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  Table of Contents    
ORIGINAL ARTICLE  
Year : 2013  |  Volume : 56  |  Issue : 3  |  Page : 231-237
Role of 30 kDa antigen of enteric bacterial pathogens as a possible arthritogenic factor in post-dysenteric reactive arthritis


1 Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
2 Department of Experimental Medicine and Biotechnology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
3 Department of Internal Medicine, Postgraduate Institute of Medical Education and Research, Chandigarh, India

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Date of Web Publication24-Oct-2013
 

   Abstract 

Background: Reactive arthritis (ReA)/Reiter's syndrome (RS) may be caused as a sequel of infections caused by enteric bacterial pathogens, although the mechanisms through, which different pathogens cause similar disease are not clear. Aim: This study was done to look for the presence and role of any common bacterial antigen among the pathogens isolated from such patients. Materials and Methods: A total of 51 patients of ReA and 75 controls (three groups of 25 subjects each: Group 1: Patients who did not develop arthritic complications within 3 months after bacillary dysentery/diarrhea; Group 2: Patients with other arthritic diseases and Group 3: Normal healthy subjects) were included. The isolated enteric pathogens were tested to detect the immunodominant antigens. Results and Conclusions: A common 30 kDa antigen was found to be specifically present among seven arthritogenic enteric bacterial strains belonging to three genera, Salmonella, Shigella and Hafnia. Post-dysenteric ReA patients' sera show higher levels of immunoglobulin G, immunoglobulin M and immunoglobulin A antibodies against this antigen as compared to the controls. Lymphocytes of ReA patients recognize this antigen, proliferate and produce interleukin-2 in response to this antigen more than the lymphocytes of controls. 30 kDa antigen may be a common arthritogenic factor associated with post-dysenteric ReA/RS. The association of Hafnia alvei with post-dysenteric ReA is described for the first time. Four cases of mycobacterial ReA had an association with this antigen, suggesting that the arthritogenic antigen of mycobacteria and enteric bacteria may be of a similar nature.

Keywords: Enteric infections, post-dysenteric arthritis, reactive arthritis

How to cite this article:
Singh M, Ganguli N K, Singh H, Deodhar S D, Sethi S, Sharma M. Role of 30 kDa antigen of enteric bacterial pathogens as a possible arthritogenic factor in post-dysenteric reactive arthritis . Indian J Pathol Microbiol 2013;56:231-7

How to cite this URL:
Singh M, Ganguli N K, Singh H, Deodhar S D, Sethi S, Sharma M. Role of 30 kDa antigen of enteric bacterial pathogens as a possible arthritogenic factor in post-dysenteric reactive arthritis . Indian J Pathol Microbiol [serial online] 2013 [cited 2020 May 25];56:231-7. Available from: http://www.ijpmonline.org/text.asp?2013/56/3/231/120373



   Introduction Top


A characteristic joint disease known as reactive arthritis (ReA) (sometimes complicates certain gastrointestinal tract infections caused by bacteria like Salmonella, Shigella and Yersinia spp., [1] and respiratory and genito-urinary tract infections caused by Chlamydia spp. [2] However, ReA is also seen in individuals with no evidence of initiating infection. Enteric and diarrheal diseases are widely prevalent in India; hence, the possibility of post-dysenteric ReA in India is quite high. [3] Approximately, one-third of ReA cases lead to a more complicated form of arthritis known as Reiter's syndrome (RS), which is a triad of arthritis, conjunctivitis and urethritis. The risk of developing arthritis after an episode of bacillary dysentery has been shown to be 1-4%, but it increases to 20-30% in genetically susceptible individuals (HLA-B27 positive). [4],[5] Despite extensive research over recent years, the role of HLA-B27 in the pathogenesis of ReA remains unanswered. The association between HLA-B27 and ReA suggests interplay between host factors and microbes in the pathogenesis of ReA. Another feature of this disease is that irrespective the site of infection or the infecting organism, the symptoms of the disease remains similar. It is not clear, how the infecting organisms belonging to different genera cause a similar post-infection squeal. It could be possible that arthritogenic microorganisms express some common antigenic polypeptide that might be playing a role in the pathogenesis of ReA. Hence, this study was undertaken to study the immunodominant antigens, if any, in enteric bacterial isolates from ReA/RS patients from India.


