IgG subclass antibodies to human and bacterial HSP60 are not associated with disease activity and progression over time in axial spondyloarthritis

For more than two decades, heat shock proteins (HSPs) have been known for their phylogenetically conserved composition and immune-modulating activities [1]. They are ubiquitous in cellular life and exist in both eukaryotic and prokaryotic cells, where their major role is to act as molecular chaperones. Over the past few years, it has become evident that, in addition to their function as intracellular chaperones, HSPs are also found in the cell membrane and outside the cell, presumably acting as indicators of the stress conditions and activating other cells, particularly cells of the immune system [2]. As a response to stress conditions, their expression level is increased to prevent aggregation of misfolded proteins [3]. Such conditions are prevalent during intracellular bacterial infections, where the highly conserved HSPs from the 60 kDa family (HSP60; also known as GroEL) act as potent stimulators of both the innate and adaptive immune systems [4].

Some rheumatic diseases, such as reactive arthritis (ReA) and Lyme disease, are associated with bacterial infections. In connection with this, it has been suggested that bacteria-related autoimmunity may be an important factor in the etiology of such diseases [5]. One hypothesis to explain the pathogenic mechanism after a bacterial infection is molecular mimicry, that is, sharing of linear or conformational epitopes common to microbial antigens and host cell molecules, giving rise to an inappropriate immune response [6]. Human HSP60, recently renamed HSPD1 in a proposed new nomenclature [7], shares more than 50% of its sequence with bacterial HSP60, and, consequently, antibody and T-cell recognition of human HSP60 have been investigated extensively in a number of studies. Such antibodies are found both in patients with different inflammatory diseases and in healthy individuals, indicating that shared epitopes between bacterial and human HSP60 may exist [8–13]. However, studies have yet to determine the pathogenic role for such autoantibodies and whether these are in fact cross-reactive [14].

SpA comprises a heterogeneous group of immune-mediated inflammatory diseases, which includes the bacteria-triggered (that is, Chlamydia trachomatis, Salmonella, Yersenia and Campylobacter jejuni) disease reactive arthritis (ReA). Even though a link to an antecedent infection is less clear for the other forms of SpA diseases [15], the group shares clinical, pathological and genetic features, and especially the link to human leukocyte antigen B27 (HLA-B27) is well-documented [16]. However, knowledge about how HLA-B27 may contribute to disease development is missing [17]. Moreover, the frequency of the HLA-B27 genotype varies in the different SpA diseases [16].

The most significant results revealing the importance of the host-pathogen interaction in SpA disease came from studies done in rodents [18]. Such studies demonstrated that HLA-B27-transgenic rats do not develop inflammatory pathology in the intestine or the joints as long as they are kept in a germ-free environment [19]. Human studies include transfection of human cell lines with HLA-B27 and investigation of a possible interaction with pathogenic arthritis-related bacteria through changes in cytokine profiles, T-cell responses or HLA-B27 expression. However, results from these studies seem to differ, depending on the investigated pathogens [20–22]. As in ReA, autoimmunity against HSP60 has also been speculated as a trigger mechanism in SpA disease development, but such studies are few, and often the patient material was split up into disease subgroups [13, 23, 24]. In addition, existing serological analyses targeting arthritis-related bacterial HSP60 in SpA patients were without subclass specificity and follow-up [13, 23, 24]. Furthermore, in these studies, it was not clear whether the purification method of the HSP60 antigens retained the in vivo conformation. Improvements of these parameters would increase not only the specificity in the IgG response but also the analysis of cross-reactivity and the ability to determine whether such antibodies are of importance in SpA pathogenesis. Recently, a serology study on females diagnosed with tubal factor infertility (TFI) was performed, and it was shown by ELISA that IgG antibodies to C. trachomatis HSP60 were of the IgG1 and IgG3 subclass and that no antibodies against human HSP60 could be detected [25]. In that study, it was demonstrated that determination of IgG1 subclass antibodies to chlamydial HSP60 increased the diagnostic value of the ELISA in identifying TFI.

Therefore, we adopted this approach and performed a study to analyze the quantified serum levels of IgG subclass antibodies to native bacterial and human HSP60 in a cohort of well-characterized SpA patients compared with an age- and gender-matched control group. We evaluated the association between antibody levels and disease severity assessed by clinical scoring comprising the Bath Ankylosing Spondylitis Metrology Index (BASMI), Bath Ankylosing Spondylitis Functional Index (BASFI), Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) and MRI changes in the spine and sacroiliac joints (SIJs) in addition to HLA-B27 status.

