1Toronto Western Research Institute, University Health Network, 2Department of Medicine, University of Toronto and 3Department of Medicine, Memorial University, St John's, Newfoundland, Canada.
Correspondence to: R. D. Inman, Arthritis Center of Excellence, Division of Rheumatology, Toronto Western Hospital, Toronto, Ontario M5T 2S8, Canada. E-mail: robert.inman{at}uhn.on.ca
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Abstract |
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Methods. Twenty-five families with two or more individuals affected with AS and 34 normal healthy controls matched with the affected family members for age, sex and ethnicity were enrolled in the study. All affected (n = 57) and unaffected (n = 37) family members had a detailed clinical evaluation. Peripheral blood was drawn to determine T-lymphocyte proliferation and the IgG and IgA (by ELISA analysis) immune responses to K. pneumoniae, Salmonella typhimurium, Yersinia enterocolitica and Chlamydia trachomatis. Immune responses to each of the four candidate organisms were compared in affected and unaffected individuals. Each individual was classified by the predominant antigenic immune response that they showed when comparison was made among the same concentrations of the four candidate microbial antigens. This stratification was then used (i) to compare immune responses in affected and unaffected family members and (ii) to compare clinical characteristics of affected family members.
Results. There was no difference in mean stimulation indices or antibody responses between affected and unaffected family members for each of the candidate organisms. In terms of predominant cellular immune responses to these organisms, there was no difference between affected and unaffected family members with respect to K. pneumoniae, C. trachomatis or Y. enterocolitica. However, a higher percentage of affected family members (25.9%) exhibited a predominant response to S. typhimurium compared with unaffected family members (5.9%, P < 0.02). In assessing antibody titres, K. pneumoniae was the predominant amongst these four organisms, but there was no difference between affected family members, unaffected family members and normal healthy controls. There was no relationship between immune responses and clinical characteristics.
Conclusion. Our analysis of affected and unaffected family members in familial AS demonstrated no significant differences with respect to cellular or humoral immune responses to K. pneumoniae and three control microbes. In addition, K. pneumoniae did not exhibit a predominant immune response in affected individuals. Thus we find no supportive evidence to implicate a causal role for K. pneumoniae in familial AS.
KEY WORDS: Ankylosing spondylitis, Klebsiella pneumoniae, HLA-B27, Immune responses.
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Introduction |
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This potential infectious relationship has led to several theories to explain a possible link between K. pneumoniae and AS. First, the theory of molecular mimicry postulated antigenic cross-reactivity of microbial and host determinants. It was demonstrated, for example, by Schwimmbeck et al. [5] that there was a degree of sequence homology between HLA-B27 and the nitrogenase enzyme of K. pneumoniae. However, molecular mimicry, while a common biochemical reality, has been difficult to prove as a pathogenic mechanism [6, 7]. Secondly, K. pneumoniae antigens may be capable of specifically modifying HLA-B27 or a hypothetical B27-associated receptor, and the resulting tissue damage is caused by cytolytic attack against the infecting organism [8]. Support for this notion came from the observation that cytotoxic T lymphocytes might recognize certain secreted bacterial antigens in association with HLA-B27, suggesting that this interaction may contribute directly to an immune-mediated inflammation during the initial stages of the disease [9, 10]. Again, these events have proved difficult to prove rigorously as pathogenic mechanisms.
Indirect evidence implicating K. pneumoniae in AS has come largely from studies of host immune response [1116]. Such an approach has in some studies shown increased serum antibody responses to K. pneumoniae [17, 18]. However, there remains significant controversy in the literature over the specificity of antibodies against K. pneumoniae. A recent study found no evidence for AS-specific serological responses to multiple serotypes of K. pneumoniae in a cohort of 187 AS patients compared with 195 controls [19]. In addition, the role of Klebsiella has proved difficult to integrate into a testable hypothesis of microbial pathogenesis. Attempts to identify Klebsiella DNA by the polymerase chain reaction in sacroiliac biopsies of AS patients have not been successful, so there is little to support localization of the organism in target joints [20]. In addition, no improvement in disease activity has been demonstrated with antibiotic therapy [21], which further weakens the argument for the role of at least an active infection in AS.
The objective of this casecontrol study was to compare the immune profile of AS patients in multiplex families with that of unaffected family members and normal healthy controls in order to find out whether there was evidence of a predominant immune response to K. pneumoniae in AS.
