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. 2001 Jan;123(1):140–146. doi: 10.1046/j.1365-2249.2001.01419.x

Reduced systemic IgG levels against peptidoglycan in rheumatoid arthritis (RA) patients

I A Schrijver *, Y A De Man *, M -J Melief *, J M Van Laar *, H M Markusse †, I S Klasen ‡, M P Hazenberg *, J D Laman *
PMCID: PMC1905964  PMID: 11168011

Abstract

The gut flora is believed to play a role in the pathogenesis of RA. Peptidoglycan, a major cell wall component of Gram-positive bacteria, is a candidate antigen because of its capability to trigger production of proinflammatory cytokines, to induce arthritis in rodents, and because of its presence in antigen-presenting cells in RA joints. We investigated whether the systemic and local antibody levels against a peptidoglycan–polysaccharide (PG–PS) are related to the presence and disease activity of RA. Significantly lower levels of systemic IgG directed against PG–PS were found in healthy females compared with healthy males, and systemic IgA levels specific for PG–PS were negatively correlated with age. Levels of systemic IgG directed against PG–PS were significantly reduced in RA patients compared with sex- and age-matched healthy controls. Local (synovial fluid) levels of IgG did not correlate with disease activity whereas synovial fluid levels of IgA correlated positively with disease activity. These data suggest that IgG in healthy people mediates protection against spreading of PG to non-mucosal sites.

Keywords: Gram-positive bacteria, gut flora, rheumatoid arthritis, antibodies, peptidoglycan

Introduction

The search for the trigger (auto)antigen in the aetiology of RA has been the topic of many studies, and in some of those a microbial aetiology has been suggested. In reactive arthritis, the triggering antigens are thought to be microbes that cause infections of the gut or urogenital tract, such as Chlamydia trachomatis, Yersinia, or Salmonella species [1]. Antigens of these bacteria have been detected in synovial cells [2–4] and T cells specific for these antigens have been found in the synovium [5–7]. Because RA is a chronic disease, it can be suggested that the bacterial load in the intestine with which the human body is in lifelong close contact might be important for the induction and perpetuation of RA. This is supported by the observation that some patients with Crohn's disease and ulcerative colitis, both intestinal autoimmune diseases, suffer from joint inflammation [8,9]. Furthermore, Crohn's-like inflammatory bowel disease and chronic inflammatory arthritis develop spontaneously in a knockout mouse lacking a tumour necrosis factor (TNF) AU-rich element, responsible for TNF mRNA destabilization and translational repression in haematopoietic and stromal cells [10].

We and others have previously hypothesized that peptidoglycan (PG) plays a role in the pathogenesis of RA [11–14]. PG is the major component of the cell wall of Gram-positive bacteria and is composed of long sugar chains of alternating N-acetyl glucosamine (GlcNAc) and N-acetyl muramic acid (MurNAc) residues, which are interlinked by peptide bridges, resulting in a large complex macromolecular structure [15]. PG isolated from the anaerobic bacterium Eubacterium aerofaciens, which is present in numbers > 109/g human faeces, induces a severe and chronic arthritis in rats [16–18]. Furthermore, PG could be detected in antigen-presenting cells (APC) in synovial tissues of RA patients using a specific antibody [19,20]. PG could also be isolated from sterile normal human spleen using biochemical methods [21,22]. In vitro analysis showed that this PG fraction is able to induce production of the proinflammatory cytokines TNF-α, IL-1 and IL-6 [22] which are crucially involved in RA pathogenesis [23].

PG can also be isolated from faeces in soluble form. This macromolecular peptidoglycan–polysaccharide complex (PG–PS) was shown to originate from the normal endogenous gut flora [24]. Since PG is present at 0·5 mg/g faeces (wet weight), it could be calculated that the intestinal anaerobic flora produces up to 200 mg of PG daily in healthy adults.

As high antibody titres of the IgM, IgG and IgA subclasses against PG can be found in healthy persons, it is obvious that the immune apparatus of the body is in continuous close contact with PG possessing strong arthritogenic capacity [25,26]. Several studies have shown that increased levels of antibodies against PG are present in patients with bacterial infections [27] and in patients with various rheumatic diseases like ankylosing spondylitis, Reiter's syndrome, juvenile rheumatoid arthritis and RA [28–30]. However, a study of Rantakokken et al. [31] showed that patients with RA had decreased antibody levels against streptococcal PG compared with healthy controls.