   Materials and Methods Top


Study population

A total of 51 patients clinically diagnosed as cases of post-dysenteric ReA or RS as per previously described criterion, [6],[7] attending the rheumatology clinic of our institute were included in the study to screen their intestinal flora. The study was approved by the Institute Ethics Committee and written informed consent was taken from all patients.

Three control groups were studied - Group 1: 25 patients without arthritis up to 3 months after bacterial dysentery or diarrhea; Group 2: 25 patients with other arthritic diseases confirmed by clinical and diagnostic methods (rheumatoid arthritis 8; ankylosing spondylitis 7; tubercular septic/ ReA 5; osteoarthritis 5); and Group 3: 25 normal healthy individuals.

Isolation of enteric pathogens

Stool specimens were collected on three successive days from each participant in Cary and Blair's transport medium. Blood samples were collected in plain vials from all subjects and sera separated. Each serum sample was divided into three aliquots and stored at -70°C. Stool samples were processed for isolation of enteric pathogens known to be associated with ReA/RS by standard procedures. [8],[9],[10],[11] Samples were directly inoculated onto Xylose Lysin Deoxycholate (XLD) and MacConkey Agar (MA). Stool samples were also inoculated into Salmonella-Shigella (SS) broth for enrichment. The plates and SS broth tubes were incubated at 37°C overnight. Next day, plates were observed for any suspected pathogen(s). From SS broth, sub-cultures were made on fresh XLD and MA plates, which were examined after overnight incubation at 37°C. Any colony suspected of Salmonella, Shigella and Escherichia coli either on primary culture or on sub-cultures after enrichment was identified by standard biochemical and serological methods as described earlier.

Seven enteric bacterial strains were isolated from seven of the 51 ReA patients, i.e., Salmonella typhimurium, three strains; Salmonella typhi, 1; Shigella flexneri serotype-2, 1; Shigella boydii, 1; and Hafnia alvei, 1 strain.

Detection of immunodominant antigen in isolated enteric pathogens

Preparation of antigens

The bacteria were grown at 37°C for overnight (18-24 h), in 200 ml of brain heart infusion broth on a shaker and/or on Mueller Hinton agar plates. Growth was harvested by centrifugation at, 10,000
g for 15 min from broth culture. The bacterial cells were washed twice with 0.01 M phosphate buffer saline (PBS, pH 7.2) and sonicated (SONIPREP-150) in an ice cooled cup for six cycles of 1 min each at 1 min intervals. The lysate was clarified by centrifugation at 10,000 g for 15 min and the supernatant was stored at -20°C until used. Protein concentration was determined by Lowry's method with bovine serum albumin (BSA) as standard (Lowry et al., 1951).

Protein profiles of whole cell sonicate antigens were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Bio-Rad mini protean II, California, USA). [12] The separated proteins were transferred on to 0.45 μm pore size nitrocellulose paper (NCP) in duplicate to detect the antigen band/s specifically reactive to ReA/RS patient' sera, after electrophoresis for 45 min at 100 volts (200 mA) for 1 h at 4°C (Bio-Rad minitransblot, California, USA). The unbound sites were blocked by keeping the blotted NCP in blocking buffer (0.01 M, PBS, pH 7.4 with 4% BSA) for 1 h at 37°C and then overnight at 4°C. NCP was washed 4 times (15 min each) with wash buffer (0.01 M, PBS, pH 7.4 with 0.05% Tween-20). The NCP was then reacted with 1:100 diluted patients' or controls' pooled sera. After incubation for 1 h at 37°C and then over night at 4°C, the NCP was washed 4 times (15 min each) with wash buffer. Anti-human immunoglobulin G (IgG) horse radish peroxidase conjugate (Anti-human IgG horseradish peroxidase [HRPO] conjugate, Sigma) diluted 1:5,000 was added to NCP followed by incubation at 37°C for 1 h. The dilutions of sera and conjugate were made in wash buffer with 1% BSA. Following four washings of 15 min each, the NCP was kept in the substrate solution until bands appeared. Reaction was stopped by washing and dipping NCP in distilled water.