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Methods |
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There were two control groups. Control group A consisted of 39 unaffected family members and control group B consisted of 34 normal healthy individuals. Group A was defined as unaffected by the following criteria: no history of inflammatory back pain, and no radiographic changes consistent with sacroiliitis for which a history suggestive of inflammatory back pain was elicited. Unaffected family members were chosen as controls for shared environmental influences in the respective families. Group B was a cohort of 34 individuals who were matched for age, sex and ethnicity with the family members. These normal subjects were not subjected to a physical examination, but a detailed history was taken, and any individual with a personal or family history of arthritis was excluded.
Lymphocyte stimulation
For each patient, peripheral blood was collected into sterile heparinized tubes. Peripheral blood mononuclear cells (PBMC) were isolated by FicollHypaque density gradient (Pharmacia, Uppsala, Sweden), washed three times in phosphate-buffered saline (PBS) and resuspended in RPMI 1640 medium (Gibco-BRL, Rockville, MD, USA) containing 10% heat-inactivated fetal calf serum (Gibco-BRL). The number of viable lymphoid cells used for the stimulation studies was 0.5 x 106 cells/ml. Lymphocytes were cultured in 96-well flat-bottom tissue culture plates. The candidate microbe in question, K. pneumoniae serotype K43, was obtained from Dr A. Geczy (Melbourne, Australia). As control microbes, Chlamydia trachomatis (L2 strain), Salmonella typhimurium and Yersinia enterocolitica were used. The last two were clinical isolates from patients with reactive arthritis. Bacteria were cultured under routine conditions then heat-killed, and the protein concentration was determined. The microbial antigens were added at concentrations of 1, 5 and 10 µg/well for each respective organism. Phytohaemagglutin (PHA) was included as a mitogen control at 1 µg/ml. All determinations were performed in triplicate. The plates were incubated at 37°C in 5% CO2 for 4 days, then the lymphocytes were pulsed with 3H-thymidine (Amersham, Amersham, UK) at 1 µCi/well. After 18 h of incubation the cells were harvested onto glass-fibre filters and the incorporated radioactivity was determined in a liquid scintillation counter (Beckman LS-800). Results are expressed in counts per minute (c.p.m.) and presented using the stimulation index (SI), which is defined as the ratio of c.p.m. in response to candidate microbes to c.p.m. induced with medium alone.
ELISA studies
The enzyme-linked immunosorbent assay (ELISA) studies were carried out using standard methods as published by our laboratory [26]. The heat-killed organisms outlined above were coated onto ELISA plates (Costar-Fisher Scientific, Nepean, Ontario, Canada) at 1, 5 and 10 µg/well and kept at 4°C with 5% CO2. The plates were then washed six times with PBS, 0.2 ml of blocking solution (PBS and 1% bovine serum albumin) was added and the plates were incubated at room temperature for 1 h. The blocking solution was then discarded. Test serum (0.2 ml of 1:100 dilution) was added to each well in triplicate and the plates were incubated at 37°C for 1.5 h. The plates were then washed six times with PBS containing 0.05% Tween 20. Horseradish peroxidase-conjugated secondary antibody (Fab'2; Serotec, Raleigh, NC, USA) was added at a dilution of 1:10 000 (0.2 ml per well) and the plates were incubated at 37°C for 1.5 h. They were then washed six times and 0.2 ml of the substrate o-phenylenediamine was added to develop the plates. The optical density (OD) at 490 mm was determined with an ELISA reader.
Statistical analysis
Comparison of immune responses in affected and unaffected individuals
The cellular immune responses of affected and unaffected family members for each of the candidate organisms at each concentration (1, 5 and 10 µg/dl) were compared using a two-sample t-test. In the primary analysis, no threshold was set for the SI, so that small differences were not overlooked, but for secondary analysis a threshold was set at an SI of 3, as has been used in other studies, so that small differences might not be overinterpreted [27]. A similar analysis was performed for the humoral immune responses, comparing affected and unaffected family members and normal healthy controls.
