Journal of Histochemistry and Cytochemistry, Vol. 49, 989-1002, August 2001, Copyright © 2001, The Histochemical Society, Inc.


ARTICLE

The Role of Tumor Necrosis Factor-alpha (TNF-{alpha}) in Skeletal Muscle Regeneration: Studies in TNF-{alpha}(-/-) and TNF-{alpha}(-/-)/LT-{alpha}(-/-) Mice

Rachel A. Collins1,a and Miranda D. Groundsa
a Department of Anatomy & Human Biology, The University of Western Australia, Nedlands, Western Australia

Correspondence to: Miranda D. Grounds, Dept. of Anatomy & Human Biology, The University of Western Australia, Nedlands, Western Australia 6907. E-mail: mgrounds@anhb.uwa.edu.au


  Summary
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Summary
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Materials and Methods
Results
Discussion
Literature Cited

The role of tumor necrosis factor-alpha (TNF-{alpha}), an important mediator of the inflammatory response after injury, was investigated in regenerating skeletal muscle. The pattern of expression of TNF-{alpha} during muscle regeneration was examined by immunohistochemistry in tissue sections of crush-injured or transplanted muscle autografts and in primary cultures of adult skeletal muscle. TNF-{alpha} was highly expressed in injured myofibers, inflammatory cells, endothelial cells, fibroblasts, and mast cells. Myoblasts and myotubes also expressed TNF-{alpha} in primary muscle cultures and tissue sections. The essential role of TNF-{alpha} and its homologue lymphotoxin-alpha (LT-{alpha}) during muscle regeneration was assessed by basic histology in TNF-{alpha}(-/-) and TNF-{alpha}(-/-)/LT-{alpha}(-/-) mice. No difference was apparent in the onset or pattern of muscle regeneration (i.e., inflammatory response, activation and fusion of myoblasts) between the two strains of null mice or between nulls and normal control mice. However, both strains of null mice appeared more prone to bystander damage of host muscle and regeneration distant from the site of injury/transplantation. Although expression of TNF-{alpha} may play an important role in muscle regeneration, the studies in the null mice show that redundancy within the cytokine system (or some other response) can effectively compensate for the absence of TNF-{alpha} in vivo.

(J Histochem Cytochem 49:989–1001, 2001)

Key Words: tumor necrosis factor-{alpha}, lymphotoxin-{alpha}, muscle regeneration, immunohistochemistry, null mice, cytokine, histology


  Introduction
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

MUSCLE REPAIR is a complex process, and it is of considerable interest to understand what factors are critical for successful muscle regeneration (Cullen 1997 ; Grounds 1999 ). Although different cytokines have been implicated in inflammatory myopathies such as polymyositis, dermatomyositis, and inclusion body myositis (Tews and Goebel 1996 ; Lundberg and Nyberg 1998 ; De Bleecker et al. 1999 ), there is little information on changes in the pattern of expression of cytokines in response to acute muscle damage. Inflammation is a key response to muscle injury and is essential for muscle regeneration (Tidball 1995 ). The infiltration of inflammatory cells soon after muscle damage results in local availability of many cytokines, and inflammation is crucial to new muscle formation. A central mediator of the inflammatory response is tumor necrosis factor-alpha (TNF-{alpha}), which is produced by the majority of activated leukocytes (Tracey and Cerami 1990 ). TNF-{alpha} plays several important roles in inflammation, including activation and chemotaxis of leukocytes, expression of adhesion molecules on neutrophils and endothelial cells, and regulation of the secretion of other proinflammatory cytokines (Beutler and Cerami 1988 ). TNF-{alpha} is part of a complex network of cytokines and is capable of initiating cytokine cascades involving both synergistic and inhibitory reactions, which control the synthesis and expression of other cytokines, hormones, and their receptors (Cannon and St Pierre 1998 ; Illei and Lipsky 2000 ). For example, TNF-{alpha} induces the expression of interleukin-1 (IL-1) and interferon-gamma (IFN-{gamma}), and these cytokines are of particular interest in muscle regeneration because of their ability to induce inflammation (Tracey 1994 ; Hodge-Dufour et al. 1998 ).

The cytokine lymphotoxin-alpha (LT-{alpha}) was also examined in this study because of its structural homology to TNF-{alpha} and its ability to bind the TNF receptors (TNF-Rs) (see Fig 1A). LT-{alpha} has similar proinflammatory effects to TNF-{alpha} but exerts only a partial agonistic effect (Decoster et al. 1998 ). LT-{alpha} is of primarily B- and T-lymphocyte origin, and can exist in a soluble homotrimeric form or in membrane-bound heterotrimeric form in complex with lymphotoxin-beta (LT-ß) (Decoster et al. 1998 ) (see Fig 1B).