Recovery of 30 kDa protein from SDS-PAGE gels and raising hyper-immune rabbit serum

All the seven bacterial lysates, were run separately in SDS-PAGE gels and 30 kDa antigens were extracted by the method previously described by Kabir, with minor modifications. [13] Hyper immune sera against 30 kDa antigens were raised individually, in seven New Zealand white healthy rabbits [14] and IgG antibody levels in rabbits were detected by enzyme-linked immunosorbent assay (ELISA).

Checking the Cross Reactivity of Eluted Antigens

All the seven 30 kDa proteins eluted from enteric pathogens were run in separate SDS-PAGE gels with 12% separating gel and 4% staking gel. These proteins were then blotted on NCP strips using Bio-Rad minitransblot apparatus at 100 V (250-350 mA) for 1 h and then western blot assay was performed by reacting each strip with seven heterologous hyperimmune rabbit sera using a Bio-Rad mini-Protean II multiscreen apparatus.

As previously described the unbound sites were blocked by 4% BSA in PBS (pH 7.2). Following 4 time washing (15 min each) with wash buffer, the strips were reacted with goat anti-rabbit IgG antibodies, for 2 h at 37°C. Then 4 times washing was followed by reaction with anti-rabbit IgG HRPO conjugate (1:5,000 dilutions) for 1 h at 37°C. Then strips were washed 4 times in wash buffer and developed by substrate solution containing ortho-phenylene-diamine (OPD). The reaction was stopped by dipping in the strips in Double Distilled Water.

Patients' immunological reactivity to 30 kDa cross-reactive antigen

Antibody class determination, lymphocyte proliferation assay and interleukin-2 (IL-2) production assay were performed to check humoral and cell mediated immune response of patients and controls in response to 30 kDa cross-reactive antigen.

Determination of IgG, immunoglobulin M (IgM) and immunoglobulin A (IgA) antibodies in patients' and controls' sera samples

Antibody levels were determined by ELISA in patient' and control's sera as described previously. [15] The polystyrene flat bottom microtiter plates (Maxisorb, NUNC Denvar) were coated with 100 μl of antigen (protein contents 1.25 μg in the carbonate-bicarbonate buffer (pH 9.6) per well at 4°C for 24 h after washing thrice with 0.01 M PBS-Tween 20 (0.05%) pH 7.2 (washing buffer) using Microtiter plate washer (8-12 SW1-SLT instruments, Austria). To each well 100 μl of 1% BSA in 0.01 M PBS, pH 7.2 was added and incubated for 2 h at 37°C to block non-specific binding sites. Plates were washed 5 times with wash buffer and then 100 μl of 1:10 diluted serum samples of patients and controls were added. The serum samples were diluted in 0.01 M PBS-T20 containing 1% BSA. Incubation for 2 h at 37°C was followed by washing the plates 5 times with 0.01 M PBS-T20 pH 7.2 and addition of 100 μl of 1:40,000 diluted anti-human IgG or IgM or IgA-horse radish peroxidase conjugate (Sigma) to each well. The dilutions were made in the same buffer as serum according to the instructions of Sigma. After incubation for 1 h at 37°C plates were again washed 5 times in wash buffer. Freshly prepared 100 μl of substrate solution (40 μg OPD [Sigma] dissolved in 100 ml of 0.1 M citrate buffer [pH 5.4] and 20 μl of 3% H 2 O 2 ) was added to each well and incubated at room temperature in dark for 15-20 min. Then the reaction was stopped by adding 50 μl of 2.5 M H 2 SO 4 to each well. The plates were read in an ELISA reader (ATC 340, SLT instrument, Austria) at 492 nm against a reference filter of 620 nm and the optical density (OD) values were obtained.

Lymphocyte proliferation assay

The ability of peripheral blood lymphocytes (PBL) of patients and controls to undergo proliferation following stimulation by 30 kDa cross-reactive antigen was studied in 16 patients since fresh blood samples were not available from all patients of ReA/RS. Different concentrations of the antigen (2.5 μg, 5 μg, 10 μg and 20 μg/ml) were used to standardize optimum proliferation of the lymphocytes. The lymphocytes were isolated by the ficoll hypaque density gradient, [16] and were subjected to viability check as previously described. [17] Lymphocytes from each patient and control were cultured in triplicate, in 96 well microtiter plates (COSTAR), in the presence of antigen and standard mitogen phytohemagglutinin (PHA). Unstimulated cultures had put in triplicate with each sample as controls. Three extra wells of lymphocyte culture stimulated with 30 kDa cross-reactive antigen had also put for IL-2 detection assay. A 100 μl suspension of lymphocytes (10 5 cells) in RPMI-1640 with 10% fetal calf serum and antibiotic cocktail was added to each well of Microtiter plate, followed by the addition of 100 μl of test stimulant (10 μg/ml 30 kDa cross-reactive antigen and 0.5 μg/ml PHA).