Defining the predominant immune response
Next, each patient was classified by the predominant antigenic immune response elicited by one of the four candidate microbial antigens. For the proliferation analysis, this was obtained by comparing the SI for each patient in response to the same concentration of each of the four organisms (10 µg/dl). The predominant response was thus defined as the microbial antigen (of the four examined) that induced the maximal stimulation index. Thus, the predominant microbial response for each sample was mutually exclusive. There was an a priori proviso that the maximal stimulation index must exceed the next highest by 5%. In cases where there was <5% difference between the stimulation indices, a predominant microbial response was not assigned to that cell sample and the immune profile was defined as indeterminate. The analysis was repeated, as described above, after a threshold had been set for the SI of >3, so that small differences might not be overinterpreted. The predominant immune response was determined similarly for each sample for both T-cell proliferation analysis and antibody studies. However, in the case of the predominant antibody response, the maximal OD was selected from amongst the four microbial antigens (concentration 10 µg/dl). The percentage of affected and unaffected individuals who exhibited a predominant immune response for each of the four candidate organisms was recorded and the analysis described below was based on this.
Analysis based on the predominant immune response
The 2 test was used to detect differences between the percentages of affected individuals who exhibited a predominant T-cell immune response to K. pneumoniae and the percentage of unaffected individuals who exhibited a predominant T-cell immune response to this microbe. Fisher's exact test was used where the frequency in a particular cell was <5. A similar analysis was performed to detect a difference in predominant T-cell response between affected and unaffected individuals for each of the control microbes (C. trachomatis, Y. enterocolitica and S. typhimurium). The odds ratio (adjusted for family) with 95% confidence intervals (CI) was reported for the likelihood of affected status according to predominant selection for one of the candidate microbes.
Associations between clinical characteristics and immune responses
To explore any possible associations with clinical variables and a particular antimicrobial immune response, two separate analyses were performed. The first analysis included all AS patients and the second included only patients having primary AS. Primary AS was defined as AS in the absence of coexisting extra-articular features, such as IBD, psoriasis or reactive arthritis. Parametric (two-sample t-test) and non-parametric tests (Wilcoxon rank sum test and KruskalWallis test) were used to compare clinical variables in patients stratified by the predominant antigenic T-cell proliferative immune response. Finally, multiple logistic regression was performed for each of the four candidate microbes to determine if affected status could be predicted by the predominant antigenic T-cell proliferative immune response. The regression model was adjusted for age, sex and HLA-B27 status. The model was fitted manually. The small sample size dictated our restriction to three variables. Statistical analysis was performed using SAS (version 8.02; SAS Institute, Cary, NC, USA) and P < 0.05 was considered significant.
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Results |
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Cellular immune response
Eight patients had an indeterminate T-cell response; therefore in the final analysis results are presented on 54 affected and 34 unaffected family members. There was no difference between affected and unaffected individuals either in mean baseline proliferation (affected, 2051 ± 2682 c.p.m.; unaffected 2315 ±5748 c.p.m.; P = 0.3) or in proliferation on stimulation with PHA (affected, 86 792 ± 49 824 c.p.m.; unaffected, 99 097 ± 68 604 c.p.m.; P = 0.6). There was no difference between affected and unaffected individuals when actual stimulation indices were compared for any of the candidate microbial antigens (Table 1, Fig. 1).
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Humoral immune response
There was no difference in humoral immune responses between affected and unaffected family members for any of the four candidate antigens. Neither was there a difference in responses between unaffected family members and healthy controls. However, affected family members exhibited a greater immunoglobulin (Ig) G immune response to all four microbes compared with normal healthy controls (Fig. 2).
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Discussion |
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As outlined above there has been much debate in the literature over the possible aetiopathogenic link between AS and K. pneumoniae [1, 8, 9, 11, 12, 28, 29]. Controversy persists as investigators have continued to report elevated concentrations of antibodies to this organism in the serum of patients with AS [11, 15, 30]. Most studies addressing microbial pathogenesis in AS have used only serum antibody profiles to implicate potential pathogens. There are far fewer studies evaluating T-cell responses to candidate organisms in AS, yet this may represent a more comprehensive approach to detecting an immune fingerprint of a recent or remote infection in an individual with AS.