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Figure 1. (A) Binding patterns for TNF-{alpha} and LT-{alpha} homotrimers and for LT-{alpha}ß heterotrimers with receptors LT-ßR, TNF-RI, and TNF-RII. Modified from von Boehmer 1997 . (B) Progression of LT{alpha} and LTß through the secretory pathway. LT{alpha} monomers either self-associate in the lumen of the endoplasmic reticulum to form homotrimers (LT{alpha}3), which are secreted from the cell on fusion of the secretory vesicle with the cell membrane, or complex with LTß to form heterotrimers of either LT{alpha}1ß2 or LT{alpha}2ß1, which remain membrane-associated. Modified from Androlewicz et al. 1992 .

Increased expression of TNF-{alpha} was observed in muscle fibers biopsied from patients with Duchenne's muscular dystrophy (DMD) (Tews and Goebel 1996 ), and in a small number of regenerating muscle fibers in inflammatory myopathies (De Bleecker et al. 1999 ). In tissue culture, TNF-{alpha} enhanced myoblast proliferation and inhibited differentiation and fusion of mouse C2 cells (Szalay and Duda 1997 ), whereas TNF-{alpha} inhibited both proliferation and fusion of rat L8 myoblasts (Ji et al. 1998 ). There is strong evidence that TNF-{alpha} induces muscle proteolysis and is involved in (cachexia) muscle wasting (Goodman 1991 ; Zamir et al. 1992 ; Cassatella 1995 ; Argiles et al. 1998 ; Ji et al. 1998 ). TNF-{alpha} has also been implicated in a number of non-muscle inflammatory diseases, including rheumatoid arthritis (Douni et al. 1996 ), inflammatory bowel disease (Noguchi et al. 1998 ), and multiple sclerosis (Sharief and Hentges 1991 ). The expression of TNF-{alpha} and the inflammatory response are also regulated by glucocorticoids (Ksontini et al. 1998 ), and this may account (at least in part) for the current clinical use of deflazacort or prednisolone as a treatment for DMD (Anderson et al. 2000 ). Furthermore, IP injection of neutralizing antibodies to TNF-{alpha} before partial hepatectomy has been shown to inhibit liver regeneration in rats (Ackerman et al. 1992 ), and antibodies to TNF-{alpha} have also been used clinically (Illei and Lipsky 2000 ), emphasizing TNF-{alpha}'s role in inflammation and regeneration. These combined observations demonstrate the crucial role of TNF-{alpha} in processes such as tissue repair and strongly suggest that TNF-{alpha} may play a critical role in normal skeletal muscle necrosis and regeneration.

This study describes the pattern of TNF-{alpha} expression in uninjured and regenerating skeletal muscle in three mouse strains, C57BL/10ScSn (the parental strain for mdx and the control strain for the knockout mice), SJL/J, and BALB/c, and in dystrophic mdx muscle. The SJL/J and BALB/c strains are compared because of the well-documented differences in their capacity for muscle regeneration (Mitchell et al. 1992 ; Maley et al. 1995 ; Roberts et al. 1997 ). In addition, the pattern of muscle regeneration in normal mice is compared with that seen in TNF-{alpha}(-/-) and TNF-{alpha}(-/-)/LT-{alpha}(-/-) mice. The results indicate that although TNF-{alpha} plays a role in muscle regeneration, there is no absolute requirement for it to be present.


  Materials and Methods
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Materials and Methods
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Animals
Animals were fed and housed according to guidelines of the National Health & Medical Research Council (NH&MRC) of Australia, and all procedures were approved by the Animal Experimentation Ethics Committee at the University of Western Australia. All mice, apart from the null strains, were obtained from the Animal Resource Centre (Murdoch University, Western Australia). C57/BL10ScSn mice (hereafter referred to as C57/BL) were used as controls for TNF-{alpha} null and TNF-{alpha}/LT-{alpha} null mice. The TNF-{alpha} knockout (Sloan–Kettering; New York, NY) (Marino et al. 1997 ) and TNF-{alpha}/LT-{alpha} (Hoffman–LaRoche; Basel, Switzerland) double-knockout mice were bred on a C57BL/129 background and were kindly donated by Hugh Reid (LaTrobe University; Victoria, Australia). Uninjured and experimentally injured adult C57/BL, SJL/J, BALB/c, TNF-{alpha}(-/-) null, and TNF-{alpha}(-/-)/LT-{alpha}(-/-) null mice were used for basic histology and immunohistochemistry. Uninjured dystrophic mdx mice were also used to model the endogenous muscle injury in DMD. Ten TNF-{alpha}(-/-) and 14 TNF-{alpha}(-/-)/LT-{alpha}(-/-) mice of a range of ages and sexes were used, as shown in Table 1. The relatively small number of these knockout mice was due to their very limited availability.