In the present study, we investigated the hypothesis of molecular mimicry as a pathogenic trigger in axial SpA. Potential cross-reaction regions were visualized in an alignment of the amino acid sequences of human and bacterial HSP60, which showed five regions with five to eight identical amino acid residues for all of the investigated HSP60 proteins, indicating the presence of potential epitopes (Figure 1). However, these protein regions may not represent epitopes for infection-induced antibodies, as most antibodies recognize conformational epitopes presented in the three-dimensional shape of the protein [37], and, even if cross-reactive antibodies were present, it would not prove causation of disease. Therefore, due to the lack of similar studies that include analysis of IgG subclass response to bacterial and human HSP60, this hypothesis is a target for further analysis [11, 23, 24, 38].

In our study, we found elevated levels of both IgG1 and IgG3 antibodies to human HSP60, indicating an increased autoimmune activity in the 39 axial SpA patients (Figure 4A). We found no correlations of IgG antibodies between bacterial and human HSP60, and there was no general elevation in levels of antibodies to bacterial HSP60 (Figures 4B-4D). In the present study, generation of antibodies to human HSP60 was independent of the presence of antibodies to bacterial HSP60, and cross-reactivity was not found (Figure 7). In addition, different subclasses (that is, IgG1 and IgG3) were found to be predominant in the antibody response to bacterial and human HSP60, thus strengthening the ability of the humoral immune response to maintain its specificity, even between similar antigenic molecules.

A significant elevation in levels of IgG1 antibodies against HSP60 from S. enterica Enteritidis, but not from C. jejuni and C. trachomatis, was also observed in the group of SpA patients compared with the control group (Figure 4). These findings suggest that more SpA patients have had an infection with Salmonella than with an infection with C. trachomatis or C. jejuni. They are in agreement with a high incidence in S. enterica Enteritidis infections in Denmark that increased from 1997 to 1999 [39]. The observation that a higher number of patients are positive for S. enterica Enteritidis than for C. trachomatis (Figures 4B, 5B and 5C) could be explained by the overweight of females (56%, Table 1), as postdysentery ReA is equally common in males and females, whereas postchlamydial ReA is much more common in males [40].

To investigate the IgG subclass response against bacterial antigens, we determined the IgG subclass response to C. pneumoniae and C. trachomatis (Figure 5). We found that the IgG antibody response against C. pneumoniae was of the IgG1 subclass (Figure 5A). Thus, the production of IgG3 antibodies was not a general response to bacterial antigens in SpA patients. Furthermore, as recently suggested by Carter et al., a previous infection with C. pneumoniae may be associated with SpA [41]. We therefore evaluated the association between the SpA disease and levels of antibodies to C. pneumoniae [34]. However, there was no statistical difference in the IgG1 antibody levels of SpA patients and controls. In 1999, Hannu et al. used microimmunofluorescence microscopy to detect antibodies to C. pneumoniae, and they found that 4 (approximately 10%) of 35 patients with ReA were highly positive. Of these four patients, three had had lower respiratory tract infection prior to the development of ReA, indicating a possible trigger of the disease [42]. We found that 84% of the SpA patients and 76.9% of controls had IgG1 antibodies to C. pneumoniae, but such antibodies were not correlated with the presence of chlamydial HSP60 antibodies. This finding indicates that a C. pneumoniae infection does not trigger production of antibodies to chlamydial HSP60, even though HSP60 of C. pneumoniae is highly similar to HSP60 of C. trachomatis (90%, Figure 1). This is in contrast to what was found when serum from TFI patients was analyzed for the presence of subclass antibodies [25]. Antibodies to both C. trachomatis and to C. trachomatis HSP60 were found in serum from TFI patients. However, although antibodies to chlamydial HSP60 were primarily IgG1, antibodies to C. trachomatis were predominantly IgG3. None of the TFI patients had antibodies to human HSP60, indicating that in this study no cross-reactive antibodies between human and bacterial HSP60 were found [25].

