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From the Duchenne Muscular Dystrophy Research Center, University of California, Los Angeles, California
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T-cell-promoted eosinophilia has been demonstrated in experimental delayed-type hypersensitivity in which CD8+ T cells are injected into the footpads of mice.8 The eosinophilia produced in this experimental delayed-type hypersensitivity is attributable to specific cytotoxic actions of T cells because T cells from perforin-null mutants caused less hypersensitivity than wild-type T cells. Although the mechanism through which CD8+ T cells induce eosinophilia is unknown, the finding that CD8+ cells which express the Th2 repertoire of cytokines cause more eosinophilia than CD8+ cells that express the Th1 repertoire, suggests that cytokines involved in humoral responses may underlie the T cell promotion of eosinophilia.
Recent provocative findings have also shown a potentially significant functional link between eosinophilia and T cell involvement in autoimmune diseases. Experimental autoimmune encephalomyopathy (EAE), the mouse model of the human autoimmune disease multiple sclerosis, results from a T-cell-mediated autoimmune response to myelin basic protein.9,10 Eosinophils have been noted previously in the immune cell population present in multiple sclerosis tissue,11 although their presence has been generally neglected as a substantial contributor to the pathology. However, adoptive transfer experiments in which activated splenocytes from EAE mice were transplanted into control mice showed that not only did EAE pathology appear in the optic nerves of the recipient animals, but eosinophilia of the optic nerve also resulted.12 This finding suggests that eosinophilia in autoimmune disease may result from T-cell-mediated activities.
Muscle pathology in the mdx mouse model of Duchenne muscular dystrophy (DMD) has been shown to involve a substantial and important contribution of CTLs to the mdx pathology. Evidence that supports this role for CTLs in muscular dystrophy includes the finding that antibody depletions of CTLs from dystrophic muscle produce large reductions in histologically-discernible pathology.13 Furthermore, a significant portion of the cytotoxicity induced by T cells has been shown to occur through perforin-mediated mechanisms that involve both apoptotic and necrotic cell death.13 However, it is unknown whether perforin-mediated pathology may also promote eosinophilia in dystrophic muscle.
In the present investigation, we test the hypothesis that eosinophilia is a component of the pathology of muscular dystrophy and that it is induced by perforin-mediated actions of T lymphocytes. We test this hypothesis by quantifying the presence of eosinophils in mdx hindlimb muscles, which undergo a single prominent bout of necrosis at 4 weeks of age, and in mdx diaphragms, which undergo progressive necrosis through the animals’ lives, in comparison to age-matched controls. We also assay for specific cell-cell contacts between eosinophils and mdx muscle fibers that indicate eosinophil-mediated cytotoxicity. We then assess whether muscle eosinophilia can be induced in wild-type animals by splenocyte transplantation from mdx animals at the peak of pathology. The possibility that mdx muscle eosinophilia is promoted by perforin-dependent mechanisms is tested by assaying whether double-mutant mice, that lack both dystrophin and perforin, exhibit lower eosinophil concentrations than mdx mice. Finally, we examine whether treating dystrophic mice with a prednisone derivative, which is one of the few known, useful therapies for dystrophin-deficient muscular dystrophy, may influence eosinophilia, and thereby support the proposal that reduction of eosinophil populations may ameliorate the pathology of muscular dystrophy.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Dystrophin-null mutant mice (mdx) and wild-type controls (C57) were obtained from Jackson Laboratories (Bar Harbor, ME). Perforin-null mutant mice were provided by Dr. William R. Clark (UCLA). All mice were maintained in the UCLA vivarium until killing for tissue collection. The Animal Research Committee at UCLA approved all animal treatments. Perforin-deficient, dystrophin-deficient mice were generated by crossing mdx mice with perforin-null mutants, according to previously described breeding and screening strategies.13 Animals were used at 4 weeks or at 30 to 32 weeks of age. The hindlimb musculature is at the peak of necrosis in 4-week-old animals and the muscles have regenerated at 30 to 32 weeks of age.14 Diaphragm pathology is also apparent at 4 weeks of age, but it continues to progress throughout the life of the animal.