 
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Table 1. Knockout mice: surgical procedure (crush/whole muscle graft), time of sampling after injury, sex, and body weight

Surgical Procedures
All surgical procedures were carried out under halothane anesthesia and in strict accordance with NH&MRC guidelines. After surgery, mice were transferred to standard cages with food and water ad libitum and allowed to recover in a temperature- and light-sensitive environment.

Crush Injury. Mice received a single severe transverse crush injury to the mid-region of the tibialis anterior (TA) muscle of both legs using a pair of artery forceps, as described previously (McGeachie and Grounds 1987 ). Mice subjected to crush injury were sacrificed at 1, 3, 5, 7, or 10 days after surgery. One or two knockout mice were sampled at each time point. Duplicate mice were sampled at each time point for all other strains.

Whole Muscle Graft. Autografts of intact whole extensor digitorum longus (EDL) muscle were transplanted over the TA muscle as described in detail elsewhere (Roberts and McGeachie 1990 ; White et al. 2000 ). Autografts were performed in both legs of each mouse. Transplanted mice were sacrificed at 3, 5, 7, or 14 days after surgery. One knockout mouse and duplicate mice of all other strains were sampled at each time point (see Table 1).

Sham Injury. Specimens of TA muscles from C57/BL mice that had undergone the surgical procedure, including incision and suturing of the skin but without muscle crush or graft, were used to check the host muscle for TNF-{alpha} staining in response to the basic surgical procedure.

Denervation. Muscles in the hindlimb of BALB/c mice were denervated by removal of a section of the sciatic nerve, as described by McGeachie and Allbrook 1978 . Muscles were sampled at 7 and 14 days after denervation.

Primary Cultures of Myoblasts
Muscle was taken from the hindlimbs and lower back region of 4–6-week-old C57/BL and TNF-{alpha}(-/-)/LT-{alpha}(-/-) mice for primary culture, as previously described (Maley et al. 1994 ). Briefly, skeletal muscle was removed from the hindlimbs and lower back region and myoblasts were isolated by enzymic digestion [collagenase (200 U/ml)/dispase (1 mg/ml), 2 ml/g tissue] and filtration through nylon gauze. Primary cultures were maintained according to standard tissue culture procedures and according to the methods of Maley and colleagues 1994 .

Tissues
Muscle samples were fixed in paraformaldehyde [4% (v/v) in PBS, pH 7.6] for 6 hr and processed for paraffin embedding. Sections of 5 µm were stained with hematoxylin and eosin (for histological analysis) or with antibodies to TNF-{alpha} (for immunohistochemistry).

Immunohistochemistry
The TNF-{alpha} staining protocol was modified from Heng 1996 and followed standard immunoperoxidase procedures. Endogenous peroxidases were quenched by immersion in H2O2 [3% (v/v) in DDW] for 10 min, followed by blocking for 1 hr in fetal calf serum (FCS, 10%). Sections were incubated with rabbit polyclonal anti-TNF-{alpha} antibody (1:400; Genzyme, Cambridge, MA) or PBS only (negative control) at 4C overnight. Sections were incubated for 30 min with biotinylated goat anti-rabbit IgG (1:200; Jackson Laboratories, West Chester, PA), followed by incubation with avidin D–peroxidase (1:100) for 30 min. Diaminobenzidine (DAB) color reagent [1:10 (v/v) in peroxide buffer; Pierce, Rockford, IL] was applied for 4 min. Sections were counterstained with Gill's hematoxylin. TNF-{alpha} protein stained brown (DAB) and cell nuclei stained blue (hematoxylin).

Normal mouse thymus was used as a positive control for TNF-{alpha} staining (Giror et al. 1992 ). TNF-{alpha}(-/-) and TNF-{alpha}(-/-)/LT-{alpha}(-/-) muscle was stained with TNF-{alpha} antibody as a negative control for immunohistochemistry. TNF-{alpha} staining in paraformaldehyde-fixed cultured cells followed the same protocol minus quenching of endogenous peroxidases, and blocking time in FCS was reduced to 20 min. TNF-{alpha} (-/-)/LT-{alpha} (-/-) cultures were used as negative controls.


  Results
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Materials and Methods
Results
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Uninjured, Dystrophic, and Denervated Muscle
The histological appearance of uninjured skeletal muscle from BALB/c, C57/BL, and SJL/J mice was similar. Furthermore, no differences were noted among muscles from these mice aged 3 weeks, 10 weeks, 1 and 3 years of age (data not shown). Dystrophic mdx muscle showed variation in muscle fiber size, many mature muscle fibers with centrally placed nuclei (characteristic of regenerated mouse muscle), focal areas of necrosis with infiltrating phagocytic cells, and areas of regeneration (not shown), as has been well described elsewhere (McGeachie et al. 1993 ). Denervated muscle exhibited muscle fiber atrophy and central nuclei at 4 weeks, with no necrosis or inflammation, as previously described (McGeachie and Grounds 1989 ).

