Yersinia-triggered Reactive Arthritis Research Articles

ECA-immunogenicity of Proteus mirabilis strains

IntroductionBacteria of the genus Proteus are opportunistic pathogens and cause mainly urinary tract infections. They also play a role in the pathogenesis of reactive arthritis (RA). Patients suffering from Yersinia-triggered RA often carry high titers of antibodies specific to enterobacterial common antigen (ECA). The immunogenicity of ECA has not received much attention thus far and studies have focused mainly on the ECA of Escherichia coli and Yersinia enterocolitica. In this paper the ECA-immunogenicity of Proteus mirabilis is elucidated using two wild-type strains (S1959 and O28) as well as their rough (R) derivative strains R110/1959, which expresses lipopolysaccharide (LPS) with a full core, and R4/O28, which expresses LPS with only an inner core.Materials and MethodsRabbit polyclonal antisera were produced by immunization with boiled suspensions of the four P. mirabilis strains. The antisera were tested for the presence of antibodies specific to ECA by Western blotting using glycerophospholipid- linked ECA (ECAPG) of Salmonella montevideo as antigen. Lipopolysaccharide (LPS) was isolated from the four strains by the hot phenol/water procedure in which ECAPG is co-extracted with LPS and by the phenol/chloroform/petroleum ether extraction that results in the isolation of LPS and/or LPS-linked ECA (ECALPS) free of ECAPG. The LPS preparations were tested for the presence of ECA by Western blotting using ECA-specific antibodies.ResultsThe results demonstrated that all four P. mirabilis strains were ECA immunogenic. The rabbit antisera immunized by the four strains all contained ECA-specific antibodies. Analysis of the LPS preparations demonstrated that the P. mirabilis wild-type strains O28 and S1959 and the Ra mutant strain R110/1959 expressed ECALPS, suggesting that it induced the anti-ECA antibody responses. Only the presence of ECAPG could be demonstrated in the Rc mutant strain R4/O28.ConclusionsThese results therefore suggest that, similar to E. coli, LPS with a full core is also required as the acceptor of ECA for P. mirabilis strains to produce ECALPS. Since ECAPG is not immunogenic unless combined with some proteins, it is likely that ECAPG-protein complexes formed during the intravenous immunization with the Rc mutant strain R4/O28.

Cross-reaction between Yersinia outer membrane proteins and anti-Borreli antibodies in sera of patients with Lyme disease

Diagnosis of Yersinia infections accompanied by reactive arthritis could be complicated by cross-reaction with other arthritogenic bacteria. The possible cross-reaction between Yersinia antigens and anti-Borrelia antibodies in blood sera of patients with Lyme disease was studied. The occurrence of specific IgA, IgG and IgM antibodies was analyzed in serum samples from 30 patients with Yersinia-triggered reactive arthritis, 30 patients with Lyme disease and five samples from healthy blood donors. For anti-Borrelia IgG antibodies, cross-reaction was detected with YopH, YopB, V-ag, YopD, YopN, YopP and YopE, and for IgA with YopD. For IgM, no cross-reaction was detected. Owing to cross-reactivity with Borrelia, the diagnosis of Yersinia-triggered reactive arthritis should be based on a combination of serological and clinical findings.

Neutrophils of healthy subjects with a history of reactive arthritis show enhanced responsiveness, as defined by CD11b expression in adherent and non-adherent whole blood cultures

To study innate immune responsiveness of HLA-B27 positive subjects recovered from Yersinia-triggered reactive arthritis (B27 + ReA+). Whole blood samples from 15 B27 + ReA+, 15 B27 + ReA- and 15 B27 - ReA- subjects were heparinized, aliquoted and (i) kept at 0 degree C to preserve constitutive cell surface marker status, or (ii) cultured with or without bacterial lipopolysaccharide (LPS) supplement, in adherent and non-adherent conditions at 37 degrees C for 4 h. Neutrophil surface expression of CD11b, CD14 and CD16 was quantified flow cytometrically, and compared between the subject groups using Jonckheere-Terpstra test. The B27 + ReA+ group showed significantly higher CD11b levels than the B27 - ReA- group on non-adherent neutrophils cultured with LPS as 100 pg/ml (P = 0.027), 10 ng/ml (P = 0.048) or 1 microg/ml (P = 0.024), or on adherent neutrophils without LPS supplement (P = 0.040). CD14 and CD16 expression on cultured neutrophils and constitutive expression of all three markers were comparable between the groups. Enhanced neutrophil reactivity observed may exacerbate innate immune inflammation in HLA-B27 positive ReA patients.

