(Allen et al., 1983). Gender differences, however, are less apparent when relatively high doses of cell walls are injected. Normal euthymic and athymic Lewis rats have also been studied extensively (Case et al., 1989; Sano et al., 1990, 1992, 1993; Wilder et al., 1987). Compared to euthymic Lewis rats, athymic Lewis rats develop severe, acute transient arthritis, but chronic disease is markedly blunted, indicating that acute disease is thy-mus-independent, whereas chronic disease is highly influenced by thymus-dependent mechanisms. These differences in host susceptibility have been evaluated in the context of numerous cytokines and other inflammatory mediators (Allen et al., 1983; Bristol et al., 1993; Case et al., 1989; Crofford et al., 1992; Lafyatis et al., 1989; Sano et al., 1990, 1992, 1993; Sternberg et al., 1989a; Wilder et al., 1987).
Although chronic SCW arthritis is clearly generated by immunopathogenic mechanisms, a major unresolved issue is the identity of an-tigenic targets of the immunopathologic process. Numerous lines of evidence support the view that CD4+ T cells are involved (e.g., Van Den Broek et al., 1992a,b). Moreover, one study indicated that the T cells ultimately target an articular autoantigen because T cell lines derived from cell wall-injected animals can induce arthritis in cell wall-naive recipients (DeJoy et al., 1989). These pathogenic T cell lines also proliferate in response to both SCW PG-PS and Mycobacterium tuberculosis. It was hypothesized that molecular mimicry between SCW PG-PS antigens and cartilage proteogly-can, an autoantigen, was involved in producing chronic arthritis, but Van De Langerijt et al. (1994) reported data not supporting this hypothesis. Thus, it is still not known whether chronic arthritis is generated by a SCW-in-duced immunopathologic response to an articular autoantigen or to persistent cell wall antigen in the joint tissues. Moreover, it is also not known to what extent nonspecific or innate inflammatory mechanisms, which are induced by the persistent cell walls ingested by macrophages, participate in or are required for chronic arthritis. These are areas of intense investigation.
SCW arthritis has been and continues to be used actively in the exploration of new therapies for chronic disease. Because the disease process is, usually, highly reproducible, it is relatively easy to evaluate therapies that modify the disease process. Monitoring of disease activity is most easily done by clinical observa tion (see Basic Protocol, step 8) and is probably more sensitive than measurement of joint swelling. Other commonly used methods for assessing disease activity include histological examination of decalcified joint tissues and radiological examination. Frequently, these studies have provided important insights into pathogenic mechanisms (Allen et al., 1993; McCartney-Francis et al., 1993; Van Den Broek et al., 1992a,b; Wahl et al., 1994; Wilder et al., 1987; Yocum et al., 1986, 1988; Yoshino et al., 1991).
Successful results with the SCW arthritis model require attention to several factors related to the SCW preparation and the host, as well as to several technical issues. SCW arthritis is generated over a relatively narrow dose range of injected 10S PG-PS (10 to 60 pg/g of body weight). Doses above this range result in very high rates of mortality and doses below this range result in very low incidence of arthritis. Initial PG-PS dose-finding studies are highly recommended before embarking on large-scale, complicated experimental protocols. Particle size distribution is also critical. Very large particles (e.g., >500 x 106 Da) are minimally arthritogenic. These particles may be removed by centrifugation at 10,000 x g for 30 min. Therefore, the 10S PG-PS preparations, which contains particles <500 x 106 Da, are ideal. However, too much sonication of SCW PG-PS preparations may generate fragments that are predominantly <5 x 106 Da. These fragments are highly arthritogenic, but they only induce transient, acute arthritis. Very small fragments will not pellet after centrifugation at 100,000 x g for 30 min. If only acute arthritis develops, but not chronic arthritis, check the ratio of PG-PS in the pellet to that in the supernatant (by measuring the amount of rhamnose) after centrifugation at 100,000 x g for 30 min. Ideally, at least 50% of the PG-PS will pellet at 100,000 x g, indicating that the preparation contains particles between 5 and 500 x 106 Da, which are most active stimulators of chronic arthritis. Sterility of the preparation is also important. Contamination of the preparation with endotoxin generates a highly lethal material.