In the present study we analysed systemic and local antibody levels against PG–PS isolated from the human gut in a large panel of healthy donors versus early and long-standing RA patients to assess whether antibodies specific for PG–PS may be involved in the pathogenesis of RA.

Patients and methods

Sera

Control sera

Sera of 98 healthy blood bank donors were obtained as a kind gift of the Department of Neurology of the Erasmus University and University Hospital-Dijzigt Rotterdam and 27 additional sera of healthy donors were a kind gift of the Department of Epidemiology from the Erasmus University and University Hospital-Dijzigt Rotterdam (Table 1). Additional sera of six healthy volunteers (two females, age 32 and 37 years and four males age 26, 27, 27 and 31 years) used for determination of optimal serum dilutions were obtained from the Department of Immunology of the Erasmus University Rotterdam.

Table 1.

General features of the donor groups

Group Source Number male/female Age, years Duration (months) DAS score* (mm/h) ESR†
Healthy controls Serum 67 52 (30–83) ND ND ND
58 50 (19–88) ND ND ND
RA patients:
Early Serum 17 64 (28–84) < 12 ND ND
27 50 (23–82) < 12 ND ND
Long-term Serum 20 62 (33–84) 89 (8–240) 5·6 (3·3–7·4) 71 (23–113)
43 63 (31–82) 132 (13–420) 5·1 (1·9–7·2) 68 (15–140)
Synovial fluid 3/12 63 (46–82) 66 (8–180) 5·7 (3·4–7·4) ND
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*

Expressed as mean with the range shown in parentheses.

†

Erythrocyte sedimentation rate.

ND, Not done.

Patient sera

Sera of 63 RA patients diagnosed with RA fulfilling the American College of Rheumatology criteria [32] were obtained from the Zuiderhospital, Rotterdam and the Leiden University Medical Centre. The majority of the patients received no immunosuppressive medication, some patients received low doses of prednisone (up to 10 mg/day) or methotrexate (up to 30 mg/week). Sera of 44 patients with RA of less than 1 year's duration as measured from the first clinical signs of arthritis were obtained from the Leiden University Medical Centre (Table 1). The majority of the patients received no immunosuppressive medication.

Standard serum

A pooled standard serum (approx. 500 blood bank donors) selected for high PG–PS reactivity was used in each ELISA plate as a reference for calculating antibody levels against PG–PS.

Synovial fluid

Paired samples of serum and synovial fluid were obtained from 15 RA patients with long-standing RA from the Zuiderhospital Rotterdam.

Clinical scoring

RA disease activity of 39 patients at time of donation (including the synovial fluid patients) was determined by the disease activity score (DAS) 28 score [33]: 0·56 √TJC + 0·28 √SJC + 0·7 log ESR + 0·014 GH, wereTJC = number of painful joints (out of 28), SJC = number of swollen joints (out of 28), GH = general health score on a visual analogue scale (0–100) (Table 1). Erythrocyte sedimentation rate (ESR) (mm/h) was used as a measure of disease activity of all other RA patients (Table 1).

Isolation of PG from human faeces

PG–PS from faeces of a healthy subject was prepared as described previously [24]. Briefly, faeces was diluted in distilled water (40 g in 100 ml) and homogenized in a Model 400 lab Blender (Stomacher, Colworth, UK). After cambric gauze filtration the suspension was centrifuged for 45 min at 5000 g. Four volumes of 96% ethanol were added to 1 volume of supernatant. After 2 h at 4°C, the precipitate was centrifuged for 15 min at 5000 g. The pellet was dissolved in and dialysed against milliQ water for 48 h. Next, the suspension was centrifuged for 1 h at 100 000 g and the clear supernatant was collected. Size exclusion chromatography was performed using dilutions of 15–60 mg/30 ml (depending on the viscosity of the solution), with a TSK HW75 column. After passage of 100 ml void volume, fractions (8 ml/5 min) were collected and assayed for their protein and carbohydrate contents. High molecular weight fractions containing carbohydrates but no protein were pooled, dialysed, and lyophilized. From 100 g of faeces about 50 mg PG could be retrieved by this procedure.