Antibodies to TNF-{alpha} showed little staining of uninjured muscle (not shown). No differences were visible in the staining among strains or among muscle at 3 weeks, 10 weeks, 1 and 3 years of age (not shown). TNF-{alpha} staining was not seen in mast cells of uninjured muscle, although mast cells were clearly present, as demonstrated by Toluidine blue staining (Nahirney et al. 1997 ) (not shown). TNF-{alpha} staining was increased in regions of necrosis and inflammation (see Fig 2), and strong TNF-{alpha} staining was seen in mast cell granules in mdx muscles (similar to staining in Fig 2F).



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Figure 2. TNF-{alpha} (brown) staining in injured and regenerating C57/BL muscle. (A) Negative control, primary antibody omitted, transverse section through Day 3 crush-injured muscle. *, central necrotic core. (B) Longitudinal section through crush-injured muscle at 3 days, showing intense staining in many cells. (C) TNF-{alpha}-stained control section of crush-injured TNF-{alpha}/LT-{alpha} null muscle, showing some nonspecific staining on the edges of muscle fibers but no staining in inflammatory cells. (D) Transverse section showing intense staining of whole EDL muscle graft and adjacent host TA muscle 3 days after transplant. (E) High-power view showing strong staining of a macrophage (m) and neutrophil (n) in Day 3 crush-injured muscle; inset, strong staining of neutrophils in Day 1 crush-injured muscle. (F) Intensely TNF-{alpha}-positive mast cells in a Day 5 graft. (G) Strong staining is seen in persisting necrotic myofibers and variable TNF-{alpha} staining in myotubes (arrows). (H) A muscle spindle in the host TA muscle of a Day 5 graft, showing TNF-{alpha}-positive staining around intrafusal muscle fibers (IF) in what appears to be the inner capsule membrane, and also in the outer spindle capsule (C). Bars = 0.1 mm.

At 1 week after denervation, TNF-{alpha} staining was similar to uninjured muscle, but at 4 weeks there was increased TNF-{alpha} staining in atrophic muscle fibers and in muscle spindles (muscle spindle staining as in Fig 2H). Muscle spindles were not stained in uninjured muscle (not shown). TNF-{alpha} staining in mast cells did not change as a result of denervation.

Muscle Regeneration After Experimental Injury for Control Strains (C57/BL, SJL/J, and BALB/c)
Crush Injury. The overall histological events of regeneration after crush injury were similar in all strains. In accordance with the literature (Mitchell et al. 1992 ), slight differences were seen between the efficiency and timing of regeneration, with BALB/c muscle being the least effective at new muscle formation, SJL/J the most effective, and C57/BL about midway between the two. The histological events are well described by our laboratory in the literature (Mitchell et al. 1992 ): Day 1, the onset of necrosis of damaged myofibers, accompanied by leukocyte infiltration and myoblast proliferation; Day 3, extensive phagocytosis of necrotic muscle by macrophages and fusion of myoblasts to form myotubes; Day 5, the injured muscle is progressively replaced by regularly aligned myotubes and some connective tissue; Day 10, replacement of damaged muscle tissue by new myotubes is essentially complete.

Whole Muscle Graft. Very small differences in the timing of regeneration (i.e., onset of inflammation and myotube formation) were seen between whole muscle autografts in BALB/c and SJL/J mice, as has been described previously (Roberts et al. 1997 ). The key histological events in the regeneration of whole muscle grafts are well described elsewhere (Lawson-Smith and McGeachie 1997 ) and are as follows: Day 2, the central core of the graft becomes necrotic and devoid of nuclei, although occasional myofibers survive at the periphery; Day 3, macrophage infiltration and phagocytosis of necrotic myofibers at the periphery of the graft are associated with revascularization and myoblast proliferation; Day 4, small myotubes have formed in the peripheral zone and regeneration proceeds towards the center; Day 10, all necrotic myofibers are replaced by myotubes, and revascularization is complete.

TNF-{alpha} Staining of Muscles Regenerating After Crush Injury or Grafting
No differences were observed between the intensity of staining with TNF-{alpha} antibodies in regenerating muscles (crushed or grafted) of BALB/c, C57/BL, and SJL/J mice. Necrotic myofibers stained strongly TNF-{alpha}-positive and the sarcoplasm of the uninjured portion of crush-injured muscle also stained TNF-{alpha}-positive (Fig 2B). Sections of single- and double-knockout muscle stained with TNF-{alpha} antibody (as a negative control) showed some staining of damaged myofibers but no staining of inflammatory cells (Fig 2C), demonstrating that although some nonspecific binding occurs, the increased TNF-{alpha} staining seen in injured TNF-{alpha} (+/+) muscles is real. The host TA muscle underlying the EDL autografts also showed strong TNF-{alpha} staining across all time points examined (Fig 2D). However, sham-operated TA muscle was TNF-{alpha}-negative, indicating that the presence of the graft is necessary to increase TNF-{alpha} expression in the host muscle (not shown).

