TNF-Alpha in the Locomotor System beyond Joints: High Degree of Involvement in Myositis in a Rabbit Model

Abstract

The importance of TNF-alpha in arthritis is well documented. It may be that TNF-alpha is also markedly involved in muscle inflammation (myositis). An animal model where this can be investigated is needed. A newly developed rabbit myositis model involving pronounced muscle overuse and local injections of substances having proinflammatory effects was therefore used in the present study. The aim was to investigate the patterns of TNF-alpha expression in the developing myositis and to evaluate the usefulness of this myositis model for further TNF-alpha research. Human rheumatoid arthritis (RA) synovial tissue was examined as a reference. TNF-alpha immunoexpression and TNF-alpha mRNA, visualized via in situ hybridization, were detected in cells in the inflammatory infiltrates of the affected muscle (soleus muscle). Coexistence of TNF-alpha and CD68 immunoreactions was noted, suggesting that the TNF-alpha reactive cells are macrophages. Expression of TNF-alpha mRNA was also noted in muscle fibers and blood vessel walls in areas with inflammation. These findings demonstrate that TNF-alpha is highly involved in the myositis process. The model can be used in further studies evaluating the importance of TNF-alpha in developing myositis.

Figure 1

Soleus muscle of a specimen of the myositis group in a section stained with htx eosin. There is a marked inflammatory infiltrate (middle part): myofibers to the left and to the right.

Figure 2

(a, b) Sections of synovial tissue of a patient with rheumatoid arthritis. The sections were stained for demonstration of TNF-alpha (a) and for TNF-alpha after preabsorption with TNF-alpha antigen (b). Inflammatory cells show specific immunoreaction in (a) (arrows). There are no specific immunoreactions in these in (b) (arrows). The inflammatory cells had in parallel sections been identified via staining for routine morphology (htx eosin).

Figure 3

Staining for demonstration of TNF-alpha in a synovial tissue specimen of a rheumatoid arthritis patient. There are specific reactions in inflammatory cells (arrows). The reactions show a granular appearance.

Figure 4

Sections of a myositis specimen stained for TNF-alpha (a) and for TNF-alpha after preabsorption with TNF-alpha antigen (b). The cells of an inflammatory infiltrate show immunoreactions for TNF-alpha (a) (arrows). There are no specific reactions in (b) (arrows at some of the cells).

Figure 5

(a, b) Sections of myositis specimens after processing for TNF-alpha. Cells show specific immunoreactions (arrows). The reactions show a granular appearance.

Figure 6

(a, b) Inflammatory infiltrate in a soleus muscle (myositis specimen). Double-staining for demonstration of TNF-alpha (a) and CD68 (b). Immunoreactions are seen in the same cells (arrows).

Figure 7

In situ hybridization for the demonstration of TNF-alpha mRNA in inflammatory cells. Adjacent sections are shown: antisense staining (a), and sense staining (b). There are reactions in (a) but not in (b). The arrows point at inflammatory cells.

Figure 8

In situ hybridization for the demonstration of TNF-alpha mRNA in fibroblasts. Adjacent sections are shown: antisense staining (a), and sense staining (b). There are reactions in (a) but not in (b). The arrows point at fibroblasts.

Figure 9

In situ hybridization for the demonstration of TNF-alpha mRNA in a small blood vessel: antisense staining (a), and sense staining (b). The vessel was located in the proximity of an inflammatory infiltrate. Arrows point at the wall. There are reactions in the wall in (a) but not in (b). Asterisks are in the lumen.

Figure 10

In situ hybridization for the demonstration of TNF-alpha mRNA in the muscle tissue. Adjacent sections are shown, one processed with antisense probe (a), the other with sense probe (b). There are reactions in muscle fibers in (a) but not in (b). Arrowheads indicate corresponding muscle fibers in (a) and (b). As verified via examinations of parallel sections processed for morphology, the reactive muscle fibers are infiltrated by inflammatory cells. Certain muscle fibers appear to be unaffected (asterisk).