Inbred female Lewis rats from several sources, weighing between 100 and 200 g, are generally highly susceptible to arthritis. Occasionally, however, arthritis does not develop even in Lewis rats. In addition to the cell wall-related factors cited above, this failure to de velop arthritis may also be related to an inapparent infection or to unusually "stressed" animals. Therefore, maintenance of a specific-pathogen-free environment and minimal stress generates the most consistent arthritic responses. Cage overcrowding should be avoided. The gut flora also appear to affect disease expression, so it is important to obtain the animals from a single source to maintain consistency. In this context, it is interesting that "germ-free" F344 rats are relatively susceptible to arthritis, whereas specific-pathogen-free or conventional F344 rats are relatively resistant to disease (Van Den Broek et al., 1992a,b). The mechanisms underlying these observations are not well defined, but are a subject of continuing investigation.
Group A SCW PG-PS-induced arthritis in female Lewis rats exhibits a biphasic course over time (Wilder et al., 1982, 1987). An acute arthritis, manifested by redness and swelling of the distal joints of the extremities, typically develops within 48 hr and is maximally severe 3 to 5 days after the injection. This inflammatory response develops coincidentally with deposition of PG-PS fragments in the peripheral joints. PG-PS deposition is also noted in the spleen, liver, and bone marrow (Anderle et al., 1985). The initial inflammatory response is characterized histologically by edema, extensive fibrin deposition, and a mixed polymor-phonuclear and mononuclear leukocytic infiltrate, and is mediated by local activation of the complement system and other humoral inflammatory effector mechanisms (Greenblatt et al., 1978; Schwab et al., 1982). The acute response typically diminishes in severity, and may totally resolve, over the first week, but increased swelling is generally noted again 10 to 21 days after injection. This later-developing chronic phase persists for months, although it may fluctuate markedly in severity. The chronic phase of disease is regulated, in part, by thymic-depend-ent cellular immune-mediated processes. This phase of disease is characterized histologically by pronounced hyperplasia of synovial lining cells, infiltration of the sublining synovial tissues by mononuclear cells, proliferation of fi-broblast-like cells, angiogenesis, and extensive cartilage destruction and bone erosion. The number of osteoclasts in the subchondral bone marrow increase markedly during this phase of the disease (Allen et al., 1985; Case et al., 1989; Clark et al., 1979; Dalldorf et al., 1988; Sano et al., 1993; Wilder et al., 1987; Yocum et al.,
1988; Yoshino et al., 1991). The chronic phase of disease may also be accompanied by the development of T cell-dependent hepatic granulomas, associated with the deposition of PG-PS in the liver (Allen et al., 1985; Wahl et al., 1986; Yocum et al., 1986).
The incidence and severity of SCW arthritis is influenced by cell wall-related factors (e.g., type, strain, group of bacteria, cell wall dose, and particle size distribution), host-related factors (e.g., source, strain, sex, age, health, and care-related factors), and technical factors (e.g., injection into stomach or cecum instead of peritoneal space). Scrupulous attention to these factors is required to obtain reproducible results.
With a proper preparation of SCW 10S PG-PS fragments and healthy female Lewis rats, the incidence of acute and chronic arthritis should approach 100%. Occasional animals may not develop disease because of the factors listed above, or because of the inadvertent injection of the cell walls into the stomach, cecum, or bladder. Injection into the left lower quadrant of the abdomen should minimize the possibility of injecting the cell walls into the stomach or cecum.
The major time factor in the generation of SCW arthritis is the preparation of cell walls. If only a few animals are to be studied, cell walls can be prepared in less than a week. If large numbers of animals are to be studied (e.g., several hundred), then the preparation cell walls is a major task. It can easily require 4 to 6 weeks to grow the bacteria and prepare the 10S PG-PS. The duration of arthritis depends on the experimental objectives. It is common to observe the disease for 6 to 8 weeks.
Abd., A.H., Hume, D.A., Halliday, W.J., and Davis, G.H.G. 1990. Immunopathological investigations during the course of arthritis induced in rats by Streptococcus agalactiae. Med. Microbiol. Immunol. 179:13-23.
Allen, J.B., Blatter, C., Calandra, G.B., and Wilder, R.L. 1983. Sex hormonal effects on the severity of streptococcal cell wall-induced polyarthritis in the rat. Arthritis Rheum. 26:560-563.
Allen, J.B., Malone, D.G., Wahl, S.M., Calandra, G.B., and Wilder, R.L. 1985. The role of the thymus in streptococcal cell wall-induced arthritis and hepatic granuloma formation: Comparative studies of pathology and cell wall distribution in athymic and euthymic rats. J. Clin. Invest. 76:1042-1056.
Animal Models for Autoimmune and
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