Elevated levels of IgM and IgA antibodies to Proteus mirabilis and IgM antibodies to Escherichia coli are associated with early rheumatoid factor (RF)-positive rheumatoid arthritis

M. M. Newkirk*, R. Goldbach-Mansky*,1, B. W. Senior2, J. Klippel3, H. R. Schumacher, Jr4 and H. S. El-Gabalawy5

McGill University Health Centre, Montreal, Quebec, 1 Arthritis and Rheumatism Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD, 2 Department of Molecular and Cellular Pathology, University of Dundee, Dundee, UK, 3 Arthritis Foundation, Atlanta, GA, 4 University of Pennsylvania and VA Medical Center, Philadelphia, PA and 5 Division of Rheumatology, University of Manitoba, Winnipeg, Manitoba, Canada.

Correspondence to: M. M. Newkirk, McGill University Health Centre, 1650 Cedar Ave., Montreal, QC, Canada H3G 1A4. E-mail: Marianna.newkirk{at}mcgill.ca


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objective. Antibodies to Proteus mirabilis were previously detected in patients with established rheumatoid arthritis (RA). We examined the prevalence of antibodies to P. mirabilis and their associations with RA in early synovitis patients.

Methods. Two hundred and forty-six patients with inflammatory arthritis for less than 1 yr were prospectively evaluated for 1 yr. Of these patients, 30% had rheumatoid factor (RF)-positive RA, 16% RF-negative RA, 17% a spondyloarthropathy and 37% undifferentiated arthritis. Serum antibodies to P. mirabilis, Escherichia coli and other potentially arthritogenic organisms (Chlamydia, Salmonella, Shigella, Campylobacter, Yersinia and parvovirus B19) and for antibodies specific for immunoglobulin (Ig) G damaged with advanced glycation end-products (anti-IgG-AGE) were measured.

Results. IgM and IgA anti-Proteus antibodies were significantly higher in patients with RF-positive RA compared with all other patient groups (P<0.0005 and P<0.005). Anti-P. mirabilis IgG, and IgG, IgA, and IgM antibodies to other potentially arthritogenic pathogens did not differ in the patient groups. IgM antibodies to E. coli were elevated in RF-positive RA patients. Anti-P. mirabilis IgM and IgA results were not explained by false-positive reactions, because after absorption of RF there was no decrease in antibodies to Proteus in 10 of 12 patients. Proteus and E. coli antibodies were highest in patients positive for both RF and anti-IgG-AGE antibodies (P<0.001). Patients with erosions tended to have higher IgA anti-Proteus titres, but no association with the shared HLA epitope or treatment was detected.

Conclusion. Anti-P. mirabilis IgM and IgA and anti-E. coli IgM antibody elevations are associated with early seropositive RA and the presence of anti-IgG-AGE antibodies. The role that P. mirabilis or E. coli plays in early RF-positive RA requires further investigation.

KEY WORDS: Proteus antibodies, Early synovitis, Rheumatoid arthritis, Rheumatoid factor, Spondylarthritis


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Rheumatoid arthritis (RA) is a systemic autoimmune disease that affects 0.5–1% of the general population, predominantly women, and is characterized by persistent inflammation and proliferation of the synovial tissue [1]. The resulting destruction of the inflamed joints leads to early disability and increased morbidity [2, 3]. The clinical heterogeneity of this disease is thought to be a reflection of the complex interactions of genetic and environmental factors that are involved in the susceptibility, course and outcome of this disease. Many infectious agents have been suggested to be involved in the aetiology of RA. However, to date their role in the direct pathogenic mechanisms that lead to RA has not been conclusively documented [4, 5].

Several studies of RA patients from different ethnic backgrounds have documented the presence of elevated levels of serum antibodies to Proteus mirabilis [6–11]. Raised levels of antibodies specific to P. mirabilis were observed in RA patients when compared with normal controls and patients with ankylosing spondylitis [12–14]. Furthermore, urine samples from patients with RA showed the presence of infection with P. mirabilis strains [15] of particular proticine types [16] more frequently than in those from healthy controls. A significant positive correlation between high anti-Proteus antibody levels in serum and the number of Proteus colony-forming units in urine specimens of RA patients supports the view that the urinary tract may be a source of the bacterial infection that contributes to the aetiology or pathogenesis of RA [17]. Although a pathogenic role for Proteus in the development of RA has not been defined, it has been suggested that molecular mimicry between an epitope shared either between HLA DRB1*0401 and P. mirabilis haemolysin or between type XI collagen and P. mirabilis urease could be a possible mechanism to activate autoimmune responses in the joint [18]. However, the lack of conclusive epidemiological data in the course of early RA and the lack of appropriate inflammatory controls in some of the other studies prompted us to examine the prevalence of antibodies to P. mirabilis, another Gram-negative bacteria associated with bacteruria, namely Escherichia coli, as well as to a number of other arthritogenic organisms in a heterogeneous cohort of early synovitis patients and to examine the potential diagnostic and prognostic value of the detection of elevated anti-Proteus antibodies. We also measured antibodies to immunoglobulin (Ig) G damaged by advanced glycation end-products (AGE) [19], and found that the RA patients with this reactivity were also positive for the anti-Proteus and anti-E. coli response. Our data suggest that recent infections with Proteus and or E. coli, as measured by IgM elevations, can be detected in patients with seropositive RA early in their disease.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients
A cohort of 246 patients with inflammatory arthritis for less than 1 yr involving one or more swollen joints was enrolled into an early synovitis study at the National Institute of Arthritis and Musculoskeletal and Skin Diseases (protocol 94-AR-0194). Patients had persistent synovitis of at least one peripheral joint for more than 6 weeks. Patients with traumatic, septic and crystal-induced arthritis were excluded. As previously reported [19], the patients were evaluated clinically, serologically and radiographically at the initial visit, and later at 6 weeks, 6 months and 1 yr. Diagnosis was established at the end of year 1. The number of swollen joints was determined by evaluating for the presence of effusion or synovial thickening or both in 66 peripheral joints, excluding hip joints. Anteroposterior and lateral radiographs of the hands and feet were taken at the initial visit and at the 1-yr follow-up visit and were evaluated for the presence of erosions. Erosions were defined as the presence of radiographic erosions of any involved joint, detected by 1 yr on either the initial or the 1-yr follow-up radiographs. The American College of Rheumatology (ACR) criteria for RA [20], and the European Spondylarthropathy Study Group (ESSG) criteria for spondylarthropathy [21] were applied to each member of the cohort based on the clinical, radiographic and laboratory data obtained. All patients not fulfilling either set of criteria were classified as having undifferentiated arthropathy (UA) for the purposes of this study.

