Facultad de Química, Bioquímica y Farmacia, Universidad Nacional de San Luis, Chacabuco y Pedernera, 5700 San Luis, Argentina
Correspondence to:
A. M. S. de Guzmán.
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Abstract |
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Methods. Male hamsters of the Syria strain were intramuscularly injected into each of the hind paws with two doses of Y. enterocolitica LPS O:8 or O:5. The measurement of swelling using a plethysmometer, the analysis of histological changes by routine techniques and the kinetics of LPS-specific antibodies and autoantibodies evaluated by enzyme-linked immunosorbent assay (ELISA) were performed.
Results. LPS O:8 was demonstrated to be more arthritogenic than LPS O:5, inducing acute arthritis on day 3 post-injection as well as more significant and longer lasting joint swelling after a second dose. LPS O:8 caused severe histopathological changes in the joints. Important LPS O:8-specific IgG responses and antibodies against type I and II collagen were observed.
Conclusion. LPS O:8 administered alone has arthritogenic power and induces activation of autoreactive clones. This study supports the key role of LPS in the development of reactive arthritis.
KEY WORDS: Reactive arthritis, Lipopolysaccharide, Hamsters, Autoantibody
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Introduction |
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LPS has been found to play a major role as an arthritis-triggering antigen in rats [5, 6], mice [7], rabbits [8] and hamsters [9, 10]. The choice of the hamster as an LPS-induced arthritis model in the present work was made taking into consideration that the specific features of the host as well as of the pathogen must coincide [11, 12]. Lim et al. proposed the hamster as an animal model for the study of Borrelia burgdorferi-induced Lyme arthritis since its propensity for the development of arthritis may lead to a better understanding of the immune mechanisms responsible for this disease [13]. In previous studies, hamsters have been used for the study of ReA induced by virulence plasmid-bearing Y. enterocolitica B1 O:5 Lis Xz and Y. enterocolitica 1821 O:8 [14, 15]. Animals were injected intramuscularly (i.m.) into their hind paws with 108109 colony-forming units (c.f.u.)/animal. The development of arthritis was estimated by measuring inflammation and correlating with histological studies and serological assessment of the immune response. In these studies a constant immunoglobulin response against LPS was observed. This response reached a peak in the moment of development of chronic joint inflammation [14, 15]. In addition, no viable bacteria were detected in the joints of Y. enterocolitica O:8- or O:5-infected hamsters during the joint chronic inflammatory peak, but bacterial antigens were detected by immunofluorescence during this peak of arthritis [15].
The aim of the present work was to study O:8 and O:5 LPS arthritogenicity administered separately in hamsters. Histopathological changes, antibodies in serum and polyclonal activation were analysed.
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Materials and methods |
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Bacteria
Yersinia enterocolitica strain 1821 serotype O:8 pYV (+) kindly provided by Dr Kapperud (Department of Bacteriology, Oslo, Norway), and virulent plasmid-bearing Y. enterocolitica B1 O:5 Lis Xz isolated from food samples [16] were used.
Cultures
The bacteria were cultured at room temperature in tryptic soy broth (TSB) on an orbital shaker (3 Hz frequency) for 18 h. Bacterial cells were collected by centrifugation and washed with 0.9% NaCl. Counts of viable cells were performed on tryptic soy agar (TSA).
LPS
LPS was obtained by extraction with hot phenolwater from Y. enterocolitica O:8 and O:5 using the method of Westphal et al. [17]. LPS contained less than 2% protein, as determined by Lowry's procedure [18], and 34% 2-keto-3-deoxyoctulosonic acid [19] and showed a ladder-like appearance in silver-stained polyacrylamide gels [20, 21].
Animal injection
Five hamsters were injected i.m. into each of the hind paws with a 0.2 ml single dose of 300 µg of LPS O:8 or O:5. A second dose of LPS O:8 was administered on day 160. Five control animals received equal doses of 0.9% NaCl. All the experiments were repeated at least twice.
Assessment of arthritis
Hind paw inflammation was measured with a plethysmometer (Ugo Basile-7150). The mean value from two hind paws was used as an index of the severity of the swelling from arthritis. The values were compared with those from non-injected animals on different days post-injection (p.i.).
Analysis of histological changes
LPS-injected and control hamsters were killed on day 3 p.i. One of the hind paws was amputated, fixed, decalcified and processed according to routine histological techniques. Sections were stained with haematoxylineosin.
Antibody response by enzyme-linked immunosorbent assay (ELISA)
Polystyrene plates were coated overnight at 4°C with either Y. enterocolitica O:8 or O:5 LPS. The hamster sera obtained on different days p.i. were diluted and incubated for 1 h at 37°C. Hamster IgG was detected with rabbit anti-hamster IgG followed by incubation with peroxidase-conjugated goat anti-rabbit immunoglobulins, and 1,2-o-phenylenediamine was used as the chromogen. Absorbance was determined in an ELISA photometer (Bio-Rad, Hercules, CA, USA).