IL-2 production assay

IL-2 producing cells in response to 30 kDa cross-reactive antigen were detected as described previously using flow Cytometery. [18] During lymphocyte proliferation studies, lymphocytes were cultured in three additional wells for each sample, in the presence of 10 μg/ml antigen in RPMI-1640 with 10% fetal calf serum and antibiotic cocktail for 48 h. 6 h prior to cell harvesting, monensin 2 μM/ml had added to the growing cells. The cells had harvested and fixed in 4% paraformaldehyde for 20 min at 4°C and then were permeabilized using 0.1% saponin. Further, the cells were incubated with antihuman IL-2 antibodies conjugated to fluorescine isothiocyanate (Serotech, UK) for 90 min at room temperature in dark, followed by washing in PBS. The cells had then resuspended in PBS and analyzed by flow cytometry.

Lymphocyte subpopulation studies

Lymphocyte subpopulation studies were performed by flow cytometry as described previously. [19]

Statistical analysis

Student's t-test was used for comparing the mean values of immunological parameters between the subject and control groups 1,2 and 3. Standard error of mean (SEM) was calculated as the ratio of standard deviation (SD) and √n (n, number of samples).


   Results Top


Detection of immunodominant polypeptide in arthritogenic enteric bacterial isolates

Among 51 patients of Post-dysenteric ReA/RS, there were 26 males and 25 females, with a mean age of 31 ± 11 years. In a preliminary experiment, sonicated lysate of seven bacterial strains were subjected to SDS-PAGE and blotted to NCP strips in duplicate. One strip was reacted with pooled normal healthy control sera and the other with pooled ReA/RS patients' sera. A 30 kDa protein band was highlighted by ReA/RS patients' sera but not with pooled sera from patients with other arthritic diseases and pooled normal healthy control sera. This protein was not expressed by S. typhimurium strains isolated from cases who did not develop ReA up to 3 months after an episode of diarrhea and also was not present in S. sonnei strains, which are known to be non-arthritogenic. Outer membrane proteins were separated from three enteropathogens isolated from ReA patients and were reacted with pooled ReA patients' sera and normal healthy control sera, but no protein band was found at the level of 30 kDa [Figure 1]. The 30 kDa protein was then eluted from all seven different bacterial strains. [Figure 2] shows the protein profile of all seven isolates. This protein was expressed more by Salmonellaand Shigella strains as compared to Haffnia.
Figure 1: Outer membrane Protein profi le from sodium dodecyl sulfate polyacrylamide gel electrophoresis blott ed to Nitrocellulose paper showing reactivity of 30 kDa protein in outer membrane protein with patient and control sera. Lane A: Molecular weight marker, Lane B: Shigella boydii (Reacti ve arthriti s [ReA]-46), Lane C: Salmonella typhimurium (ReA-19), Lane D: S. typhimurium (ReA-32), Lane E-G: Nitrocellulose paper strip (NCP) with blott ed outer membrane proteins (OMPs) of three pathogens reacted with pooled sera of ReA patients, Lane H-J: NCP strip with blott ed OMPs of three pathogens reacted with pooled sera of normal healthy controls

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Figure 2: Protein profi le (coomassie blue stained sodium dodecyl sulfate polyacrylamide gel electrophoresis gel) of seven enteric pathogens isolated from reactive arthritis patients

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Testing cross reactivity amongst 30 kDa antigens eluted from all the seven isolates