This study was approved by the local medical ethics committee and patients gave their written informed consent.

Measurement of antibodies to P. mirabilis and other microbial organisms
Serum taken at the patients’ initial visit was assayed by enzyme-linked immunosorbent assay (ELISA) for antibodies to microbial organisms, including P. mirabilis, as previously described [15]. Eleven strains of P. mirabilis representing the 11 commonest O serotypes of strains associated with a P. mirabilis urinary tract infection [22], namely serotypes O3, O6, O10, O11, O13, O23, O24, O27, O28, O29 and O30, were each cultured overnight at 37°C on plates of cystine lactose electrolyte-deficient (CLED) medium (CM 301; Oxoid, Basingstoke, UK). Equal numbers of cells of all the 11 different O serotypes were pooled together and used as the source of Proteus antigen. The cells of each serotype were harvested and suspended in phosphate-buffered saline (PBS) pH 7.2 containing sodium azide 0.04% (PBS-Az) and then washed in this solution. The washed cells were resuspended in PBS-Az to an optical density (OD) (550 nm) of 1 and stored as a standard suspension at 4°C. Equal volumes of each of the standard 11 O serotype suspensions of P. mirabilis were then pooled. Aliquots (100 µl) of a 1 in 10 dilution of the pooled standard suspension in PBS-Az were added to flat-bottomed wells of microtitration plates (Maxisorb F8; Nunc, Nottingham, UK), which were covered and left overnight at room temperature. The contents of the wells were then discarded and the wells washed three times in water. PBS-Az containing Tween-20 0.05% (PBST) supplemented with bovine serum albumin 0.05% (200 µl) was added to each well and incubated for 1 h at room temperature. The contents of the wells were then discarded and the wells washed three times in water. One hundred microlitres of either serum diluted in PBST (for IgG, dilution was 1 in 1000; for both IgA and IgM, dilution was 1 in 100) or of PBST alone (control) was added to each well in triplicate and the plates were incubated for 2 h at room temperature. All sera were assayed for anti-Proteus antibodies with the laboratory blinded to the clinical status. The well contents were then discarded and the wells washed three times in water. Two hundred microlitres of goat anti-human alkaline phosphatase-conjugated IgG or IgA or IgM (Sigma, St Louis, MO, USA) diluted 1 in 1000 in PBST was added to the each well. After incubation for 1.5 h at room temperature, the contents of the wells were discarded and the wells washed three times in water. Two hundred microlitres of p-nitrophenyl phosphate 1 mg/ml in substrate buffer (100 mM sodium bicarbonate, 1 mM magnesium chloride, pH 10.2) was added to each well. After incubation at 37°C for 30 min the OD405 of each well of the plate was immediately measured by a Dias multiplate reader (Dynatech Medical Products, Guernsey, Channel Islands, UK). Results are expressed as the mean of three OD405 readings. Comparisons are made across all of the patient groups, thus providing groups of appropriately matched patients who served as controls. The reliability of the assay was as previously reported [15].

Antibodies (IgG, IgA and IgM) to E. coli were measured by ELISA. In brief, E. coli (ATCC 3521) washed in PBS were made to a stock solution at an OD550 of 1.0. ELISA plates (ICN, Montreal, QC, Canada) were coated with the E. coli diluted 1 part stock to 10 parts of a sodium carbonate/bicarbonate buffer, pH 9.4 at 4°C for 16 h. After three washes with PBS/0.1% Tween 20, sera were added at the appropriate dilution (for IgG, 1/2000; for IgA and IgM, 1/100) and incubated for 2 h at 37°C. Plates were washed three times and the appropriate horseradish peroxidase-conjugated (Fab'2) fragments of anti-human IgG, IgA or IgM (BioCan; Jackson Immunolabs, Mississauga, ON, Canada) were added. Dilutions in PBS/Tween of the antisera were 1/4000 (IgA, IgM) or 1/8000 (IgG). After 1 h incubation at 37°C, the plates were washed and the substrate o-phenylene diamine (2 mM in 0.02 M citric acid, 0.05 M Na2HPO4, 0.012% H2O2) was added. The reaction was stopped by the addition of 4 M H2SO4 approximately 30 min later. The OD492 (reference OD690) was measured using an ELISA plate reader (SLT Lab Instruments, Fisher, Montreal, QC, Canada). Antibody levels in sera were considered significantly elevated and positive if they were above the 95% confidence level of the entire cohort for the antibody in question. Since normal individuals have antibodies to these bacteria, it is not possible to assign positivity based on the mean plus 2 S.D. of normal controls.