Assays to Confirm Identity of Perforin-Deficient mdx Mice
Presence of the perforin mutation was confirmed by polymerase chain reaction (PCR) analysis of genomic DNA isolated from tail tissue. Primer pairs and PCR conditions used to identify the homozygous mutants have been described previously.13 Mice in the F2 and F3 generations in the breeding strategy used were dystrophin-deficient13 although this was confirmed in select mice by immunoblot analysis.15
Adoptive Transfer Procedures
Splenocytes were isolated from 4-week-old, female mdx mice by sterile dissection of spleens after which the spleens were forced through a stainless steel mesh in PBS (50 mmol/L sodium phosphate buffer at pH 7.4 containing 150 mmol/L sodium chloride). The filtrate was then pressed through a 70-µm nylon filter and red cells were lysed with Tris-ammonium chloride. The splenocytes in the filtrate were then counted with a hemocytometer.
Four-week-old female C57 mice that had been 
-irradiated with 800
rads received an injection of 1.4 x 107
mdx
splenocytes into the tail vein 4 days after irradiation. In addition,
the mice received intraperitoneal (ip) injections of sterile extracts
of mdx muscle to prime transplanted T cells. Control mice were
irradiated and received ip injections of muscle extract but received
tail vein injections of PBS only. The host animals’ hindlimb muscles
and diaphragms were then analyzed 14 days later for eosinophilia.
Electron Microscopy
Hindlimb muscles from 4-week-old C57 and mdx mice were fixed in situ at physiological length in 1.4% glutaraldehyde in 0.2 mol/L sodium cacodylate, pH 7.4, for 30 minutes and dissected free from the hindlimb and fixed for an additional 30 minutes. The muscles were then sliced into small strips and placed in 1.0% osmium tetroxide for 40 minutes, after which they were rinsed in 0.2 mol/L sodium cacodylate, pH 7.4, dehydrated in a series of ethanols, followed by propylene oxide, and then embedded in epoxy resin. Thin sections were cut at 70 nm, stained with uranyl acetate and lead citrate and viewed in a transmission electron microscope.
Prednisolone Treatments
mdx mice received ip injections of 0.75 mg of prednisolone (a derivative of prednisone) per kilogram mouse body mass in sterile PBS daily, beginning at 2 weeks of age and continuing until sacrifice for tissue collection at 4 weeks of age. Control mdx mice received injections of equal volumes of PBS ip.
Assay for Eosinophils
Hindlimb muscles and diaphragms were collected from mice and prepared and sectioned as described previously. All sections used for analysis were cross-sections taken at the mid-belly of the hindlimb muscle or through the coronal plane of the diaphragm muscles. Cationic vesicles that are characteristic of eosinophils were identified by fluorescein binding, as described by Nonaka et al.16 The concentration of eosinophils in each sample was determined by quantitative microscopic techniques in which the number of eosinophils per total cross-section of muscle was measured and then the number of eosinophils per unit volume was calculated for 10-µm-thick sections.13
Statistical Analysis
All quantitative data were analyzed using Student’s t-test with confidence level at 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fluorescein binding assays show that eosinophilia is a prominent
feature of the necrotic phase of muscular dystrophy in mdx mice (Figure 1)
. At the peak of hindlimb pathology in
4-week-old mice, mdx quadriceps contains ~4033
eosinophils/mm3
(SE = 467; n
= 22) which is a highly significant (P <
0.001), nearly 20-fold increase over the concentration present in
age-matched C57 mice (228 eosinophils/mm3;
SE = 190; n = 11) (Figures 1 and 2)
. This eosinophil concentration in mdx
muscle decreases significantly during the regenerative period of
the pathology to ~1184 eosinophils/mm3
(SE = 190; n = 11), which is still greater
than the concentration present in age-matched C57 mice (167
eosinophils/mm3; SE = 44; n
= 5).
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. The concentration of eosinophils in
the mdx diaphragm was significantly lower than the concentration in mdx
hindlimbs at both 4 weeks and 30 to 32 weeks of age. However, we also
found that control C57 diaphragms contain significantly fewer
eosinophils than present in C57 hindlimbs at both 4 weeks and 30 to 32
weeks of age.