TNF-{alpha} antibodies stained all neutrophils in crush-injured muscle at Day 1 (Fig 2E, inset), and the staining intensity and number of positive neutrophils decreased after this time. Macrophages in crush-injured and grafted muscles stained strongly for TNF-{alpha} (Fig 2E). The small numbers of lymphocytes present in injured muscle and endothelial cells in small blood vessels were also TNF-{alpha}-positive.

Lymphocytes were identified as cells with a small rim of cytoplasm surrounding a dense, non-lobulated nucleus. Mast cells in injured muscle were strongly TNF-{alpha}-positive, with staining localized to mast cell granules (Fig 2F). TNF-{alpha}-positive muscle spindles were present in crush-injured muscles and, after muscle transplantation, in both the EDL graft and host TA muscle (Fig 2H). Both the inner and outer spindle capsule membranes stained TNF-{alpha}-positive [see Maier 1997 for spindle morphology], and the intrafusal muscle fibers within the spindles were variably TNF-{alpha}-positive.

Myoblasts were difficult to identify in sections of crush-injured muscle. Crescent-shaped cells, presumed to be myoblasts (activated satellite cells), were seen within the contour of the basement membrane of necrotic myofibers in grafts. These "cuffing" myoblasts were TNF-{alpha}-negative to moderately TNF-{alpha}-positive. Myotubes in crush-injured and grafted muscles ranged from TNF-{alpha}-negative to moderately TNF-{alpha}-positive, and the latter were usually larger more mature myotubes. In general, TNF-{alpha} staining was confined to the cytoplasm of myotubes (Fig 2G). The regenerated zone of the muscle usually had a much lower intensity of TNF-{alpha} staining than freshly injured and necrotic muscle.

Tissue Culture Studies
To specifically address the production of TNF-{alpha} in myoblasts and myotubes, immunostaining was also carried out on primary cultures of skeletal muscle. Muscle cultures from BALB/c, C57/BL, and SJL/J mice showed intense cytoplasmic staining with TNF-{alpha} antibodies in mononucleated cells and myotubes, often present in mononuclear cells in a granular distribution (Fig 3C and Fig 3D). Negative controls, in which the primary antibody was omitted, showed some scattered nonspecific staining (Fig 3A). In addition, muscle cultures from TNF-{alpha}(-/-)/LT-{alpha}(-/-) double-knockout mice served as negative controls. The presence of some TNF-{alpha} staining in adherent mononuclear cells confirmed some nonspecific staining in myoblasts (Fig 3B).



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Figure 3. TNF-{alpha} staining of cultured muscle. (A and inset) Negative control, primary antibody omitted; C57 culture showing some nonspecific staining. (B) TNF-{alpha} staining of TNF-{alpha}/LT-{alpha} double-knockout muscle, showing weak staining in adherent cells. (C) TNF-{alpha}-stained SJL/J culture, showing strongly positive myotubes. (D) TNF-{alpha}-stained C57 culture, showing staining of adherent cells and intense staining of rounded cells. Bars = 0.1 mm.

Regeneration in Single [TNF-{alpha}(-/-)]- and Double [TNF-{alpha}(-/-)/LT-{alpha}(-/-)]-Knockout Mice
It was noted that surgery was difficult on the TNF-{alpha} single- and double-knockout mice because of their thin, weak skin, soft muscle, and unexpectedly high number of capillaries in the muscle. The soft muscle and connective tissue of the knockout mice was also noted during the preparation of cultures from double-knockout mice. The single-knockout (and to a lesser extent the double-knockout) mice were hyperactive and appeared highly stressed, as emphasized by their prominent hair loss. Possible reasons for these observations are outlined in the Discussion.

The pattern of muscle regeneration after crush injury (12 muscles sampled per strain) or in whole muscle grafts (eight muscles sampled per strain) in both single [TNF-{alpha}(-/-)- and double (TNF-{alpha}(-/-)/LT-{alpha}(-/-)]-knockout mice appeared histologically normal at all times. Detailed examination revealed no apparent differences in the timing or extent of inflammation or new muscle formation between (a) single- and double-knockout muscle or (b) C57/BL control and single- or double-knockout muscle (data not shown).