Antibodies to a panel of possible arthritogenic organisms (Chlamydia trachomatis, Salmonella typhi, Shigella flexneri, Campylobacter jejuni, Yersinia enterocolitica and parvovirus B19) were measured by ELISA at a clinical laboratory (Specialty Laboratories, OncQuest, Santa Monica, CA, USA). Positive tests were defined as follows: C. trachomatis IgM, >0.45 EIA units; C. trachomatis IgG, >0.18 EIA units; C. jejuni, >3 S.D. above the mean of a reference group of normal subjects; Salmonella IgG, >10 U; Salmonella IgM, >20 U; Salmonella IgA, >20 U; Shigella IgG, >20 U; Yersinia IgG, >3 S.D. above the mean of a reference group of normal subjects; Yersinia IgM, >3 S.D. above the mean of a reference group of normal subjects; Yersinia IgA, >3 deviation above the mean of a reference group of normal subjects; parvovirus B19 IgG, >10 EIA units; parvovirus B19 IgM, >10 EIA units.

Detection of RF
RF was measured by nephelometry. In addition, in a subset of patients in one experiment the RF was also assayed by an alternative method in which 50 µl of serum diluted 20-fold in glycine–saline buffer, pH 8.2, was mixed with 50 µl of RF latex reagent (IgG-coated latex particles; Biostat Diagnostics, Stockport, UK), rocked for 2 min and then examined for agglutination of the latex particles. Positive and negative RF sera were included as controls in each batch of tests. Agglutination indicated the serum had an RF content of ≥20 IU/ml. Doubling dilutions of such positive sera were retested. The RF concentration was equal to the highest dilution of serum giving visible agglutination of the latex particles [15]. RF was removed from sera by repeated adsorption with latex beads coated in human IgG, and centrifugation to deposit the latex beads until the RF level of the serum supernatant was <20 IU/ml. The supernatant was then measured for IgM antibodies to P. mirabilis by ELISA.

Measurement of antibodies to IgG-AGE
IgM and IgA anti-IgG-AGE antibodies were detected in serum or plasma by ELISA as previously described [19, 23, 24], the testing laboratory being blinded to the diagnosis. IgGs of all four subclasses, which were fully glycated in vitro, were used at a concentration of 2 µg/ml (100 µl/well) to coat the wells of an ELISA plate (EIA; ICN, Montreal, QC, Canada). After washing the plates, 100 µl of the sera or plasma diluted 1 in 1000 were applied in duplicate to each well, and were incubated in the AGE-modified Ig-coated wells for 2 h at 37°C. After washing the plates in PBS/0.1% Tween 20, the bound antibodies were detected with peroxidase-conjugated F(ab')2 fragments of anti-human IgM, or IgA (Jackson Immunolabs) diluted 1 in 10 000 in PBS/0.1% Tween. To follow the reactivity over time and to maintain consistent results, a control serum from a normal individual (approximately the mean reactivity) and a positive control were tested each time the assay was performed. After washing the plates three times with PBS/0.1% Tween, 100 µl of the substrate o-phenylene diamine (2 mM in 0.02 M citric acid, 0.05 M Na2HPO4, 0.012% H2O2) was added. The reaction was stopped by the addition of 4 M H2SO4 approximately 30 min later. The OD492 (reference OD690; the reference wavelength is used as it gives better signal-to-noise ratios for the assay) was measured, using an ELISA plate reader (SLT Lab Instruments). Cut-off values were determined from the sera of 20 normal controls, and were the mean + 2 S.D.

HLA typing
Patients were HLA-typed for MHC class I and DR alleles by the molecular polymerase chain reaction-sequence specific primer method using sequence-specific primers, as previously described [25].

Statistical analysis
Statistical analyses were performed using SAS (Bethesda, MD, USA) and EpiInfo 16 statistical software for parametric and non-parametric comparisons between groups (Centers for Disease Control and Prevention, Atlanta; http://www.cdc.gov/epiinfo). Patient groups were compared using parametric and non-parametric statistics. Analysis of variance (ANOVA) and the Kruskal–Wallis test were used for continuous variables, and the {chi}2 test for proportions. Bonferroni correction was made for multiple comparisons, where appropriate.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
As previously reported, of the 246 patients with synovitis of recent onset in the cohort, 113 (46%) met the ACR criteria for RA [19]. Of the 246 patients, 43 (17%) fulfilled the ESSG criteria for a spondyloarthropathy (SpA), and 90 (37%) patients had undifferentiated arthritis (UA), respectively. The SpA group appeared atypical (Table 1), but this may reflect the fact that the ESSG criteria do not discriminate as well in an early synovitis cohort. Table 1 shows the demographic and clinical data of the patients studied. Radiographic erosions were either present at entry or developed during the year of follow-up. Of the patients with RF-positive RA, 40% had erosions that were either present at study entry or developed within the year of study follow-up, and 37% of RF-negative RA patients had erosions either at study entry or at the 1 yr follow-up evaluation. By comparison, only 12 and 15%, respectively, of patients with SpA or UA had radiographic erosions at 1 yr. As expected, a significantly larger number of patients with RA received treatment with prednisolone and/or DMARDs (P<0.005 and P<0.0001 respectively). All patient groups received similar levels of NSAID therapy.


View this table:
[in this window]
[in a new window]
 
TABLE 1. Demographic, clinical and treatment characteristics of the patients (for additional details of the cohort see references 19 and 25)

 
Anti-IgA and IgM P. mirabilis-specific serum antibody titres are elevated in patients with RF-positive RA
Figure 1 shows the anti-P. mirabilis antibody levels in the sera of all the patients at their initial visit to the National Institutes of Health. No significant differences in the means of the sera Proteus IgG antibodies were detected in patients with RF-positive RA, RF-negative RA, SpA or UA. In contrast, the means of sera anti-Proteus IgM and IgA antibody levels were both significantly higher in patients with RF-positive RA compared with all other patient groups (OD405 0.74 ± 0.29 vs 0.58 ± 0.20 (P<0.0003), and OD405 0.144 ± 0.057 vs 0.121 ± 0.049 (P<0.015), respectively).