These proteins were highly immunogenic as antibody titer in all the post-immunization rabbit sera was above 1:6,400 as detected by ELISA. All the seven proteins were cross-reactive to each other as all were highlighted by seven heterologous rabbit antisera [Figure 3].
Figure 3: Cross reacti vity among seven 30 kDa proteins by reacting with heterologous hyperimmune rabbit sera (1-7). Strips A to G contain 30 kDa antigen eluted from enteric bacterial pathogens from arthritis patients, reactive arthritis-13, 19, 32, 46, 61, 62 and 78 respectively

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Testing antibody levels in blood samples

Moreover, IgG, IgM and IgA antibody levels were significantly high as compared to control Groups 1-3 [Figure 4]. The mean OD for IgG antibodies shown by ReA/RS patients' sera was 1.800 ± 0.085 (Mean ± SEM) as compared to 0.600 ± 0.028 (control Group 1), 0.680 ± 0.090 (control Group 2) and 0.670 ± 0.040 (control Group 3). Mean OD for IgM antibodies shown by ReA/RS patients' sera was 0.650 ± 0.040 as compared to 0.120 ± 0.001 (control Group 1), 0.190 ± 0.030 (control Group 2) and 0.080 ± 0.008 (control Group 3). Mean OD for IgA antibodies shown by ReA/RS patients' sera was 0.330 ± 0.020 as compared to 0.140 ± 0.010 (control Group 1), 0.230 ± 0.040 (control Group 2) and 0.090 ± 0.009 (control Group 3). Sera from four patients suffering from tubercular ReA showed high OD values for IgG (almost equal to post-dysenteric ReA patients' sera). Out of these four, a known case of ileocaecal tuberculosis with ReA showed high values for IgM and IgA antibodies.
Figure 4: Mean immunoglobulin G, immunoglobulin M and immunoglobulin A levels in serum samples of reactive arthritis patients and different control groups

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Lymphocytes proliferation assay

The maximum proliferation of PBLs was observed at 10 μg/ml of the 30 kDa cross-reactive antigen. The proliferation of PBL of all the 16 ReA/RS patients in response to this antigen was significantly higher than controls (P < 0.0001). The stimulation index values in response to specific stimulant (30 kDa antigen) and non-specific stimulant (PHA) is shown in [Figure 5]. Tubercular arthritis patients in the control Group 2 showed higher response than other controls when stimulated with 30 kDa antigen, as well as with PHA.
Figure 5: Stimulation index values in response to specific stimulant (30 kDa anti gen) and non-specific stimulant phytohemagglutinin

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IL-2 production assay

The percentage of IL-2 producing cells in response to 30 kDa antigen was significantly higher (29.77 ± 14.10%) than the control Group 1 (1.48 ± 1.76%), control Group 2 (3.92 ± 3.3%) and control Group 3 (1.10 ± 1.07%) (P < 0.001). In tubercular arthritis patients IL-2 producing cells in response to 30 kDa antigen were higher than the other controls [Figure 6] and [Figure 7].
Figure 6: Determination of interleukin-2 producing lymphocytes (mean ± standard error of mean) of patients and controls in response to 30 kDa cross-reactive (common) antigen

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Figure 7: Determination of interleukin-2 producing lymphocytes (mean ± standard error of mean) of patients and controls in response to 30 kDa cross-reacti ve (common) antigen (Bar diagram with p values)

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Lymphocytes sub-population studies

Lymphocytes sub-population studies for CD3 + , CD4 + and CD8 + markers showed that CD8 + lymphocytes were less (mean 21.73%) in ReA patients than the control Group 1 (27.3%) control Group 2 (25%) and control Group 3 (37%) [Table 1]. CD8 + cells in ReA patients were significantly less (P < 0.05) than normal healthy control group subjects. The helper suppressor (CD4 + :CD8 + ) ratio was also higher in ReA patients (1.49 ± 0.59 SD) than the control Group 1 (1.19 ± 0.23 SD), control Group 2 (1.08 ± 0.36) and control Group 3 (1.00 ± 0.31).
Table 1: Peripheral blood lymphocyte sub-population studies