Multispecific CD4+ T Cell Response to a Single 12-mer Epitope of the Immunodominant Heat-Shock Protein 60 ofYersinia enterocoliticainYersinia-Triggered Reactive Arthritis: Overlap with the B27-Restricted CD8 Epitope, Functional Properties, and Epitope Presentation by Multiple DR Alleles

Yersinia heat-shock protein 60 (Ye-hsp60) has recently been found to be a dominant CD4 and CD8 T cell Ag in Yersinia-triggered reactive arthritis. The nature of this response with respect to the epitopes recognized and functional characteristics of the T cells is largely unknown. CD4+ T cell clones specific for Ye-hsp60 were raised from synovial fluid mononuclear cells from a patient with Yersinia-triggered reactive arthritis. and their specificity was determined using three recombinant Ye-hsp60 fragments, overlapping 18-mer synthetic peptides as well as truncated peptides. Functional characteristics were assessed by cytokine secretion analysis in culture supernatants after specific antigenic stimulation. Amino acid positions relevant for T cell activation were detected by single alanine substitutions within the epitopes. Fragment II comprising amino acid sequence 182-371 was recognized by the majority of clones. All these clones were specific for peptide 319-342. Th1 clones and IL-10-secreting clones occurred in parallel, sometimes with the same fine specificity. The 12-mer core epitope 322-333 is a degenerate MHC binder and is presented to some T cell clones in a "promiscuous" manner. This epitope is almost identical with a B27-restricted CTL epitope of Ye-hsp60. Cross-reactivity of Ye-hsp60-specific T cell clones with self-hsp60 was not observed. In conclusion, an interesting Ye-hsp60 T cell epitope has been identified and characterized. It remains to be determined whether this epitope is also relevant in other reactive arthritis patients.

Persistence of Yersinia antigens in peripheral blood cells from patients with Yersinia enterocolitica O:3 infection with or without reactive arthritis.

To assess the persistence of bacterial antigens in peripheral blood cells from patients with Yersinia enterocolitica O:3-triggered reactive arthritis (ReA). Peripheral blood samples were obtained from 20 patients with Y. enterocolitica O:3 infection (11 with ReA and 9 without). These samples were studied by immunochemical techniques for the presence of Yersinia antigens at the beginning of infection and up to 4 years thereafter. Synovial fluid samples from 6 of the 11 ReA patients were also studied. The Yersinia antigens lipopolysaccharide and heat-shock protein (HSP) were detected in peripheral blood mononuclear cells and polymorphonuclear phagocytes from all patients studied at the early phase of the disease. They were also found in the synovial fluid cells of patients with Yersinia-triggered ReA. At 4 years after the onset of infection, these bacterial antigens were still detected in the peripheral blood cells of most of the ReA patients studied. This study has, for the first time, directly demonstrated that bacterial antigens persist for a long time in patients who develop ReA after Y. enterocolitica O:3 infection. The finding of bacterial HSP in synovial fluid cells could provide a link to the pathogenesis of ReA, since T cell responses of synovial cells have been shown to be directed against that structure. A close similarity between the bacterial and host HSP might contribute to the development of the relatively common, chronic form of this complication.

Experimental Yersinia-triggered reactive arthritis: effect of a 3-week course of ciprofloxacin.