Figure 1
View larger version (21K):
[in this window]
[in a new window]
 
FIG. 1. Levels of anti-Proteus IgG, IgM and IgA antibodies as determined by ELISA in the different patient groups. Grey box represents range; + within box represents mean; horizontal bar represents median; solid black circles indicate outliers. 95% confidence levels and outliers are indicated. *P = 0.005; **P = 0.02; comparisons were made between all four disease groups.

 
Proteus antibodies associate with markers of disease severity only in patients with RA
The results in Table 2 show that anti-Proteus IgA, IgM and IgG antibody levels correlated moderately with total serum IgA, IgM and IgG levels (r = 0.49, 0.45 and 0.28, respectively; P<0.0001). A moderate correlation between the titre of the IgM antibodies to P. mirabilis and the RF titre was observed (r = 0.46; P<0.0001). RF levels were more weakly correlated with the anti-Proteus IgA levels (r = 0.21; P<0.01). In contrast, anti-Proteus IgG levels did not correlate with the RF. In addition to the association with RF, anti-Proteus IgA was weakly associated with the ESR (r = 0.29; P<0.0001), total joint count (r = 0.2; P<0.05) and swollen joint count (r = 0.2; P<0.05). No association between anti-Proteus IgA, IgM or IgG levels and the presence of shared epitope alleles was noted (data not shown). Drug treatment with either DMARDs or prednisolone did not influence Proteus antibody titres (data not shown). There was no difference in antibiotic used in either group. Interestingly, there was a tendency for higher anti-Proteus IgA levels to be found in patients who had radiographic erosions at 1 yr when compared with patients who had no radiographic erosions [mean 0.14 ± 0.05 and 0.12 ± 0.05; median 0.13 (range 0.2) and 0.12 (range 0.26); P = 0.065]. No differences in IgG and IgM anti-Proteus antibody levels were seen in patients with and without erosions.


View this table:
[in this window]
[in a new window]
 
TABLE 2. Correlation coefficients (r) for markers of disease activity with Proteus antibody serum levels

 
Because of the correlation between IgM anti-Proteus antibody levels and RF, it was necessary to examine whether the IgM RF was causing a false-positive result in the Proteus assay. Therefore, sera from 12 patients with the highest levels of RF who also had high anti-Proteus IgM antibody titres were selected and RF was removed by repeated absorption with latex beads coated with human IgG. In 10 patients’ sera this resulted in no significant drop in the anti-Proteus IgM antibody level. In one patient's serum (patient 8) there was a significant drop in anti-Proteus IgM titre after removal of RF (Table 3). To further exclude the possibility that the Proteus assay was measuring the RF, the mean OD for the anti-Proteus IgM antibody of the RF positive patients was used as an arbitrary cut-off level to define high-positive anti-Proteus IgM patients. This new category comprised 58 patients, of whom 34 (59%) were patients with RA and 24 (41%) were patients with other forms of arthritis. Of the patients with high anti-Proteus levels, 38 (66%) were RF-positive and 20 (34%) patients were RF-negative. Similar results were obtained when anti-Proteus positivity was defined on the basis of elevated IgA levels or a combination of either IgA or IgM levels (data not shown). If RF was causing false-positive measures of IgM anti-Proteus, we would anticipate that all RF-positive individuals would be IgM anti-Proteus-positive, and this was not the case. Of the 41 RA patients with the lowest levels of IgM anti-Proteus antibodies, 11 had markedly elevated RF, with levels of 80–598 IU/ml.


View this table:
[in this window]
[in a new window]
 
TABLE 3. Effect of removal of RF on IgM antibody levels to P. mirabilis in sera

 
Anti-Proteus IgM and IgA antibodies were highest in sera positive for RF and anti-IgG-AGE antibodies
Because the immune response to IgG damaged by AGE has been shown to be linked to the RF response [23, 24] in other cohorts, it was of interest to divide the patients in the present cohort according to those that were positive for both RF and anti-IgG-AGE antibodies; those positive for the RF alone; those positive for anti-IgG-AGE antibodies alone; and those negative for both RF and anti-IgG-AGE antibodies. Table 4 summarizes the results obtained from this stratified analysis. Anti-Proteus IgM and IgA antibody levels were more significantly elevated in patients who were positive for both RF and anti-IgG-AGE antibodies than in any of the other patient groups (Fig. 2). In contrast, anti-Proteus IgG antibody levels did not differ significantly in the different groups.


View this table:
[in this window]
[in a new window]
 
TABLE 4. Analysis of the cohort of patients with RF and/or anti-IgG-AGE antibodies for the ability to make anti-Proteus antibodies

 

Figure 2
View larger version (22K):
[in this window]
[in a new window]
 
FIG. 2. Levels of anti-Proteus IgG, IgM and IgA antibodies as determined by ELISA in patient groups subdivided according to positivity for RF and/or anti-IgG-AGE antibodies. Grey box represents range; + within box represents mean; horizontal bar represents median; solid black circles indicate outliers. 95% confidence levels and outliers are indicated. *P = 0.0002; **P = 0.015 comparisons were made between all four groups.