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. Hindlimb muscles from each of five
mdx mice at 4 weeks of age showed eosinophils at the surface of
necrotic fibers. No eosinophils were observed by electron microscopy in
C57 hindlimb muscles. Electron microscopic observations showed three
different relationships between eosinophils and mdx muscle fibers: 1)
some eosinophils were separated from the surface of apparently healthy
muscle fibers by basement membrane and other connective tissue
components (Figure 4)
; 2) other eosinophils were closely apposed to the
intact surface membrane of intact mdx fibers (Figure 5)
; or 3) other eosinophils were located
at the surface of mdx fibers that had already experienced cell
membrane dissolution (Figure 6)
. In the
first instance, no obvious signs of pathology were evident in
the fibers near the eosinophils, and the MBP rods tended to be
oriented along the long axis of the eosinophils (Figure 4)
. In the
second instance, the eosinophil and muscle cell membranes were
closely apposed at sites where the eosinophil had crossed the muscle
cell’s basement membrane. At these sites of close apposition, MBP rods
in the eosinophils were oriented approximately normal to the membranes,
and the eosinophil membrane protruded toward the muscle fiber membrane,
and impinged on the muscle cell surface (Figure 5)
. In the latter case
when the muscle fibers were frankly necrotic, the eosinophils remained
at the surface of the necrotic fiber, but the fiber to which they were
adjacent showed membrane lysis and disruption of organelles (Figure 6)
.
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If eosinophilia of mdx muscle were a specific response
to the actions of autoreactive T cells to dystrophic muscle, we
postulated that the transplantation into wild-type mice of splenocytes
from mdx mice collected at the peak of pathology would induce
eosinophilia in the muscles of the recipient animals. Injection of mdx
splenocytes into the tail veins of irradiated C57 mice that had also
received a sterile injection of mdx muscle extract produced a
significant increase in eosinophils in the recipient mouse hindlimb
muscles at 2 weeks after injection (49
eosinophils/mm3; SE = 13; n
= 14) (Figure 7)
. C57 mice that received
muscle extract injections ip and but received buffer only in tail vein
injections, showed no eosinophils in their muscles at 2 weeks after
injection (0 eosinophils/mm3; SE = 50;
n = 6). Both the control mice and the mice undergoing
splenocyte transplantation contained eosinophil concentrations that
were significantly much lower than nonirradiated, age-matched C57 mouse
muscles, but this is attributable to the irradiation of the
experimental animals and their buffer injected controls.
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All animals used in this analysis were shown to be
homozygous for the perforin mutation in that the PCR analysis yielded
PCR products at 350 and 1600 bp, whereas the wild-type PCR product is
at 500 bp.13
Quantitative histochemical analysis of
4-week-old double-mutant mice showed that they contain significantly
fewer eosinophils than mdx muscles, although the eosinophil
concentration in double-mutant mice remained much higher than that
observed in age-matched C57 (Figures 1–3)
. The reduction in eosinophil
concentration in 4-week hindlimb muscle of double-mutants was ~30%,
whereas their reduction in diaphragm was ~65% compared to
age-matched mdx muscles. Eosinophil concentrations in 30- to
32-week-old diaphragms of double-mutant mice were significantly
(~65%) less than the concentration in age-matched mdx diaphragms,
and did not differ significantly from age-matched C57 diaphragms.
However, the concentration of eosinophils in the hindlimb musculature
of 30- to 32-week-old double-mutants did not differ from that found in
mdx.
Prednisolone Treatments Reduce Eosinophilia in mdx Mice
Administration of prednisolone to mdx mice beginning in
prenecrotic, 2-week-old animals until the peak of necrosis at 4 weeks
of age caused a significant reduction in eosinophilia (Figure 2)
.
Quadriceps muscle of prednisolone-treated animals showed a significant,
30% decrease in the concentration of eosinophils compared to
age-matched, nontreated controls. The concentration of eosinophils in
prednisolone-treated animals did not differ from the concentration
present in age-matched, perforin-deficient mdx hindlimb muscles.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The electron microscopic observations reported here that show close associations between eosinophil and muscle cell membranes also provide strong evidence that eosinophils specifically recognize the dystrophic muscle cell surface. Muscle fibers are ensheathed in basement membranes that form a barrier between their cell membrane and those of all other cell types except satellite cells in healthy tissue,19 and immune cells during disease states in which specific receptor-ligand interactions between the muscle and immune cells are expected to occur.20 The ability of immune cells to cross this connective tissue barrier indicates specific chemoattractant activity that may result from muscle injury.21 Furthermore, the close apposition between the membranes of the two cells reflects specific cell-cell interactions, and is not observed to occur between cells that do not associate via specific molecular interactions. The 15-nm gap observed between the eosinophil and muscle membrane at sites of close cellular apposition is similar to that observed at adherens junctions (about 15 to 20 nm22 ) or desmosomes (about 20 to 30 nm23 ), and is substantially narrower than the gap that occurs between the membranes of adjacent cells that do not associate via specific molecular associations (50 nm or more). This spacing between neighboring cells that do not share direct adhesive associations is largely attributable to the negative charge carried by cell membranes that precludes close apposition of membranes in the absence of specific interactions between surface molecules.24 For example, the distance between apposing membranes of CTLs and their targets does not reach values less than 10 nm which is attributable to glycoconjugates present on the cells’ surfaces.24 Finally, the restricted distribution of dense material in the space between the two apposing cell membranes is a consistent feature of sites of specific cell-cell adhesion.22,23
Previous electron microscopic analyses have shown that the rods present in eosinophils consist of MBP,17,18 which is a cytolytic protein that directly disrupts plasma membrane structure25,26 and accounts for much of the eosinophilia of the vesicles in which it is stored. Eosinophil cationic protein is colocalized in the same vesicles that contain the MBP and is also capable of contributing to the cytolytic actions of eosinophils in that eosinophil cationic protein can form pores in target cell membranes.27 Together, eosinophil cationic protein and MBP-induced damage to target cell membranes result in swelling of the target cell, influx of Ca2+, and loss of cytosolic proteins into the extracellular space.25 All of these pathological changes are consistent with previous reports of the pathological changes that occur in dystrophin-deficient muscle during its necrosis.14,15 The finding in the present investigation that each of the sites of close apposition between eosinophil and muscle cell membrane is associated with a MBP rod that impinges on the muscle cell surface, indicates that cytolysis of the muscle cell may occur at these sites of cell adhesion.