ELISA

To measure anti-PG–PS antibodies in serum and synovial fluid an ELISA was performed. PG–PS (50 μl; 10 μg/ml) was coated overnight at 50°C in 96-well polystyrene microtitre plates. The plates were washed three times with PBS−0·02% Tween 20. One hundred microlitres serum or synovial fluid diluted in PBS−0·2% Tween were added to the wells and after 1 h at 37°C unbound antibody was removed by three wash steps. As detecting antibody, peroxidase-conjugated rabbit anti-human IgM, IgG or IgA (Jackson Immunoresearch, Inc., Westgrove, PA) diluted in PBS–Tween 0·2% was used during 1 h at 37°C. After washing three times the development of the colourimetric assay took place at 37°C for 30–45 min after the addition of 100 μl of orthophenylene diamine/H2O2. The reaction was stopped by adding 50 μl 4 m H2SO4 and the optical density (OD) was measured at a wavelength of 492 nm with a Titertek Multiskan (Flow Labs, Irvine, UK). Results were expressed as OD units. On each plate the standard serum was included and each sample was tested in triplicate. Background absorption of the conjugate was also tested in triplicate. The average OD reading of the sera and standard serum minus the background staining was used to calculate concentrations of antibodies in a ratio versus the standard serum according to the following formula: ratio = ODsample −ODbackground/ODstandard serum − ODbackground. This ELISA has been validated previously [19]. We excluded cross-reactivity with rheumatoid factor (RF) by preincubating sera with known amounts of RF with PG. This did not interfere with subsequent detection of RF.

Determination of optimal dilutions for measuring antibody levels against PG–PS

Standard serum, sera of six healthy volunteers, as well as synovial fluids of five RA patients were titrated from 1:2 to 1:6400 and added to the wells coated with either 5, 10 or 20 μg/ml of PG–PS to determine the optimal concentration of serum and antigen. Optimal serum concentration was the concentration in the linear area in the dilution curve. Based on checkerboard titrations, anti-PG–PS antibodies were measured after coating plates with 10 μg/ml PG–PS and serum dilutions of 1:400, 1:1600 and 1:200 for IgM, IgG, and IgA, respectively. To analyse PG–PS antibodies in synovial fluid, dilutions of 1:20, 1:200 and 1:80 were used, respectively, for IgM, IgG and IgA.

Total immunoglobulin determinations

Of 11 randomly chosen sera of RA patients total IgM, IgG and IgA concentrations were measured using immunonephelometry [34] to examine whether total immunoglobulin concentrations were in the normal range.

Statistical analysis

The ratios of IgM, IgG and IgA measured in the PG–PS ELISA of RA patients versus standard serum and controls were compared with Student's t-test to analyse significant differences between the two groups. Linear regression was used to detect any age-related influence on the levels of immunoglobulin specific for PG–PS in healthy controls and to evaluate correlation between disease activity and anti-PG–PS antibodies in synovial fluid.

Results

Systemic antibody levels against PG in healthy females compared with healthy males

Antibody levels against PG–PS were analysed in the healthy control group to examine whether levels against PG–PS differed between male and female donors. Figure 1 shows that females had a significantly lower mean ratio of anti-PG–PS IgG antibodies to standard serum, compared with males (P < 0·01). Levels of IgA and IgM antibodies against PG–PS were not different in females compared with males (Fig. 1). These results show that IgG antibody levels against PG–PS differ in healthy male and female donors and have to be analysed separately.

Fig. 1.

Fig. 1

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Systemic antibody responses against peptidoglycan (PG) in healthy females (□) compared with males (▪). Levels of IgM, IgG and IgA against PG–polysaccharide (PS) were measured in 67 healthy men and 58 healthy women (Table 1). Levels of IgG were significantly reduced in women compared with men (P < 0·01). Results are indicated as mean values + s.d.

Systemic antibody levels against PG in healthy donors in relation to age

To examine whether antibody levels against PG–PS are related to age, the same sera used for determination of the sex difference in systemic antibodies against PG–PS were analysed. Figure 2 shows that in females a decline occurred in antibody levels of all three isotypes with advancing age. This correlation was only significant for IgA antibodies. In males, a minor decline could be observed in IgA titres with age but this difference was not significant. These results imply that age-matched controls have to be used for the examination of PG–PS antibodies in female RA patients compared with healthy controls.

Fig. 2.

Fig. 2

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Systemic antibody responses against peptidoglycan–polysaccharide (PG–PS) in relation to age of healthy females and males. Levels of IgM, IgG and IgA against PG–PS measured in 67 healthy males and 58 healthy females were related with age. The decline of IgA antibodies in females was significantly related to increasing age (P < 0·01) (r2 = 0·24).

Systemic antibody levels against PG in RA patients

To determine whether the systemic antibody levels against PG–PS are different in RA patients compared with healthy controls, sera of 101 healthy controls and of 61 RA patients were compared. Males and females were compared separately and RA females were compared with age-matched controls. Analysis of 67 male donors (mean age 52 years, range 30–83 years) and 21 RA patients (mean age 62 years, range 33–82 years) showed that RA patients had lower IgG antibody levels against PG–PS compared with healthy controls (Fig. 3).