 
Anti-IgM E. coli-specific antibody titres are observed in patients with RF-positive RA
We investigated the immune response to E. coli, the principal bacterium associated with bacteruria in the population at large. As can be seen from Table 5, there were no significant differences in the IgG, IgM antibody responses to E. coli for any patient group. The IgA response was slightly elevated in the RA group when compared with the UA patients, but not when compared with the SpA group. Interestingly, however, the RF-positive RA subgroup had a significantly elevated reactivity for IgM anti-E. coli compared with the RF-negative RA group [median (95% CI): 0.488 (0.593–1.021) and 0.288 (0.337–0.614); P = 0.023]. Of the RF-positive RA patients, 26 of 57 (46%) were IgM anti-E. coli positive compared with 13 of 49 (27%) for the RF-negative group (P = 0.047; Fisher's exact test). There was a weak correlation between the total IgM and the IgM anti-E. coli response (r = 0.336; P = 0.0018), whereas the correlations between total IgA or IgG and the respective anti-E. coli subclasses were negligible (r = 0.26; P = 0.016 for IgA, and r = 0.159 for IgG). There was a correlation between RF and the IgM anti-E. coli response (r = 0.373; P<0.0001); however, by using F(ab'2) fragments of the anti-IgM used in the ELISA assay we had lowered the possibility of false positives caused by the RFs. Indeed, several individuals with very high RF titres were negative for IgM anti-E. coli antibodies. There was a significant correlation between the IgM and IgA anti-E. coli responses, with r = 0.55; P<0.0001, which is expected given the mucosal site of infection by these bacteria. There was no correlation observed between the IgA anti-E. coli response and the IgM RF.


View this table:
[in this window]
[in a new window]
 
TABLE 5. The median immune response to E. coli (95% CI) in the patient groups as measured by ELISA

 
Interestingly, there was a significant correlation between the IgM anti-P. mirabilis response and the IgM anti-E. coli response (r = 0.49; P = 0.0001), but only in the RF-positive RA patient subgroup, linking them as infectious organisms that colonize the mucosal surfaces. There was no significant correlation between the IgA anti-E. coli and IgA anti-P. mirabilis responses (r = 0.073). The elevations in the IgM response may suggest recent infections. As with the Proteus infections, there was a highly significant correlation between the IgM response to E. coli and the presence of anti-IgG-AGE antibodies [median (95% CI) for anti-E. coli response in those that had anti-IgG-AGE antibodies, 0.724 (0.662–1.133); in those that lacked anti-IgG-AGE antibodies, 0.288 (0.347–0.652); P = 0.0003]. There were no significant correlations between any disease activity scores, markers of inflammation or erosions or the presence of the shared epitope with the IgM immune response to E. coli. There was a weak association between the IgA anti-E. coli response and the use of prednisolone (r = 0.245; P = 0.0112). However, prednisolone use did not affect the IgM anti-E. coli response or the IgA or IgM anti-P. mirabilis response. We investigated the frequency of the antibody response to either or both of E. coli and P. mirabilis in the three patient groups, and found that 55% of RA patients had an IgM response to either or both bacteria, whereas the frequencies were lower in both the SpA (36%) and UA (34%) patients. Interestingly, in the RA patients there was a trend for elevated IgM responses being higher in females (39/70 or 56%) compared with males (18/37 or 49%). This is consistent with the fact that bacteruria and/or urinary tract infection is much more common in females than males.

Differences in antibody responses to other potential arthritogenic pathogens were not seen in the different disease groups
Analysis of the presence of antibodies to other potential arthritogenic pathogens, including C. trachomatis (IgG and IgM), C. jejuni (total Ig), S. typhi (IgG, IgM, IgA), S. flexneri (total Ig), Y. enterocolitica (IgG, IgM, IgA), and parvovirus B19 (IgG, IgM), was made to determine whether antibody titre differences could be detected in the different disease groups: RF-positive RA, RF-negative RA, SpA and UA. No significant differences in the frequency of antibody presence or levels (data not shown for antibody levels) were observed for any of the anti-pathogen antibodies listed (Table 6).


View this table:
[in this window]
[in a new window]
 
TABLE 6. Frequency of antibody responses to potentially arthritogenic pathogens in the different patient groups

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Multiple cross-sectional studies in various ethnic populations have demonstrated that elevated antibody titres to P. mirabilis are seen in patients with RA [6–13]. However, the patients examined in these studies had longstanding disease, and control subjects were either healthy or comprised patients with reactive arthritis or osteoarthritis. Thus, the patient populations were not appropriate for the evaluation of the diagnostic and prognostic significance of raised antibody production to P. mirabilis. We therefore examined the prevalence of anti-Proteus antibodies and their prognostic and diagnostic value in a diverse cohort of patients with recent onset of inflammatory arthritis.

In our cohort of patients with recent onset of synovitis, we demonstrate an association between RF-positive RA and anti-Proteus antibodies, specifically IgM and IgA but not IgG. We also examined the titres of the IgG anti-Proteus antibodies (data not shown) and there was no significant association found in this early synovitis cohort for the different patient groups or subgroups, unlike what was reported previously [6, 9]. Because IgG has a long half-life in serum (about a month) compared with IgM (about a week), it is not as reflective as the IgM levels of acute changes. Although the detection of IgM anti-Proteus antibodies could indicate a recent infection, elevations of both IgM and IgA antibodies are associated with an immune response in the mucosa. Elevations of both IgA and IgM in our patient cohort may therefore reflect an infection at a mucosal surface. Since patients from all groups had similar levels of anti-P. mirabilis IgG antibodies, it is evident that most of the individuals in the cohort had previously encountered this ubiquitous bacterium.

Since both P. mirabilis and E. coli are indigenous enterobacteria that inhabit our colons, a perturbation at that site could stimulate the antibody response. However, it also may be that the elevated serum levels of these antibodies in patients with RA may reflect an asymptomatic bacteruria. Indeed, it has been shown that P. mirabilis can be isolated more frequently from the urinary tract of women and men with RA than from controls [15, 17]. Furthermore, a significant positive correlation (r = 0.714) between anti-Proteus antibody levels in the serum and Proteus colony-forming units in the urine of RA patients has been reported [17]. Of RA patients with bacteria in the urine, P. mirabilis was twice as frequently present as E. coli, and represented 52% of the infections [15]. This is in contrast to urinary tract infections in the population at large, where E. coli is the most frequent pathogen [26]. These bacteriurial infections in the patients with RA were asymptomatic [17].

