 1999;
revised version accepted 20 September 1999.