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   Discussion Top


In this study, we evaluated the predominant immunodominant antigen present in enteric bacterial isolates from ReA/RS patients. Majority of patients in the study were in the age group of 20-30 years, the peak age for the onset of ReA in India, when immune reactivity is also at the peak. [20] There is a strong evidence that Salmonella, Shigella and Yersinia enterocolitica persist in the intestinal mucosa of post-dysenteric ReA patients and Salmonella and Shigella had been isolated from intestinal specimens of ReA/RS patient. [21] In the present study, seven enteric bacterial species were isolated from stool samples of seven out of 51 ReA/RS patients. The isolation rate was approximately 13.72% by collecting one sample each on 3 successive days. The 30 kDa protein was found to be expressed specifically by enteric pathogens isolated from stool samples of post-dysenteric ReA patients but was not by non-arthritogenic enteric pathogens. Similarly, in Chlamydia triggered ReA patients anti-57 kDa heat shock protein antibodies have been detected from sera and as well as synovial fluid specimens. [22] Salmonella Lipopolysaccaride have also been thought to be arthritogenic factor, [21] and 16 kDa antigen of Y. enterocolitica and 20 kDa antigen of S. flexneri have been shown to cross-react with HLA-B27. [4]

The 30 kDa proteins from seven different species were found to be highly immunogenic when hyperimmune rabbit sera were raised in rabbits. Higher levels of serum antibodies to arthritogenic 30 kDa protein have been detected in ReA patients than controls. [5],[21] In patients with Salmonella triggered ReA, IgG, IgM and IgA and in Yersinia triggered ReA patients, IgA antibody levels have been found to be higher than controls. [23] In this study, IgG, IgM and IgA antibody levels against 30 kDa cross-reactive antigen were significantly high. It is possible that in ReA patients high levels of antibodies are produced against 30 kDa cross-reactive antigen leading to the formation of circulatory immune complexes (CICs), which might be having predilection for synovium causing complement mediated damage. CICs had been detected in serum as well as synovium of Yersinia and Salmonella triggered ReA patients. [24]

Decreased lymphocyte proliferation and impaired IL-2 productions in responses to arthritogenic bacteria in ReA patients has been reported. [25] In another study, PBL responses were shown to be enhanced in response to arthritogenic bacteria in ReA and RS patients, [26] and some others have shown unchanged responses. [27] In the present study, PBL responses were observed in response to purified 30 kDa antigen of arthritogenic enteric bacteria and PBL responsiveness to this purified antigen was significantly high in ReA/RS patients than in controls.

Yersinia-reactive T-cells predominantly produced interferon gamma (IFNγ) and IL-2 but not IL-4, IL-5 or IL-10 upon antigen specific and non-specific activation thus representing a Th-1 type of cytokine secretion. [28] The presence of arthritogenic antigen as well as the expansion of Th-1 type CD4 + T-cells within the joints probably play an important role in the generation and propagation of ReA. In this study, it was observed that IL-2 production by lymphocytes of ReA patients in response to 30 kDa antigen was significantly high than the controls. This indicates that lymphocytes of post-dysenteric ReA (and probably of mycobacterial ReA) recognize this antigen in vitro, produce IL-2 and proliferate in response to this antigen much more than lymphocytes of normal healthy controls or other controls. The 30 kDa antigen belonging to arthritogenic bacteria might be inducing Th-1 type of cytokine secretion leading to expansion of Th-1 type CD4 + cells. Th-1 type T-cells producing IFNγ will also constitute an important defense against the intracellular arthritogenic organisms and therefore may promote the effective eradication of antigenic material from the inflamed tissue. The process may be responsible for recovery of patients in self-limiting ReA patients. The clinical manifestations of ReA have been associated with low CD4 + T-cell in patients suffering from acquired immunodeficiency syndrome. [29]