An examination of the E. coli immune response in our cohort indicated that the levels of all three isotypes were very similar in the patient groups, consistent with previous reports [11, 27–30], only the IgA anti-E. coli response being higher in the RA patients when compared with the undifferentiated arthritis group in the present study. We did find, however, a significant elevation in the IgM response to E. coli in the subgroup of early RA patients that were RF-positive. Senior et al. [7] previously reported a slight elevation of antibodies to E. coli, Klebsiella and Pseudomonas in RF-positive RA patients over that found in RF-negative RA patients with longstanding disease, but the differences did not reach statistical significance, whereas the difference between the anti-Proteus antibody response between the RF-positive and the RF-negative RA patients in that study was highly significant.

Individuals with a compromised mucosal immune response may be at higher risk of bacteruria or recurrent urinary tract infection, and this might be a predisposing factor for RA. It is interesting that we detected twofold elevated levels of IgM, indicating recent infection with both bacteria in patients with RA, compared with the other patient groups. Patients with RA who have secondary Sjögren's syndrome (SS) have been reported to have a significant increase in urinary tract infection: 35% compared with 4% in RA patients without SS [31]. A number of these SS patients had a history of recurrent urinary tract infection, but the infecting bacteria were not identified. In the present study, the patients were not systematically evaluated for the presence of SS.

It appears that the RF-positive subgroup of RA patients is particularly associated with elevated levels of anti-P. mirabilis IgM and IgA antibodies. This is consistent with a previous report [7]. We now extend this to elevated levels of IgM anti-E. coli. From the adsorption studies, this association with RF does not appear to be a result of a false-positive anti-P. mirabilis antibody analysis, consistent with a previous study [32]. Indeed many RF-positive patients were negative for antibodies to P. mirabilis. Many infections, bacterial, viral and parasitic, are associated with an RF response [33]. Recently it has been shown that efficient triggering of RF B cells by an immune complex (IC) containing CpG DNA required the co-ligation of surface RF and toll-like receptor-9 (TLR9) [34, 35]. The toll-like receptors [36] expressed on the B-cell surface or on the endosome can recognize several different bacterial products, such as lipopolysaccharide, peptidoglycan and CpG DNA. These receptors play an important role in the host's innate immune response and can contribute to the inflammatory response. It is possible that IgG-coated P. mirabilis or IgG-coated E. coli containing a number of different pathogen-associated molecules could trigger one or more of the toll-like receptors expressed by B cells and, together with the cross-linking of the RF expressed on the B cell, lead to the activation of RF-positive B cells.

Since it appears that B-cell activation is an outcome of infection by one of these Gram-negative bacteria, particularly in those susceptible to RA, it is possible that the link is due to a bacterial product that directly stimulates B cells. This might be OmpA, a 39-kDa outer membrane protein of P. mirabilis, which is a murine B-cell mitogen [37]. Interestingly, a previous study has shown that lipopolysaccharide can stimulate RF production in mice [38].

The striking association of the anti-Proteus and the anti-E. coli response and IgM antibodies to IgG that were damaged by AGE, however, is novel. It is likely that these bacterial infections, even when subclinical, can stimulate a local inflammatory response, which could lead to IgG damage. We have shown previously that the anti-IgG-AGE response is linked to the RF response both in this cohort and in others [19, 23, 24].

It was previously proposed that molecular mimicry might explain the association between Proteus infection and RA. It had been suggested that an extra-articular infection might trigger arthritis through the mimicry of two different sets of bacterial proteins and host proteins [39]. The mimicked proteins postulated to play a role included the epitope motif EQRRAA of HLA DRB1*0401, which is similar to the motif ESRRAL found in P. mirabilis haemolysin, and the epitope LRREI on type XI collagen shared with P. mirabilis urease [18, 35, 40, 41]. There is as yet neither conclusive epidemiological data nor direct proof that these bacteria or bacterial proteins induce arthritis. Moreover, in the present study no association was found between the shared HLA epitope linked to RA and the presence of elevated levels of P. mirabilis antibodies.

The possibility of other potentially arthritogenic bacteria playing a role in the development of early synovitis in the cohort was investigated, but there was no evidence that prior infection with any of these was more prevalent in the RA group with and without RF than in the other groups.

Our study indicates that, in patients with RA of less than 1 yr, elevated levels of anti-Proteus IgM and IgA and anti-E. coli IgM antibodies can be detected. Since IgG antibodies specific for these bacteria are also present, we cannot conclude that it is a primary infection that is associated with the onset of RA. Rather, it may be that individuals who develop RA have a propensity for recurrent subclinical bacterial infections, but how this relates to RA pathogenesis is not known as yet. In the case of the E. coli infections in RA, prednisolone use may be a contributing factor that needs to be considered. It is of interest that the anti-Proteus IgA but not the anti-E. coli antibodies appeared to be of some clinical significance, because higher levels of such antibodies were detected in individuals who had erosions compared with those that did not. In addition, there was a weak association of the IgA but not the IgM anti-Proteus antibodies with ESR and both total joint and swollen joint counts. The active inflammation induced by the bacterial infection may be important in the light of the association with anti-IgG-AGE antibodies. How the P. mirabilis or even the E. coli infection or the antibodies, or both, contribute to the disease process is still unresolved, but such infections do appear to be an early event in the disease course of seropositive RA.
Figure 3


    Acknowledgments
 
We acknowledge the invaluable contributions of Marianna Crane, Cheryl Yarboro and Drs Thurayya Arayssi, Jose Pando, Percio Gulko, Richard Siegel, Michael Froncek and Robert Ortmann in the clinical evaluations of the patients, Sheila Laku, Nicole Saba, Sharon Suson, C. Deville and Joseph Hoxworth for their excellent technical help, and Dr Robert Wesley for help with the statistical analyses. We thank Drs Michael Ward and Peter Lipsky for their insightful review of the manuscript.