These findings contribute to the growing evidence that the immune system plays an important role in the pathophysiology of dystrophin-deficient muscular dystrophy. Death of dystrophin-deficient muscle has been primarily attributed to mechanical damage incurred by the cell membrane which lacks the support of the cytoskeletal protein, dystrophin.28 However, this mechanical defect hypothesis for dystrophic muscle death has been inadequate to explain many aspects of the pathophysiology of the disease. For example, the onset of severe muscle cell necrosis does not occur when ambulation first begins in humans or other mammals that are null mutants for dystrophin.29 Instead, overt muscle weakness and necrosis does not occur until ~3 years of age in humans and ~3.5 weeks of age in mice. The mechanical defect hypothesis is also inadequate to explain the effectiveness of nonanabolic steroids in slowing the progress of muscle weakness or necrosis, and current evidence indicates that the beneficial effects of prednisone on DMD muscle result from suppression of the activities of immune cells.30
The observation that prednisone treatments significantly reduce the concentration of eosinophils in dystrophic muscle suggests that this response may underlie some of the beneficial effects of prednisone in the treatment of DMD. Currently, prednisone treatments are the most effective therapeutic that is approved in the United States for use in slowing the progress of muscle weakness in DMD, although there is no clear, pharmacological understanding for the beneficial effects of this drug in DMD.31 Previous investigators have speculated that prednisone could increase muscle regenerative capacity, stabilize lysosomal proteases and thereby reduce wasting, or stabilize the muscle cell membrane and reduce muscle damage as a result. However, there is little support for those speculations and several investigations show that these proposals are not well-founded.30,32 Prednisone or its derivatives have been shown to reduce necrosis of dystrophin-deficient muscle fibers and to reduce the concentrations of CD8+ T cells30 and eosinophils (present study) in dystrophin-deficient muscle, although it has no significant effect on the concentration of other immune cells in DMD muscle, for example B cells, macrophages, or CD4+ T cells.30 Thus, the apparently selective effect of prednisone in diminishing CTL and eosinophil concentrations in dystrophic muscle, while improving strength of dystrophic muscle, further supports the proposal that both of these cell types contribute to the pathology of dystrophin deficiency and that CTLs promote eosinophilia in muscular dystrophy.
As examination of the potential involvement of the immune system in the pathology of dystrophin-deficient muscle proceeds, evidence continues to accumulate which indicates that the immune system may play an important role in promoting dystrophic muscle death. For example, T cell receptors of CTLs isolated from DMD patients show a conserved peptide in the hypervariable domain of the receptor that interacts with antigen, and this peptide does not occur in the receptors of CTLs obtained from patients with other neuromuscular diseases.33 This suggests that a specific antigen may activate T cells in DMD. More recently, antibody depletions of CTLs from mdx mice were found to cause significant reductions in muscle pathology,13 which shows that CTLs promote the pathology of muscular dystrophy. As the present results now show, some of this CTL-mediated pathology may occur through muscle cytolysis by eosinophils.
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Footnotes |
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Dr. Cai and Dr. Spencer contributed equally to this investigation.
Accepted for publication January 24, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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receptor. N Engl J Med 1991, 324:877-881[Abstract]
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