Lewis rats were injected i.v. with live Yersinia enterocolitica, resulting in 1-2 weeks in an arthritis greatly resembling human reactive arthritis. Starting on day 3, 5, 10 or 13 after the bacterial inoculation, the rats were treated for 3 weeks with 20 mg/kg/day of ciprofloxacin. The development of arthritis was completely prevented if the antibiotic treatment was started on day 3. In a group of rats treated with ciprofloxacin from day 5 onwards, 2/14 rats already showed mild arthritis at the time when the treatment was started. Antibiotic treatment cured the arthritis of these rats as well as that of one additional individual in this group which developed arthritis. No later exacerbations occurred. If the antibiotic treatment was started on day 10 or 13, i.e. at the time of well-developed arthritis, no effect on arthritis was observed; rather, increased faecal excretion of Yersinia occurred following the antibiotic treatment. We conclude that experimental Yersinia reactive arthritis can be cured by antibiotics introduced at an early phase of arthritic development. Regarding acute human enterogenic arthritis, the decision on antibiotic treatment is not a straightforward matter. Our experimental results indicate that the earlier the treatment is started, the better the result, whereas late treatments seem to favour bacterial persistence.

Antibiotic prophylaxis and treatment of reactive arthritis. Lessons from an animal model

To study the effect of antibiotic prophylaxis and treatment of reactive arthritis (ReA), using an experimental model. Yersinia enterocolitica O:8, when injected intravenously into Lewis rats, causes a sterile arthritis closely resembling human ReA in 70% of the animals. Arthritis develops in 1-2 weeks; in some of the animals it remains chronic, and exacerbations occur. This model was applied to study the effect of a 7-day treatment with ciprofloxacin, using 2 different dosages (20 or 100 mg/kg/day) and 4 different schedules for initiation of treatment. The effects were evaluated by determining the daily arthritis score, the number of rats developing arthritis, and fecal excretion of Yersinia. In addition, weight gain was monitored. At autopsy (35 or 60 days after inoculation with bacteria), samples were obtained for determination of Yersinia-specific antibodies in the serum. At the same time, samples were collected from mesenteric lymph nodes, lung, spleen, and liver for bacterial cultures, and from the ankle joints for histologic evaluation. In a separate experiment, ciprofloxacin concentrations in samples from serum and mesenteric lymph nodes were analyzed by high performance liquid chromatography. A 7-day course with 100 mg/kg/day of ciprofloxacin, started on day 3 after bacterial inoculation, completely prevented the development of ReA and eliminated Yersinia during the 60-day experiment. If a dosage of 20 mg/kg/day was used, development of acute arthritis was prevented, but some of the animals had positive fecal cultures at the end of experiment. If antibiotic treatment was started on day 5, the preventive effect was still observed, but was less pronounced. If the treatment was started at the peak of the development of arthritis, no effect on arthritis was observed. These results indicate that if any effect of antibiotic treatment in Yersinia-triggered ReA is to be expected, the treatment must be started early and given in sufficient dosage. However, antibiotic treatment has no effect on fully developed arthritis.

Alkali-treated LPS of Yersinia enterocolitica does not induce expression of E-selectin, ICAM-1 or VCAM-1 on endothelial cells but may mediate antibody- and complement-dependent cell injury.

Lipopolysaccharide (LPS) prepared from a rough mutant of Salmonella typhimurium and deacylated enzymatically (dLPS) does not promote neutrophil adherence to human umbilical vein endothelial cells (HUVECs). This paper reports that similarly, a smooth form of LPS prepared from Yersinia enterocolitica O:3, a serotype known to trigger reactive arthritis in humans, and treated with alkali (yersinia LPS-OH) failed to augment neutrophil adherence to HUVECs. Studies of the mechanism underlying the poor augmentation revealed that neither enzymatically deacylated LPS from Escherichia coli J5 (J5 dLPS) nor yersinia LPS-OH stimulated expression of endothelial cell adhesion molecules E-selectin, VCAM-1 and ICAM-1, whereas both intact J5 LPS and yersinia LPS were stimulatory. Impaired up-regulation could not be explained by decreased binding of yersinia LPS-OH to HUVECs. Furthermore, 51Cr-labelled HUVECs treated with different concentrations of yersinia LPS-OH released 51Cr in the presence of anti-yersinia anti-O antibody and complement. J5 dLPS and yersinia LPS-OH inhibited up-regulation of the adhesion molecules induced by J5 LPS and yersinia LPS but not that induced by tumour necrosis factor alpha. Taken together, the results suggest that although yersinia LPS-OH can depress development of acute inflammation by inhibiting up-regulation of endothelial-cell adhesion molecules, sufficient LPS-OH is bound to induce cell injury and thereby inflammation in the presence of specific antibody and complement. The findings may have pathogenetic implications in yersinia-triggered reactive arthritis characterized by dissemination of yersinia LPS throughout the body.