Fig. 3.

Fig. 3

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Systemic antibody responses against peptidoglycan–polysaccharide (PG–PS) in early (hatched) and long-term (▪) RA patients. Levels of IgM, IgG and IgA against PG–PS were compared in early and long-term RA for male and female patients. Levels of IgG were reduced in RA patients compared with healthy controls (□). This difference was significant for long-term RA males and females (P < 0·01) and for early RA male and female donors (P < 0·01/P < 0·05). Results are indicated as mean values + s.d.

Figure 3 also shows that IgG antibody levels against PG–PS were lower in 40 sera of RA females (mean age 63 years, range 30–84 years) compared with 34 sera of healthy controls (mean age 63 years, range 31–88 years). Using Student's t-test, this difference was found to be significant for both males (P < 0·01) and females (P < 0·01). No differences could be observed in IgM and IgA antibody levels against PG–PS in male or female RA patients compared with healthy controls.

As antibody levels shortly after the disease onset might be more informative than those in long-term RA, we also compared patients with newly developed disease (< 1 year) and healthy controls.

Sera of 27 females with early RA (mean age 50 years, range 23–84 years) were compared with 58 sera of healthy controls (mean age 50 years, range 19–88 years).

The level of IgG specific for PG–PS was reduced compared with healthy controls. No differences were observed between levels of IgM and IgA of females with early RA and healthy controls. Sera of 17 male donors with early RA (mean age 64 years, range 28–84 years) and of 67 healthy controls (mean age 52 years, range 30–83 years) were compared. Again IgG levels against PG–PS were reduced and no differences were observed in IgM and IgA antibodies. The reduction in levels of IgG antibodies against PG–PS was significant for both male and female donors (P < 0·01, P < 0·05, respectively).

Antibody levels specific for PG in synovial fluid of RA patients

To examine whether antibodies can also be detected locally in synovial fluids of RA patients where they might affect inflammation, antibody levels against PG–PS were determined in synovial fluid of 15 RA patients. In all synovial fluids IgM, IgG and IgA antibodies against PG–PS could be measured.

To assess whether anti-PG–PS antibody levels occurring in the joints correlated with disease activity, linear regression was performed. No correlation could be observed between disease activity and anti-PG–PS IgG. In contrast, synovial IgM and IgA titres against PG–PS were related positively with disease activity (Fig. 4), but only the correlation found for IgA against PG–PS and disease activity was significant.

Fig. 4.

Fig. 4

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Antibodies against peptidoglycan–polysaccharide (PG–PS) synovial fluid in relation to disease activity in RA. In 15 synovial fluid samples of RA patients IgM, IgG and IgA against PG–PS were measured and in relation to disease activity (DAS score). All 15 synovial fluid samples contained IgM, IgG and IgA against PG–PS. IgA levels against PG were significantly positively related with disease activity (P < 0·05).

Discussion

The present study shows that early and long-term RA patients had significantly lower systemic IgG antibody levels against PG–PS than healthy controls, and that the amount of IgA antibodies against PG–PS in synovial fluid samples of RA patients is positively correlated with disease activity. Furthermore, this study shows that IgG antibody levels against PG–PS were significantly lower in healthy females than in healthy males, and levels of IgA against PG–PS are negatively correlated with age in healthy female donors.

This study was performed to analyse systemic and local antibody levels against PG–PS in an extended panel of RA patients and healthy controls. As shown before [25,26], all healthy donors have serum antibodies against PG–PS, indicating that no tolerance at the B cell level occurs despite continuous exposure to PG–PS at the mucosal surface of the gut, which is thought to be optimally suited for tolerance induction.

A lower antibody level was found in serum of females compared with males, which was significant only for IgG (P < 0·01). We also observed a negative relationship between age and PG–PS-specific IgA antibodies in females. Although there is no evidence of causal linkage, it is of interest to note that the lower antibody levels against PG–PS in females compared with males correlates with the two-to-three-fold higher general incidence of RA in females. In addition, the decrease of antibody levels against PG with age in females parallels the rise of RA incidence with advancing age [35,36].