IgG1 and IgG3 have similar properties in terms of activating complement and binding of Fc receptors [43]. This may explain why they are often induced together, indicating a shared switch mechanism (via IL-21 stimulation), as reported by Péne et al. [44]. However, on the basis of the results of the present study, IgG3 was the predominant antibody subclass to human HSP60. Furthermore, a trend toward an inverse association between IgG1 and IgG3 antibodies to this antigen was seen (Figures 6C and 6D), indicating the presence of a fine-tuning mechanism, recently suggested to be IL-4 [45]. A study similar to ours showed the same distribution of the IgG subclass response to a self-antigen, peptidylarginine deiminase 4 (PADI4), in patients with rheumatoid arthritis (RA) [46]. However, in this study, the dominant subclass was IgG1 and not IgG3. The structure of the two molecules differs in that IgG3 is more flexible due to its longer hinge region [47]. They also differ in half-life (1 wk for IgG3 compared to 3 wk for IgG1), and their concentrations in serum vary (65% for IgG1 and 7% for IgG3) [48–50]. The presence of antibodies with a short half-life, as demonstrated in this study, indicates that the IgG3 antibodies against human HSP60 are continuously stimulated and produced. Thus, as suggested in other studies [51, 52], the response to human HSP60 seen in SpA patients (Figure 4A) seems to rely on the ability to quickly control and regulate this response. This may explain the decrease in high-level IgG3 against human HSP60 from 2006 to 2010 (Figure 6B) and the average decrease in disease parameters (Table 1). Both human HSP60 and PAD4I are found to be expressed in the synovium, and PAD4I is suggested to form complexes that activate complement proteins, thereby giving rise to the observed correlation between the detection of the protein and the intensity of tissue inflammation in RA [53]. Autoantibodies to human HSP60 may represent a response to a foreign extracellular antigen (that is, mitochondrial HSP60) arising from the ongoing inflammation in the synovial membranes. However, we found no correlation of the antibodies against bacterial and human HSP60 to any of the clinical parameters and MRI scores. Our SpA patients had, in general, low disease activity but a high level of HSP60 antibodies, suggesting that such antibodies did not reflect the disease activity. Nor was there any relation to disease progression in clinical, and MRI scores during the 4-yr observation period. In the present study, HLA-B27 status did not further explain the role of anti-human HSP60, as these antibodies were at the same level in HLA-B27-positive and HLA-B27-negative patients. This is in contrast to findings from a similar study by Dominguez-López et al., who investigated the total IgG (total) response to enterobacterial HSP60 in ankylosing spondylitis patients, one of the five disease entities defined within SpA [23]. They found that HLA-B27-positive patients and their healthy relatives had significantly higher IgG antibody levels to all enterobacterial HSP60 proteins than HLA-B27-negative healthy individuals. Furthermore, a correlation between IgG antibodies to E. coli and Shigella flexneri HSP60 and disease activity was observed in a later study [24]. In these studies, antibodies to human HSP60 were not determined, and patient disease characteristics were not included; therefore, the results cannot be directly compared to those in our study. It should be emphasized that in our study all patients analyzed had axial disease, which does not exclude subjects with concomitant peripheral disease, but it excludes those with only peripheral disease. Therefore data could be different in patients with peripheral SpA.

In summary, we found that IgG3 antibodies against human HSP60 were elevated in SpA patients and that such antibodies with a short half-life were present at both time points separated by more than 4 yr. Therefore, IgG3 against human HSP60 must be produced constantly in these patients, indicating a disease-related function. However, the antibodies were not cross-reactive to bacterial HSP60, indicating that bacteria do not seem to be involved at this stage of disease. Instead, we suggest that the autoantibodies to human HSP60 represent a response to a foreign extracellular antigen (that is, mitochondrial HSP60) arising from the ongoing inflammation in the synovial membranes of these axial SpA patients.

In a cohort of SpA patients with symptoms restricted to the axial skeleton, elevated levels of IgG1 and IgG3 antibodies to human HSP60 were determined by ELISA. These levels were significantly higher than those in a healthy control group of similar size matched for sex and age. Change in antibody levels differed for IgG1 and IgG3 over time, and there was an inverse relationship of IgG1 to IgG3 antibodies in the sera of the SpA patients. Generation of antibodies to human HSP60 was independent of the presence of antibodies to bacterial HSP60, and cross-reactivity could not be supported from the present study. Finally, we found no evidence that HSP60 antibodies reflected the disease activity in these 39 SpA patients.