Function of terminal ileum in patients with Yersinia-triggered reactive arthritis.

In order to study the function of the intestinal epithelium in the terminal ileum, the Schilling test was performed in 10 patients with Yersinia-triggered reactive arthritis, in 10 patients who had recovered from Yersinia enteritis without complications, and in five patients with rheumatoid arthritis treated with non-steroidal anti-inflammatory agents. The Schilling test indicates absorption of vitamin B12 in the terminal ileum, i.e. the area affected by Yersinia and inflamed in patients with reactive arthritis. The findings obtained demonstrate increased uptake through the epithelium in this area of the intestine in patients with Yersinia-triggered reactive arthritis. There are two possible explanations. First, Yersinia infection may have a long-term effect on the gut mucosa. Secondly, some individuals may, at the level of the terminal ileum, show enhanced absorption of vitamin B12 and/or other substances such as microbes or their components, resulting in increased susceptibility to certain infections.

Yersinia-triggered reactive arthritis. Use of polymerase chain reaction and immunocytochemical staining in the detection of bacterial components from synovial specimens.

To investigate whether microbial DNA is present in synovial specimens from patients with Yersinia-triggered reactive arthritis. Synovial specimens from 13 patients with Yersinia enterocolitica O:3-triggered reactive arthritis and from 16 control patients were studied using polymerase chain reaction and immunocytochemical staining techniques. Yersinia chromosomal DNA was not found in any of the synovial specimens from Yersinia-triggered arthritis patients or controls, whereas with immunocytochemical techniques, Yersinia antigens were observed in synovial specimens from all of the patients with Yersinia-triggered reactive arthritis. Only stable bacterial degradation products, not whole bacteria, are present at the site of inflammation in Yersinia-triggered reactive arthritis.

Yersinia enterocolitica activates a T helper type 1-like T cell subset in reactive arthritis.

The profile of lymphokines secreted by 14 T cell clones and 24 T cell lines reactive with Yersinia Ag isolated from the synovial fluid cells of two HLA-B27+ patients with Yersinia-triggered reactive arthritis was characterized. In response to Ag-specific or -nonspecific stimulation, all of the Yersinia-reactive T cell clones and lines had a pattern of lymphokine secretion resembling that of murine (Th1) cells. A total of 50% of T cell lines and clones randomly isolated from a reactive arthritis patient, without prior in vitro stimulation with Yersinia Ag, also exhibited a Th1-like profile of cytokine secretion upon nonspecific activation. This indicates that the selective expansion of this subset of T cells had already occurred in vivo. The possibility that the predominance of Th1-like T cells was an artefact generated by the T cell cloning procedure was excluded; 50% of the randomly isolated T cell clones and lines produced IL-4, IL-5, or both cytokines upon nonspecific activation. These results indicate that Yersinia Ag selectively activate a Th1-like subset of T cells in patients with Yersinia-triggered reactive arthritis. Accumulation of such cells in the synovial tissue of patients with reactive arthritis may play a key role in the pathogenesis of this disease.

Molecular mimicry in the pathogenesis of spondyloarthropathies. A critical appraisal of cross-reactivity between microbial antigens and HLA-B27.