RA patients had significantly lower IgG antibodies against PG–PS compared with healthy controls. To exclude the possibility that the decrease in antibody levels to PG–PS in RA patients was due to generalized depression of antibody levels, total IgG, IgM and IgA levels were measured in 15 randomly selected RA sera. These levels were either normal or slightly increased, indicating that no general decrease of immunoglobulin titres had occurred (data not shown). To analyse whether the decrease of immunoglobulins against PG–PS in RA was also apparent during early stages of the disease we analysed patients with early RA (< 1 year disease duration). Both male and female patients had significantly lower IgG levels specific for PG–PS, suggesting that this phenomenon is apparent already early in the disease. Of five early RA patients, we also obtained a blood sample 1 year after the first sample. The antibody levels in the paired samples were comparable, indicating that disease progression has no influence on antibody levels specific for PG–PS (data not shown). Decreased antibody levels against PG–PS possibly were not due to disease progression, as can be concluded from the fact that in early RA male patients the level of IgG specific for PG–PS was even lower than in long-standing RA patients.

The present study also showed the presence of IgM, IgG and IgA antibodies specific for PG–PS in synovial fluid of RA patients and that the level of IgA anti-PG–PS antibodies correlated positively with disease activity of the patients. At present it is unclear whether these antibodies aggravate local inflammation (e.g. due to immune complex formation or activation of cells through Fc receptor binding), or that they locally capture PG with beneficial results. Neither is it known whether antibody is produced locally by plasma cells, or leaks into the synovium from the circulation. Inflammation can enhance permeability of the synovial membrane [37]. Paired analysis of serum and synovial fluid showed a positive relationship between synovial fluid and serum IgM, IgG and IgA levels of the same patient specific for PG–PS, suggesting that at least part of the antibodies present in the synovial fluid could be derived from the circulation (data not shown). The local inflammatory response in the synovium may attract lymphocytes. It is known that especially mucosal lymphocytes are able to home to the synovium by expression of relevant adhesion molecules [38–40]. Trapping of PG in the synovium may result in the attraction of PG-specific plasma cells, due to local antigen deposition as well as antigen-driven T cell activation which can provide important ‘second signals’ for expansion and terminal differentiation of mucosal immunoglobulin-producing immunocytes at secretory effector sites [41]. In addition, plasma cells may be recruited non-specifically to the joint.

Some earlier studies focusing on antibody levels against PG in RA reported elevated and not decreased antibody titres against PG [27–29]. An explanation for this discrepancy might be that the immunoglobulin levels were measured using PG isolated from cultured bacteria, which may be structurally distinct from PG physiologically present at the mucosa. We used PG–PS derived from the indigenous intestinal flora representing PG to which the body is continuously exposed at high quantities. Other studies support our findings. For instance, Rantakokko et al. [31] found lower IgG levels against PG from cultured Streptococcus pyogenes in RA patients compared with controls. Another study showed that in vitro responses of B cells to PG were markedly depressed in RA patients compared with healthy controls [42], which might provide a mechanistic explanation for reduced antibody levels in RA patients.

How could reduced IgG levels against PG in RA patients be causally linked to joint inflammation? A possible mechanism is that more PG reaches tissues outside the mucosal sites, and is captured in the synovial tissue where it promotes proinflammatory responses [22]. Increased access of PG to non-mucosal sites may be due to at least two mechanisms. First, reduced gut barrier function may result from inflammation, leading to increased bacterial translocation. Ultrastructural lesions of the gut wall have been reported in RA patients [43]. Second, lower antibody levels against PG may not be sufficiently capable of eliminating the bacterial products from mucosal sites and/or the bloodstream. Antibodies that reach the mucosal lumina perform ‘immune exclusion’, a term coined for non-inflammatory mucosal surface protection against antigens. This function is performed by secretory IgA or IgM (S-IgA, S-IgM), but also by serum IgG antibodies [44,45].

In conclusion, the lower antibody levels against PG in RA patients suggests a protective role of IgG antibodies. The protective properties of antibodies against PG can be further investigated by measuring mucosal antibody levels in faeces and intestinal fluids. The correlation between synovial fluid IgA antibodies against PG and disease activity suggesting intra-articular production of IgA is currently being further investigated by in situ detection of PG-specific antibody-forming cells in synovial tissues using labelled PG. The study implies that restricting the access of PG to non-mucosal sites may contribute to dampening RA activity. Therapeutic agents designed to decrease mucosal permeability should therefore be investigated in in vitro and in vivo models of bacterial translocation to assess whether they might have clinical relevance.

Acknowledgments

The authors would like to thank W. Ang and Dr D. van der Kuip for providing control sera. Professor Dr R. Benner is thanked for critical reading of this manuscript and T. van Os for preparing the figures.

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