This is the first study that compares affected with unaffected family members to address these issues in evaluating the role of K. pneumoniae in the pathogenesis of AS. Since one of the unique features of our study was the familial casecontrol design, we addressed whether there was any clustering of specific antimicrobial immune responses within families, but we saw none. This analysis was somewhat limited by the sample size. With that caveat, adjusting our analysis for family did not alter our results. It is interesting to note that our study showed that the majority of affected and unaffected family members responded predominantly to K. pneumoniae in the humoral studies. However, there was no difference between affected and unaffected individuals, which suggests that this may be an in vitro laboratory phenomenon related to the affinity of the antigen with the test plate. However, affected individuals exhibited a greater IgG response compared with normal healthy controls. This highlights the importance of careful selection of environmental controls, such as the unaffected family members used in this study. In addition, this observation may shed some light on the elevated serum anti-Klebsiella antibodies reported in previous studies in which affected individuals were compared with normal healthy controls as opposed to family members. Thus, while we confirm the results of prior investigations showing that there appears to be a heightened serum antibody pattern against K. pneumoniae, we find no specificity for AS. This speaks against a pathogenic role for this organism in AS.
The finding that S. typhimurium exhibited a predominant cellular immune response in affected individuals when compared with the remaining candidate microbes warrants further mention. It is unlikely that this finding provides substantive evidence of a causal role for S. typhimurium as no difference was observed for actual stimulation indices between affected and unaffected family members (P = 0.80). Furthermore, the results of the logistic regression analysis suggest that age or sex may have been confounders in the relationship between S. typhimurium and outcome (affected status), and this may have accounted for the difference observed between affected and unaffected individuals for the cellular immune responses. This also highlights the importance of careful consideration of clinical covariates in the interpretation of the cellular immune responses in these patients. Whether a disease process that began as a post-Salmonella reactive arthritis can evolve over time into a clinical picture that meets the definition of AS is an issue that has been raised in the literature before, but the frequency of this event is unknown.
There are relatively few studies addressing T-cell responses to Klebsiella antigens in the literature. Dominguez-Lopez et al. [27] evaluated T-cell responses in a small cohort of 13 AS patients. Using PBMC, seven of 13 patients with AS and some patients with RA, but no healthy controls, demonstrated a significant cellular immune response, defined in this study as SI > 3, to a GroEL-like protein (HSP60) of K. pneumoniae. However, numbers were small in this study and the antigen preparation used was different from that used in our study. Furthermore, the key control group was rheumatoid arthritis, whereas in our study the unaffected family members served as controls. Interestingly, these same authors also showed that HLA-B27-positive individuals recognized not only the GroEL-like HSP protein from K. pneumoniae but GroEL-like proteins from other organisms, such as Mycobacterium [12]. This suggests that the humoral immune response they have detected in AS may be related to HSP from a range of microbes, but with no specificity for K. pneumoniae.
In the only prior study addressing cellular immune responses in familial AS, Hohler et al. [31] reported that there was no difference in T-cell responses between affected and unaffected twins in a study of 11 monozygotic twins. Affected twins, however, had a decrease in the calculated frequency of K. pneumoniae-responsive T cells in peripheral blood lymphocytes (PBL) compared with healthy B27+ subjects. It was speculated that this quantitative reduction in specific T cells might reflect a defective host defence against Klebsiella, thereby allowing bacterial antigens to reach the joint. In this study also, various antigen preparations of K. pneumoniae were used for ELISA antibody studies; there were no differences between affected and unaffected for K21, but there was a lower response to K43 in affected subjects. In our study we used K43 as the antigen preparation as it has been the antigen used most consistently in prior studies implicating K. pneumoniae in the pathogenesis of AS [9, 13]. Interestingly, the cellular immune responses were lower in affected than in unaffected family members, though the difference was not significant.
Hermann et al. [32] also reported a quantitative reduction in K. pneumoniae-responsive T cells in the PBL of AS patients compared with healthy controls. Because of the suggestion in this study that PBMC responses to K. pneumoniae may be depressed in comparison with concurrent synovial fluid mononuclear cell responses (SFMC), we analysed paired samples PBMC and SFMC in four patients with AS. We found there was no significant difference in the pattern of peripheral blood and synovial fluid lymphocyte responses to K. pneumoniae in these patients (data not shown). However, the methods used in the studies by Hermann et al. differed from those used in our studies and this may account in part for the some of the differences in results.
In conclusion, our study is the largest to date to examine cellular and humoral immune responses to K. pneumoniae in affected vs unaffected individuals in familial AS. We did not find evidence that affected individuals exhibit a dominant immune response to K. pneumoniae in familial AS. Thus we find no supportive evidence supporting a causal role for K. pneumoniae in familial AS.
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Acknowledgments |
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Conflict of interest
The authors have declared no conflicts of interest.
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References |
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