Of particular interest was the observation that in single- and double-knockout mice, transverse sections through host TA muscle underlying whole muscle autografts regularly contained newly formed, centrally located myotubes (Fig 4E). Such foci of necrosis/regeneration were not present in host quadriceps or gastrocnemius muscle, i.e., muscles distant from the grafted site, or in host TA muscles taken at Day 1 after muscle autografting (data not shown). It was also noted that inflammatory cell infiltration in the host of the knockout mice was conspicuous between myofibers (Fig 4F) compared with normal control host muscle. Inflammatory cells were particularly conspicuous between muscle fascicles. Such damage and subsequent regeneration (i.e., myotube formation) was never seen deep in the host muscles of control C57BL (nor in BALB/c or SJL/J) mice in response to the presence of overlying whole muscle grafts.



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Figure 4. Hematoxylin and eosin staining of single- and double-knockout muscle. (A) Longitudinal section through crush-injured TNF-{alpha}/LT-{alpha} double-knockout muscle at Day 3 after injury, showing leukocyte infiltration and necrotic muscle tissue (*). (B) Whole muscle graft, showing the central necrotic core (*) and regeneration in the graft periphery at Day 7. (C) Neutrophil infiltration of a necrotic muscle fiber, Day 1 crush. (D) Macrophages within a necrotic muscle fiber, Day 3 crush. (E) Areas containing inflammatory cells and myotubes in host TA muscle (arrows). (F) Leukocyte infiltration through muscle (arrowhead). Bars = 0.1 mm.

Antibodies to TNF-{alpha} showed some very low nonspecific staining over injured and uninjured myofibers in both strains of null mice (negative controls). However, no TNF-{alpha} staining was seen in inflammatory cells or mast cells of these null mice, emphasizing the good specificity of the TNF-{alpha} antibody (Fig 2C).


  Discussion
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Although immunostaining showed strong upregulation of TNF-{alpha} in many cells in response to skeletal muscle damage, the absence of TNF-{alpha} and its homologue LT-{alpha} had no apparent effect on the processes of regeneration and new muscle formation in TNF-{alpha}(-/-) and TNF-{alpha}(-/-)/LT-{alpha}(-/-) mice. An important finding was a high incidence of "bystander" damage to adjacent host muscles in response to overlying muscle grafts in the single- and double-knockout host mice. These observations are discussed in detail below.

Immunostaining of Normal and Regenerating Muscle
The lack of TNF-{alpha} expression in normal uninjured skeletal muscle indicates that, unlike in other tissues (Hunt et al. 1992 ), TNF-{alpha} is not involved in muscle homeostasis and is expressed only in situations of injury and inflammation. The absence of TNF-{alpha} staining in uninjured skeletal muscle is highlighted by the lack of TNF-{alpha} staining of host muscles in sham-operated mice, indicating that significant muscle disturbance or the presence of necrotic muscle in the overlying graft is required for detectable TNF-{alpha} expression in skeletal muscle.

After experimental injury, myofibers in both the directly injured and "uninjured" regions of muscle expressed TNF-{alpha}. This was seen in muscle adjacent to the crush injury and also in the "uninjured" host TA muscle underlying muscle grafts. Expression of TNF-{alpha} by damaged myofibers after acute injury has not been described previously. This observation is compatible with results showing TNF-{alpha} staining in dystrophic mdx myofibers and in muscle fibers of DMD patients (Tews and Goebel 1996 ), and in inflammatory myopathies (De Bleecker et al. 1999 ). The greater intensity and wider distribution of staining observed in the present study may be due to the relatively high antibody concentrations used (1:400 present study; 1:500 DeBleecker; 1:1000 Tews) on paraffin (rather than frozen) sections, and the acute nature of the experimental injuries. Necrotic muscle sarcoplasm stained particularly strongly for TNF-{alpha}, supporting the reported role of TNF-{alpha} in the induction of muscle proteolysis (Goodman 1991 ; Zamir et al. 1992 ; Cassatella 1995 ; Argiles et al. 1998 ), although this may be an indirect effect mediated by glucocorticoids (Ji et al. 1998 ). Muscle atrophy has also been implicated as a trigger for cytokine expression by muscle fibers (Tews and Goebel 1996 ), and expression of TNF-{alpha} was also seen in the present study in atrophic muscle fibers at 4 weeks after denervation. That staining of myotubes in vivo was due to endogenous production of TNF-{alpha} rather than to exogenous TNF-{alpha} protein binding to the cell surface is supported by the strong TNF-{alpha} staining of myotubes in tissue culture.