Many studies on synovial fluid T lymphocytes show that T-cells play a critical role in the pathogenesis of ReA. [30] In one study, there was no significant difference in T-cell sub-population among ReA patients and control groups, though the CD8 + subset population was lesser than CD4 + in PBL of ReA patients as compared to the control groups.(CD4 + :CD8 + ratio was 1.49 ± 0.59). However, peripheral blood subpopulation is not representative of synovial fluid T-cell population, so nothing could be concluded about the role of any particular lymphocyte subpopulation of peripheral blood in pathogenesis of ReA. Histopathological studies of ReA synovial membrane show a mononuclear cell infiltrate consisting mainly of memory CD4 + and CD8 + lymphocytes indicating the role of cellular immune response at the site of synovitis. [30] Among cytotoxic T-lymphocytes (CTL), several distinct cells types have been identified. Classical MHC-restricted CD8 + CTL expressing an αβ-T-cell receptors (TCR) generally recognize their specific antigen as peptide in association with polymorphic parts of MHC class I molecules. If HLA-B27 disease association is a consequence of its physiological function in antigen presentation, ReA should be mediated by CTL that recognize bacterial or self-peptides presented by HLA-B27. Proof of this arthritogenic peptide model requires isolation of B27 restricted CD8 + T-cells from arthritic joints of ReA patients. Synovial fluid CD8 + T-cells of ReA patients have been shown to have higher proliferative responses to Salmonella and Yersinia antigen than by lymphocytes of normal healthy controls. Also, these cells were cytotoxic αβ-TCR + CD8 + which were specific for Yersinia and Salmonella infected HLA-B27 positive cells.

Hence, this study suggests a role of the 30 kDa protein found specifically in arthritogenic enteric bacteria, which may be a common arthritogenic polypeptide (arthritogenic factor). Further studies need to be done to detect the presence of this antigen in CICs and in synovium of ReA patients to delineate the exact mechanism by which it plays a role in pathogenesis of the disease and to carry out the N-terminal sequencing of this protein and evaluate the immunological reactivity of ReA patients in response to this antigen. The presence and role of H. alvei of this common 30 kDa arthritogenic antigen in one patient of ReA and this association of H. alvei to ReA/RS based on the presence of the 30 kDa antigen may explain the initiation of the disease process in at least some of those post-dysenteric ReA cases where the triggering infection has not been identified. It is evident that the pathogenesis of ReA/RS may be a multifactorial process but the role of 30 kDa antigen of certain arthritogenic enteric bacteria may be crucial and central to the understanding of the pathophysiology of this disease. The critical role in the triggering mechanism of the disease process by these enteric bacteria at distant sites of ReA/RS patients is manifest in the common 30 kDa antigen found in them and perhaps by a similar process of sharing of this common antigen in mycobacterial ReA cases as it has been found in the present study.

 
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23.Granfors K, Jalkanen S, von Essen R, Lahesmaa-Rantala R, Isomäki O, Pekkola-Heino K, et al. Yersinia antigens in synovial-fluid cells from patients with reactive arthritis. N Engl J Med 1989;320:216-21.  Back to cited text no. 23
    
24.Lahesmaa-Rantala R, Granfors K, Kekomäki R, Toivanen A. Circulating Yersinia specific immune complexes after acute yersiniosis: A follow up study of patients with and without reactive arthritis. Ann Rheum Dis 1987;46:121-6.  Back to cited text no. 24
    
25.Hermann E, Grabe H, Mayet WJ, Büschenfelde KH, Fleischer B. Lower frequency of Yersinia-reactive peripheral blood T cells in acute Yersinia-induced arthritis-indication of defective first line of defense? Clin Rheumatol 1992;58(Abstract):11.  Back to cited text no. 25
    
26.Brenner MB, Kobayashi S, Wiesenhutter CW, Huberman AK, Bales P, Yu DT. In vitro T lymphocyte proliferative response to Yersinia enterocolitica in Reiter's syndrome. Lack of response in other HLA-B27 positive individuals. Arthritis Rheum 1984;27:250-7.  Back to cited text no. 26
    
27.Inman RD. The interplay of microbe and MHC in the pathogenesis of Reiter's syndrome. Clin Exp Rheumatol 1986;4:75-82.  Back to cited text no. 27
    
28.Lahesmaa R, Yssel H, Batsford S, Luukkainen R, Möttönen T, Steinman L, et al. Yersinia enterocolitica activates a T helper type 1-like T cell subset in reactive arthritis. J Immunol 1992;148:3079-85.  Back to cited text no. 28
    
29.Winchester R, Bernstein DH, Fischer HD, Enlow R, Solomon G. The co-occurrence of Reiter's syndrome and acquired immunodeficiency. Ann Intern Med 1987;106:19-26.  Back to cited text no. 29
    
30.Hermann E. T cells in reactive arthritis. APMIS 1993;101:177-86.  Back to cited text no. 30
    

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Correspondence Address:
Meera Sharma
Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.120373

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