It is well known that TNF-alpha is highly involved in arthritis, notably in RA. Accordingly, in our reference studies in the present investigation we found that TNF-alpha was expressed in mononuclear-like cells in the RA synovial tissue. Detection of TNF-alpha reactions was thus clarified from the methodological point of view, and verifications were obtained via preabsorption stainings. With this as a basis, studies on TNF-alpha in myositis were performed.
A unique model for the production of myositis in rabbit musculature (the soleus muscle) was utilized. The main finding was that cells in the inflammatory infiltrates in the myositis muscles were found to express TNF-alpha at both at the mRNA and protein levels. Colocalization between TNF-alpha and CD68 was noted for these cells. Expression of TNF-alpha in macrophages has previously been noted in other situations (e.g., [13
]), including inflammatory myopathies [17
]. In contrast, in our recent studies using the current myositis model, expression of the vesicular glutamate transporter VGluT2 was noted in white blood cells in the inflammatory infiltrates other than macrophages [24
]. A further main finding was that the muscle fibers and blood vessel walls in areas showing inflammatory infiltration exhibited TNF-alpha mRNA and that fibroblasts also were seen to exhibit TNF-alpha mRNA.
From a methodological point of view, it was clear that muscle fibers, blood vessel walls, and fibroblasts exhibited TNF-alpha mRNA but that no reactivity (muscle fibers, blood vessel walls) or very weak reactivity (fibroblasts) was noted at the protein level. The production level in these locations is therefore likely to be low, which precluded clear detection with our immunohistochemical methods. It is also possible that our in situ hybridization method detects very small quantities of TNF-alpha mRNA. Nevertheless, it has previously been shown that TNF-alpha can be expressed not only in inflammatory cells but also in injured muscle fibers and fibroblasts in response to muscle injury (crush-injury) [7
] as well as in muscle fibers and cells in the connective tissue in inflammatory myopathies [17
The patterns of morphologic appearances of the inflammatory infiltrates and other morphologic changes seen resembled the appearances that can be seen in the muscle tissue in inflammatory myopathies [38
]. Nevertheless, preliminary analysis using ELISA detecting anti-Jo-1 antibodies, which are known to correlate with disease activity for patients with inflammatory myopathy [39
], does not lend proof to the theory that the myositis in our model is autoimmune in origin (unpublished observations). However, further studies on this aspect are warranted.
As noted previously, TNF-alpha immunoreactions in inflammatory cells invading muscles affected by myositis have previously only been documented in biopsies from patients with inflammatory myopathies [17
] and in muscle of mice in response to crush-injury [7
]. Thus, in combination with this previous work, our results imply that TNF-alpha is intimately involved in the inflammatory process in myositis. Indeed, it has been suggested that TNF-alpha may have a role in the pathogenesis of the myositis in the inflammatory myopathies [40
] and that a marked inflammatory response involving TNF-alpha may be directly responsible for damaging muscle fibres in myopathic conditions [42
Whether or not the TNF-alpha produced by the cells of the inflammatory infiltrates is entirely responsible for pro-inflammatory and damaging effects remains open to speculation. It is well known that TNF-alpha administration can have pro-inflammatory and detrimental effects, for example, leading to various catabolic changes as seen in studies on cultured skeletal muscle cells [4
]. However, there is a marked discrepancy in the literature regarding the effect of TNF-alpha on the musculature. Some studies on myoblast cell culture show that TNF-alpha administration does not have catabolic effects (e.g., [45
]), and other studies documenting accumulations of inflammatory cells in skeletal muscle in response to TNF-alpha administration [4
] show no decrease in skeletal muscle proteins and no signs of muscle atrophy or injury. Perhaps these discrepancies reflect a dual role of TNF-alpha where in some circumstances inflammatory cell derived TNF-alpha can play a protective role [46
] and also be involved in the recovery of muscle function after traumatic injury [9
] and in muscle regeneration [47
]. The discrepancies may also reflect the fact that different methods have been used in the studies that have been performed. The results in preliminary studies on inflammatory myopathies suggest that TNF blocking might be useful [48
], but it is also emphasized that further studies are needed in order to clarify if this type of treatment is indeed useful [22
]. Results of in vitro studies suggest that targeting TNF-alpha might be worthwhile in myositis [49
] and studies on dystrophic mdx mice subjected to wheel exercise indicate that TNF blockade can reduce myofiber necrosis [20
]. The use of anti-TNF treatment in studies on a rat model of repetitive reaching and grasping leads to an improvement in grip strength and attenuated task-induced increases in inflammatory cytokines, including TNF-alpha [51
Although the use of other animal models have shown inflammation in muscle tissue in response to various forms of exercise [50
], the myositis model used in the current experiment is clearly distinguishable from these models. Thus, in contrast to these models, it leads to a marked presence of inflammatory infiltrates in the muscle tissue, that is, a morphology resembling that seen in inflammatory myopathies. In fact, no experimental myositis model exists which resembles the one used here and in which a marked presence of inflammatory infiltrates becomes present in the muscle tissue. Those for which such an infiltration has been demonstrated are the model of crush-injury described above [7
] and models designed to help understand the mechanisms of inflammatory myopathies that occur in man. In these latter cases, myositis is induced by various infectious agents [53
], immunization with muscle components, for example, myosin [55
], and intraperitoneal injections with lipopolysaccharide [57
]. The TNF system has not been examined in any of these myositis models replicating the inflammatory myopathies seen in man. Interestingly, there is evidence indicating a relationship between the inflammatory myopathies and another condition, muscular dystrophy, in the form of complex interactions between immunological and nonimmunological features of the diseases [58
A noteworthy aspect with the currently used model is that marked overuse is applied in the procedures. Nevertheless, a limitation of the present study is that the relative contributions of the exercise protocol and the injections of the proinflammatory substances to the observed myositis are unclear. Forthcoming studies will clarify this issue. In any case, the model will, as it is currently used, provide the opportunity to evaluate the effects of interference with TNF-alpha actions in myositis development.