TNF-{alpha} staining of infiltrating inflammatory cells in injured muscle confirmed reports of TNF-{alpha} production by activated leukocytes (Vilcek and Lee 1991 ; Korner and Sedgwick 1996 ). Macrophages have previously been described as the major systemic source of TNF-{alpha} (Beutler and Cerami 1988 ), and this is supported by the strong TNF-{alpha} staining observed in these cells. Neutrophils are also known to produce TNF-{alpha} (Schmauder-Chock et al. 1994 ), and were correspondingly shown in this study to be TNF-{alpha}-positive. The decrease in the proportion of TNF-{alpha}-positive neutrophils across the time points in crush-injured muscle indicates that peak neutrophil activation occurs at Day 1 and subsequently decreases, in agreement with other studies in regenerating muscle (Grounds and Davies 1996 ). An alternative explanation is that different subsets of leukocytes may arrive in the muscle at different times, and these may possess different abilities to produce TNF-{alpha}. The accuracy of TNF-{alpha} staining in this study was further reinforced by the strong positive TNF-{alpha} staining seen in mast cell granules. It is well documented that TNF-{alpha} is stored within cytoplasmic granules (Malaviya et al. 1996 ) and is synthesized and released when mast cells are appropriately stimulated (Gordon and Galli 1991 ; Wedemeyer et al. 2000 ). We also observed that relatively few lymphocytes are present (compared with macrophages or neutrophils) in crush-injured and grafted muscles. Although lymphocytes are known to produce TNF-{alpha} (Ware et al. 1996 ; Tateyama et al. 1997 ), these cells were not present in sufficient numbers to be a major source of TNF-{alpha} in the muscle injury models described here.

Pathways of inflammatory cell recruitment, activation, and cell death differ among different types of muscle injury (Tidball 1995 ; Goebels et al. 1998 ), and this is reflected in the differences between the TNF-{alpha} staining observed in mdx muscle and in experimentally injured muscle. The relatively low TNF-{alpha} staining intensity observed in all cells except mast cells in mdx muscle indicates that the inflammatory cell activation and migration of specific subsets of cells (Tidball 1995 ; Spencer et al. 1997 ) in the two types of injury (i.e., acute experimental vs chronic) result in different levels of TNF-{alpha} production.

Muscle Spindles
TNF-{alpha} staining was observed in muscle spindles in cross-sections of injured and denervated muscle but was not seen in normal uninjured muscle. Muscle spindles are mechanoreceptors specialized to monitor changes in muscle length (Barker and Banks 1986 ; Prochazka and Gorassini 1998 ) and contain a variable number of small-diameter intrafusal muscle fibers with their attendant sensory and motor innervation, all located within a multilayered, fusiform outer capsule (Barker et al. 1974 ). Satellite cells are known to be present within the inner capsule of the muscle spindle (Bird 1978 ), and the intrafusal muscle fibers of muscle spindles are also known to be capable of regeneration in the same manner as extrafusal muscle fibers (Maier 1997 ). Muscle spindles show selective expression of neurotrophin-3 (Copray and Brouwer 1994 ), adhesion molecules (Maier and Mayne 1995 ), and high Pax7 (Rodger et al. 1999 ), but this is the first report demonstrating the presence of TNF-{alpha}. Staining of intrafusal myofibers of muscle spindles indicates that a response of both extrafusal (i.e., "normal") and intrafusal (i.e., spindle) muscle fibers to injury is the production of TNF-{alpha}. The presence of TNF-{alpha} in the capsule cells of muscle spindles suggests that TNF-{alpha} may also play a role in regeneration of the associated neuromuscular junctions (Maier and Mayne 1995 ; Maier 1997 ).

Regeneration in TNF-{alpha} Knockout Mice
The first part of this study showed that TNF-{alpha} is highly expressed in injured and regenerating skeletal muscle. However, the absence of TNF-{alpha} had no effect on the timing and extent of inflammation and new muscle formation in regenerating skeletal muscles of null mice. No reduced macrophage activity was apparent in the TNF-{alpha}(-/-) mice, in agreement with results by Marino et al. 1997 . Furthermore, in support of our observations, Marino reports that the absence of TNF-{alpha} (in null mice) has no effect on liver regeneration, based on assessment of both cell proliferation and liver weights (Marino, personal communication). These observations contrast with impaired liver regeneration reported in situations in which TNF-{alpha} is still present but its activity is blocked by antibodies (Ackerman et al. 1992 ; Yamada et al. 1997 ) or in mice deficient in the receptors for TNF-{alpha} (TNF-RI and/or TNF-RII) (Yamada et al. 1997 ). Knight et al. 2000 found that mice deficient in TNF-RI but not TNF-RII had impaired oval cell proliferation and reduced liver tumorigenesis when fed a carcinogenic diet. The presence of normal or high levels of TNF-{alpha} protein (in both of these situations) presumably does not stimulate compensatory upregulation of other cytokines or host cellular responses (Durbin et al. 2000 ). In contrast, it appears that changes in cytokine profiles (see below) probably do occur in mice completely lacking TNF-{alpha} protein as in the present study. Our in vivo studies in single- and double-knockout mice strongly indicate that any role played by TNF-{alpha} in skeletal muscle regeneration can be performed efficiently and effectively by the upregulation of other cytokines (when TNF-{alpha} is not present).