Detection of polyclonal activation
The technique was performed as described by Falcào et al. [22]. Rabbit brain myelin basic protein (MBP), calf skin collagen type I, bovine tracheal cartilage collagen type II and rabbit muscle myosin (Sigma, St Louis, MO, USA) were used as antigens. Autoantibodies were detected by ELISA as follows. Polystyrene microtitre plates were sensitized with 100 µl of the autoantigens diluted with 0.06 M sodium carbonatebicarbonate buffer, pH 9.6 (myosin 20 µg/ml, myelin 20 µg/ml, collagen type I 20 µg/ml, and collagen type II 20 µg/ml). The plates were then incubated for 18 h at 4°C and washed three times with phosphate-buffered physiological saline containing 0.05% Tween 20 (PBS/T). After washing, 100 µl of a serum sample diluted 1/100 in PBS/T containing 1% bovine serum albumin (PBS/T/BSA) was added and incubated for 2 h at 37°C. The plates were washed and 100 µl of a peroxidase-conjugated rabbit anti-mouse immunoglobulin diluted 1/1000 in PBS/T/BSA was added. After 1 h of incubation at 37°C and another washing, 100 µl of 1 mg/ml o-phenylene diamine in 0.1 M citrate phosphate buffer, pH 5.0, containing 0.03% hydrogen peroxide was added and the plates were incubated at 35°C for 10 min. Finally, the reaction was stopped with 100 µl of 3 N hydrochloric acid. Absorbance was read with an ELISA photometer (Bio-Rad) at 450 nm. All serum samples were assayed in duplicate and the results were expressed as the mean value.
Statistics
Data were subjected to analysis of variance followed by Tukey comparison. Student's t-test was used for inhomogeneous variance. Values of P<0.05 were considered statistically significant.
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Results |
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Discussion |
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Bacterial LPS is a potent toxin released from Gram-negative bacterial cell walls, and it is notorious for the initiation of inflammation [24]. Earlier studies by our group have demonstrated the existence of Yersinia LPS in the joints of hamsters with Yersinia-triggered arthritis. In the present study, LPS alone was administered in hamsters and induced the swelling of joints as well as histological changes. Wuorela et al. [4] have suggested that LPS released from bacteria ends up in corresponding compartments of the cell as isolated LPS. The LPS-containing phagocytes are a constant source of membrane-active LPS in their microenvironment, such as in the joints of arthritic patients.
Low bactericidal capacity of the host and appropriate virulence potential of the pathogen have been suggested as a prerequisite for arthritis development [11]. Significant joint inflammation and marked histological changes were observed in joints of LPS O:8-injected hamsters. At the same concentration, LPS O:8 was more arthritogenic than LPS O:5, when both were administered alone. It is therefore the variation in antigenicity that could be the cause of the different arthritogenic power observed between LPS O:8 and LPS O:5 in the present study.
Only acute inflammation was observed after the first dose of LPS O:8, indicating that it was probably removed by monocytes and macrophages [25, 26]. However, a second dose of LPS O:8 induced more significant and longer lasting joint inflammation. Several reports have suggested that the O-polysaccharide chain of LPS is also partially degraded by these cells [27, 28], and that this portion of LPS is retained in highly mobile cells. Phagocytes could transport LPS from the site of primary infection to the other parts of the body, such as the swollen joints of arthritic patients [4]. Partially degraded LPS from the first dose probably causes the more important inflammation after the second dose. In previous studies, a chronic inflammatory peak was observed when whole bacteria were administered [14, 15], suggesting that live bacteria probably persist somewhere in the body, continuously providing antigenic material into the blood circulation and stimulating the persistent immune reactions seen in the patients [29]. Granfors et al. [23] observed that 4 yr after the onset of Y. enterocolitica O:3-induced ReA, LPS was present in peripheral blood cells in most of their patients.
Our results indicate that, unlike LPS O:5, LPS O:8 causes a transient response of specific antibodies, and activation of autoreactive clones, with production of autoantibodies against collagen I and II. These are the most abundant collagens in the joints [30]. In a study of autoantibodies in mice infected with Y. enterocolitica O:3, Falcào et al. [22] observed responses against myosin, myelin and (trinitrophenyl) TNP-BSA, while no response was obtained against the collagens. These different results might reveal that the activation of different autoreactive clones depends on the host and the serovar employed in the experimental infection. In the present work, serum antibodies decayed with time, probably due to the fact that they deposited in the joint collagens or because the degraded LPS stopped inducing polyclonal activation.
In conclusion, we have demonstrated in hamster the different arthritogenic responses to LPS from different serovars of Y. enterocolitica. LPS O:8 administered alone has arthritogenic power and induces activation of autoreactive clones. These results support the key role of LPS in the development of ReA.
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Acknowledgments |
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References |
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