Eosinophilia of dystrophin-deficient muscle is promoted by perforin-mediated cytotoxicity by T cell effectors

Abstract

Previous investigations have shown that cytotoxic T lymphocytes (CTLs) contribute to muscle pathology in the dystrophin-null mutant mouse (mdx) model of Duchenne muscular dystrophy through perforin-dependent and perforin-independent mechanisms. We have assessed whether the CTL-mediated pathology includes the promotion of eosinophilia in dystrophic muscle, and thereby provides a secondary mechanism through which CTLs contribute to muscular dystrophy. Quantitative immunohistochemistry confirmed that eosinophilia is a component of the mdx dystrophy. In addition, electron microscopic observations show that eosinophils traverse the basement membrane of mdx muscle fibers and display sites of close apposition of eosinophil and muscle membranes. The close membrane apposition is characterized by impingement of eosinophilic rods of major basic protein into the muscle cell membrane. Transfer of mdx splenocytes and mdx muscle extracts to irradiated C57 mice by intraperitoneal injection resulted in muscle eosinophilia in the recipient mice. Double-mutant mice lacking dystrophin and perforin showed less eosinophilia than was displayed by mdx mice that expressed perforin. Finally, administration of prednisolone, which has been shown previously to reduce the concentration of CTLs in dystrophic muscle, produced a significant reduction in eosinophilia. These findings indicate that eosinophilia is a component of the mdx pathology that is promoted by perforin-dependent cytotoxicity of effector T cells. However, some eosinophilia of mdx muscle is independent of perforin-mediated processes.

Figure 1.

Fluorescent microscopy of eosinophils in cross-sections of mouse quadriceps muscle. Bright cells are eosinophils. Micrographs are printed so that muscle fibers can be discerned by faint background fluorescence. A: Representative section of 4-week-old C57 quadriceps. One eosinophil is present in the field. Scale bar, 60 μm. B: Representative section of necrotic region of 4-week-old mdx quadriceps. Scale bar, 50 μm. C: Representative section of a regenerated region of 30-week-old mdx quadriceps. Scale bar, 50 μm. D: Representative section of quadriceps from a 4-week-old perforin-deficient, mdx mouse. Scale bar, 60 μm.

Figure 2.

Concentration of eosinophils in quadriceps muscle. Data from C57 (black bars), mdx (stippled bars), perforin-deficient mdx (striped bars), and prednisolone-treated mdx (white bars) are shown for 4-week-old (left) and 30- to 32-week-old (right) groups are shown. Asterisks indicate those groups that differ significantly from the age-matched mdx at P = 0.05. #, Indicates those groups that differ significantly (P = 0.05) from 4-week animals receiving the same treatment.

Figure 3.

Concentration of eosinophils in diaphragm muscle. Symbols are identical to those used in Figure 2 ▶ .

Figure 4.

Electron micrograph of 4-week-old mdx muscle showing an eosinophil lying within the endomysial connective tissue that separates two muscle fibers that show no signs of pathology. The eosinophil is separated from the muscle fiber surface by ∼0.5 μm (arrows) and the MBP rods within the eosinophil are oriented along the length of the eosinophil. Scale bar, 1.0 μm.

Figure 5.

Electron micrograph of 4-week-old mdx muscle showing invading eosinophil. A: Portions of two muscle fibers (M) between which an eosinophil (E) has invaded are shown. Scale bar, 1.0 μm. B: Higher magnification of invading eosinophil illustrated in A. Note that the extracellular matrix (ECM), which includes the basement membrane that envelops the muscle fiber, lies between the eosinophil and the upper muscle fiber. The eosinophil lies in close apposition to the lower muscle fiber, and no basement membrane or other connective tissue separates the two cells. Note that the rod-like inclusions of MBP impinge on the surface of the lower muscle fiber (arrows). Vesicles containing MBP are not similarly distributed at the opposite surface of the eosinophil, where direct contacts between the eosinophil and muscle membranes have not been formed. Scale bar, 400 nm. C: Higher magnification view of single MBP rod impinging on muscle fiber surface where the close association between the eosinophil and muscle cell membranes (arrows) can be resolved. Note that no connective tissue lies between the two cells. Scale bar, 150 nm.

Figure 6.

Electron micrograph of 4-week-old mdx muscle showing an eosinophil at the surface of a necrotic fiber. Note that the muscle cell membrane is absent or disrupted adjacent to the site of eosinophil apposition and that the region of the muscle fiber subjacent to the site of membrane loss (arrow) shows dissolution of cytosolic contents. Also note that the eosinophilic granules are aggregated at the region of the eosinophil that is closely applied to the necrotic fiber surface. Scale bar, 1.0 μm.

Figure 7.

Concentration of eosinophils in quadriceps muscles of control C57 mice (striped bars), irradiated C57 mice receiving injection of mdx muscle extract (no eosinophils were present) and irradiated C57 mice receiving injection of mdx muscle extract and mdx splenocytes. Asterisks indicate groups that differ significantly from C57 control at P = 0.05. #, significant difference (P = 0.05) from irradiated animals receiving injection of muscle extract but no splenocyte injection.