We describe an amino acid homology between a virulence plasmid encoded outer membrane protein of Yersinia, YadA (previously called Yop1) and HLA-B27. This tetrapeptide is also included in the hexapeptide, earlier found to be identical between Klebsiella nitrogenase and HLA-B27. The synthetic peptide based on the HLA-B27 homologous portion of the YadA does not stimulate lymphocytes obtained from HLA-B27+ patients with Yersinia-triggered reactive arthritis or from controls. One-third of the yersiniosis patients have antibodies against the synthetic peptide. Instead of recognizing the HLA-B27 homologous portion, the antibodies are directed against the left flanking sequence of the synthetic peptide. Similar results were obtained regarding antibody response to Klebsiella nitrogenase derived synthetic peptide included in the panel of controls; the response was not restricted to patients with reactive arthritis nor was it specifically directed against the sequence shared by HLA-B27 and Klebsiella pneumoniae nitrogenase. The present results do not support the role of molecular mimicry or cross-reactive antibodies in the pathogenesis of spondyloarthropathies.

Application of the polymerase chain reaction and immunofluorescence techniques to the detection of bacteria in Yersinia‐triggered reactive arthritis

Leukocytes in synovial fluid and peripheral blood samples from patients with Yersinia-triggered reactive arthritis were analyzed after DNA amplification using the polymerase chain reaction. The primers applied were specific for the virulence plasmid-coded 1crE genes of Yersinia enterocolitica O:3 and Yersinia pseudotuberculosis III. No Yersinia DNA was observed within the synovial fluid cells or peripheral blood cells by polymerase chain reaction techniques. However, Yersinia antigens were detected in the synovial fluid cells by immunofluorescence techniques. These results suggest that only parts of the causative agents, not the entire microbe, can enter the joint and initiate the inflammation that leads to a reactive arthritis.

A Tetrapeptide Shared byYersiniaOuter Membrane Protein Yop1 and HLA-B27Antibody Response in Patients with Yersinia-triggered Reactive Arthritis

A Tetrapeptide Shared by<i>Yersinia</i>Outer Membrane Protein Yop1 and HLA-B27<i>Antibody Response in Patients with Yersinia-triggered Reactive Arthritis</i>

Analysis of IgA antibodies to lipopolysaccharide in Yersinia-triggered reactive arthritis.

Analysis of IgA antibodies to lipopolysaccharide in Yersinia-triggered reactive arthritis.

Immunoblot analysis of human IgM, IgG and IgA responses to plasmid-encoded antigens of Yersinia enterocolitica serovar O3

Human IgM, IgG and IgA responses after infection with Yersinia enterocolitica serovar O3 were studied by immunoblotting sera against whole-cell homogenates of a plasmid-containing strain of Y. enterocolitica O3 and a plasmid-free strain derived from it; each strain was grown in conditions expressive for the plasmid. The antibodies observed were directed against several plasmid-encoded polypeptides. The response against different bacterial components decreased uniformly with time and the persisting antibody production was directed against several epitopes. Strong reactions to the prominent plasmid-specified antigens of mol. wts (10(3] 26, 34, 45 and 52.5 were found more often with IgG-class antibodies than with IgM or IgA; the latter immunoglobulins recognised, respectively, antigens of mol. wt (10(3] 26 and 45 (IgM) and 26, 34 and 52.5 (IgA). Immunoblotting of sera from patients with yersinia-triggered reactive arthritis did not reveal any antigens that were involved additionally or specifically. However, IgA-mediated recognition of certain antigens of mol. wts (10(3] 26, 34 and 52.5 tended to persist longer in the arthritic patients.

Characterization of circulating Yersinia-specific immune complexes in patients with yersiniosis

The size of immune complexes (ICs) containing Yersinia enterocolitica antigens was studied by size exclusion high-pressure liquid chromatography and sucrose density gradient ultracentrifugation in sera of patients with recent yersiniosis. The ICs detected were relatively small, i.e., of equal size to or slightly larger than the corresponding anti-Yersinia antibodies. The size of the ICs was equal in the patients with Yersinia-triggered reactive arthritis and in those recovering without complications. No changes were observed during a follow-up. The equal size of ICs in the patients with and without arthritis also suggests that antigens and antibodies involved are similar in both patient groups. Taken together with our earlier findings indicating occurrence of high concentrations of Yersinia IgM ICs in the arthritic patients, the present results suggest that Yersinia-IgM ICs have a role in the pathogenesis of Yersinia-triggered reactive arthritis.