The authors have declared no conflicts of interest.


    Notes
 
*The first two authors contributed equally to this work. Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Fox D. Etiology and pathogenesis of rheumatoid arthritis. In: Koopman WJ, ed. Arthritis and allied conditions. 14th edn. Philadelphia: Lippincott, Williams & Wilkins, 2001:1085–102.
  2. Gabriel SE, Crowson CS, O'Fallon WM. Mortality in rheumatoid arthritis: have we made an impact in 4 decades? J Rheumatol 1999;26:2529–33.[ISI][Medline]
  3. Young A, Dixey J, Kulinskaya E et al. Which patients stop working because of rheumatoid arthritis? Results of five years’ follow up in 732 patients from the Early RA Study (ERAS). Ann Rheum Dis 2002;61:335–40.[Abstract/Free Full Text]
  4. El Gabalawy HS, Duray P, Goldbach-Mansky R. Evaluating patients with arthritis of recent onset: studies in pathogenesis and prognosis. JAMA 2000;284:2368–73.[Abstract/Free Full Text]
  5. Albani S, Keystone EC, Nelson JL et al. Positive selection in autoimmunity: abnormal immune responses to a bacterial dnaJ antigenic determinant in patients with early rheumatoid. Nat Med 1995;1:448–52.[CrossRef][ISI][Medline]
  6. Rashid T, Darlington G, Kjeldsen-Kragh J, Forre O, Collado A, Ebringer A. Proteus IgG antibodies and C-reactive protein in English, Norwegian and Spanish patients with rheumatoid arthritis. Clin Rheumatol 1999;18:190–5.[CrossRef][ISI][Medline]
  7. Senior BW, McBride PD, Morley KD, Kerr MA. The detection of raised levels of IgM to Proteus mirabilis in sera from patients with rheumatoid arthritis. J Med Microbiol 1995;43:176–84.[Abstract]
  8. Blankenberg-Sprenkels SD, Fielder M, Feltkamp TW, Tiwana H, Wilson C, Ebringer A. Antibodies to Klebsiella pneumoniae in Dutch patients with ankylosing spondylitis and acute anterior uveitis and to Proteus mirabilis in rheumatoid arthritis. J Rheumatol 1998;25:743–7.[ISI][Medline]
  9. Chou CT, Uksila J, Toivanen P. Enterobacterial antibodies in Chinese patients with rheumatoid arthritis and ankylosing spondylitis. Clin Exp Rheumatol 1998;16:161–4.[ISI][Medline]
  10. Wanchu A, Deodhar SD, Sharma M, Gupta V, Bambery P, Sud A. Elevated levels of anti-proteus antibodies in patients with active rheumatoid arthritis. Indian J Med Res 1997;105:39–42.[ISI][Medline]
  11. Subair H, Tiwana H, Fielder M et al. Elevation in anti-Proteus antibodies in patients with rheumatoid arthritis from Bermuda and England. J Rheumatol 1995;22:1825–8.[ISI][Medline]
  12. Tani Y, Tiwana H, Hukuda S et al. Antibodies to Klebsiella, Proteus, and HLA-B27 peptides in Japanese patients with ankylosing spondylitis and rheumatoid arthritis. J Rheumatol 1997;24:109–14.[ISI][Medline]
  13. Khalafpour S, Ebringer A, Abuljadayel I, Corbett M. Antibodies to Klebsiella and Proteus microorganisms in ankylosing spondylitis and rheumatoid arthritis patients measured by ELISA. Br J Rheumatol 1988;27(Suppl. 2):86–9.
  14. Maki-Ikola O, Penttinen M, Von Essen R, Gripenberg-Lerche C, Isomaki H, Granfors K. IgM, IgG and IgA class enterobacterial antibodies in serum and synovial fluid in patients with ankylosing spondylitis and rheumatoid arthritis. Br J Rheumatol 1997;36:1051–3.[CrossRef][ISI][Medline]
  15. Senior BW, Anderson GA, Morley KD, Kerr MA. Evidence that patients with rheumatoid arthritis have asymptomatic ‘non-significant’ Proteus mirabilis bacteriuria more frequently than healthy controls. J Infect 1999;38:99–106.[CrossRef][ISI][Medline]
  16. Wilson C, Senior BW, Tiwana H, Capparros-Wanderley W, Ebringer A. Antibiotic sensitivity and proticine typing of Proteus mirabilis strains associated with rheumatoid arthritis. Rheumatol Int 1998;17:203–5.[CrossRef][ISI][Medline]
  17. Wilson C, Thakore A, Isenberg D, Ebringer A. Correlation between anti-Proteus antibodies and isolation rates of P. mirabilis in rheumatoid arthritis. Rheumatol Int 1997;16:187–9.[CrossRef][ISI][Medline]
  18. Tiwana H, Wilson C, Alvarez A, Abuknesha R, Bansal S, Ebringer A. Cross-reactivity between the rheumatoid arthritis-associated motif EQKRAA and structurally related sequences found in Proteus mirabilis. Infect Immun 1999;67:2769–75.[Abstract/Free Full Text]
  19. Newkirk MM, Goldbach-Mansky R, Lee J et al. Advanced glycation end-product (AGE)-damaged IgG and IgM autoantibodies to IgG-AGE in patients with early synovitis. Arthritis Res Ther 2003;5:R82–90.[CrossRef][ISI][Medline]
  20. Arnett FC, Edworthy SM, Bloch DA et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988;31:315–24.[ISI][Medline]
  21. Dougados M, van der Linden S, Juhlin R et al. The European Spondylarthropathy Study Group preliminary criteria for the classification of spondylarthropathy. Arthritis Rheum 1991;34:1218–27.[ISI][Medline]
  22. Larsson P, Olling S. O antigen distribution and sensitivity to the bactericidal effect of normal human serum of Proteus strains from clinical specimens. Med Microbiol Immunol (Berl) 1977;163:77–82.[CrossRef][ISI][Medline]
  23. Ligier S, Fortin PR, Newkirk MM. A new antibody in rheumatoid arthritis targeting glycated IgG: IgM anti-IgG-AGE. Br J Rheumatol 1998;37:1307–14.[CrossRef][ISI][Medline]
  24. Lucey MD, Newkirk MM, Neville C, LePage K, Fortin PR. Association between IgM response to IgG damaged by glyoxidation and disease activity in rheumatoid arthritis. J Rheumatol 2000;27:319–23.[ISI][Medline]
  25. El-Gabalawy HS, Goldbach-Mansky R, Smith D et al. Association of HLA alleles and clinical features in patients with synovitis of recent onset. Arthritis Rheum 1999;42:1696–705.[CrossRef][ISI][Medline]
  26. Neu HC. Urinary tract infections. Am J Med 1992;92:63S–70S.[CrossRef][Medline]
  27. Aoki S, Yoshikawa K, Yokoyama T et al. Role of enteric bacteria in the pathogenesis of rheumatoid arthritis: evidence for antibodies to enterobacterial common antigens in rheumatoid sera and synovial fluids. Ann Rheum Dis 1996;55:363–9.[Abstract]
  28. Maki-Ikola O, Hallgren R, Kanerud L, Feltelius N, Knutsson L, Granfors K. Enhanced jejunal production of antibodies to Klebsiella and other Enterobacteria in patients with ankylosing spondylitis and rheumatoid arthritis. Ann Rheum Dis 1997;56:421–5.[Abstract/Free Full Text]
  29. Deighton CM, Gray JW, Bint AJ, Walker DJ. Anti-Proteus antibodies in rheumatoid arthritis same-sexed sibships. Br J Rheumatol 1992;31:241–5.[Medline]
  30. Fielder M, Tiwana H, Youinou P et al. The specificity of the anti-Proteus antibody response in tissue-typed rheumatoid arthritis (RA) patients from Brest. Rheumatol Int 1995;15:79–82.[CrossRef][ISI][Medline]
  31. Tishler M, Caspi D, Almog Y, Segal R, Yaron M. Increased incidence of urinary tract infection in patients with rheumatoid arthritis and secondary Sjogren's syndrome. Ann Rheum Dis 1992;51:604–6.[Abstract]
  32. Otto R, Kohler W. Inhibition of the rheumatoid factor (RF) in hemagglutination and Proteus agglutination tests by bacteria and their lipopolysaccharides. Z Immunitatsforsch Allerg Klin Immunol 1967;134:235–48.[Medline]
  33. Newkirk MM. Rheumatoid factors: host resistance or autoimmunity? Clin Immunol 2002;104:1–13.[CrossRef][ISI][Medline]
  34. Leadbetter EA, Rifkin IR, Hohlbaum AM, Beaudette BC, Shlomchik MJ, Marshak-Rothstein A. Chromatin-IgG complexes activate B cells by dual engagement of IgM and Toll-like receptors. Nature 2002;416:603–7.[CrossRef][ISI][Medline]
  35. Viglianti GA, Lau CM, Hanley TM, Miko BA, Shlomchik MJ, Marshak-Rothstein A. Activation of autoreactive B cells by CpG dsDNA. Immunity 2003;19:837–47.[CrossRef][ISI][Medline]
  36. Takeda K, Kaisho T, Akira S. Toll-like receptors. Annu Rev Immunol 2003;21:335–76[CrossRef][ISI][Medline]
  37. Korn A, Kroll HP, Berger HP et al. The 39-kilodalton outer membrane protein of Proteus mirabilis is an OmpA protein and mitogen for murine B lymphocytes. Infect Immun 1993;61:4915–8.[Abstract]
  38. Izui S, Eisenberg RA, Dixon FJ. IgM rheumatoid factors in mice injected with bacterial lipopolysaccharides. J Immunol 1979;122:2096–102.[Abstract]
  39. Ebringer A, Wilson C, Tiwana H. Is rheumatoid arthritis a form of reactive arthritis? J Rheumatol 2000;27:559–63.[ISI][Medline]
  40. Wilson C, Ebringer A, Ahmadi K et al. Shared amino acid sequences between major histocompatibility complex class II glycoproteins, type XI collagen and Proteus mirabilis in rheumatoid arthritis. Ann Rheum Dis 1995;54:216–20.[Abstract]
  41. Ebringer A, Wilson C. HLA molecules, bacteria and autoimmunity. J Med Microbiol 2000;49:305–11.[Abstract/Free Full Text]
Submitted 16 May 2005; revised version accepted 24 June 2005.



This Article
Abstract
Full Text (PDF)
All Versions of this Article:
44/11/1433    most recent
kei036v1
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Search for citing articles in:
ISI Web of Science (1)
Disclaimer
Request Permissions
Google Scholar
Articles by Newkirk, M. M.
Articles by El-Gabalawy, H. S.
PubMed
PubMed Citation
Articles by Newkirk, M. M.
Articles by El-Gabalawy, H. S.
Related Collections
Rheumatoid Arthritis