The important distinction between the markedly different outcomes (seen in both skeletal muscle and liver) when TNF-{alpha} is present but its activity is blocked either by antibodies to TNF-{alpha} itself or by blocked or absent receptors, compared with situations in which TNF-{alpha} protein is absent (and the probable consequence of an altered host cytokine response), has critical implications for the design of therapeutic strategies to modulate cytokine levels.

Cytokine Interactions
TNF-{alpha} exists within a complex network of interactions with a multitude of other cytokines. In TNF-{alpha} null mice, interleukin-12 (IL-12) expression is greatly increased (Hodge-Dufour et al. 1998 ). Because IL-12 and TNF-{alpha} are co-stimulators for IFN-{gamma}, one of the essential cytokines for regulation of the inflammatory response (Storkus et al. 1998 ), the upregulation of IL-12 in the absence of TNF-{alpha}, could act in a compensatory manner to induce and maintain appropriate IFN-{gamma} levels. IFN-{gamma} induces macrophage activation, an important event in muscle regeneration (De Maeyer and De Maeyer-Guignard 1998 ). It also induces IL-12 (Hodge-Dufour et al. 1998 ) and can induce or suppress IL-1 synthesis (Schindler and Dinarello 1990 ). IL-1, like TNF-{alpha}, is a primary mediator of the inflammatory response (Schindler and Dinarello 1990 ; Fiers 1991 ) and is a strong candidate for potential upregulation in the absence of TNF-{alpha}. IL-1 is also likely to be important in muscle regeneration because of its roles in angiogenesis, fibroblast proliferation, and chemotaxis (Schindler and Dinarello 1990 ; Heckmann et al. 1993 ; Tidball 1995 ) and, like TNF-{alpha}, its direct effects on proliferation and fusion of myoblasts (Ji et al. 1998 ).

IL-6 expression increases in regenerating and diseased skeletal muscle (Kurek et al. 1996 , Kurek et al. 1998 ) and IL-6-induced protein degradation and muscle wasting may be important in DMD (Tsujinaka et al. 1998 ). IL-6 also stimulates myoblast proliferation (Austin and Burgess 1991 ) and is a candidate for upregulation in the absence of TNF-{alpha}. Another cytokine that may be of importance in muscle regeneration in the absence of TNF-{alpha} is transforming growth factor-beta (TGF-ß) (Derynck and Choy 1998 ). In particular, TGF-ß is a modulator of myoblast differentiation and fusion and in remodeling of the extracellular matrix (Murakami et al. 1999 ). TGF-ß also attenuates the inflammatory response and inhibits the activity and/or production of a number of cytokines, including TNF-{alpha}, LT-{alpha}, and IL-1 (Derynck and Choy 1998 ). Like TNF-{alpha}, TGF-ß is also elevated in skeletal muscle disorders (Murakami et al. 1999 ). However, because the muscles of TGF-ß(-/-) mice appear normal (McLennan et al. 2000 ), this indicates that other genes are capable of substituting for the missing TGF-ß in vivo.

The cytokines TNF-{alpha}, IL-1, IL-6, and IFN-{gamma} can stimulate the production of glucocorticoid hormones by the pituitary and, in turn, glucocorticoids inhibit the production of these cytokines and thus exert an immunomodulatory effect (for review see Brattsand and Linden 1996 ). Cytokine regulation by glucocorticoids concerns not only the ligands but also signal transduction by the specific receptors.

Oligomerization of receptors for TNF-{alpha} has been reported to occur in the absence of ligand binding (Engelmann et al. 1990 ), thus inducing receptor signaling characteristic of TNF-{alpha} (or LT-{alpha} or LT-{alpha}2ß1) without the specific ligand–receptor interaction. It is not known to what extent this might occur during injury. Such potential receptor signaling in the absence of TNF-{alpha} or LT-{alpha} ligand binding in the knockout mice must therefore also be considered as a possible contributing factor to the normal regeneration seen in null mice.

Studies in double-knockout mice indicate that, like TNF-{alpha}, LT-{alpha} has no essential role in muscle regeneration, and therefore LT-{alpha}1ß2 and LT-{alpha}2ß1 also have nonessential roles in the process. The similarity between regeneration in the single- and double-knockout mice indicates either that the cytokines upregulated in the absence of TNF-{alpha} are also sufficient to compensate for the lack of LT-{alpha} or that a different cytokine expression profile exists in the single-knockout mice that has essentially the same overall function.

Observations of thin, weak skin, and soft muscle with many capillaries in the TNF-{alpha} single- and double-knockout mice can be accounted for by the roles that TNF-{alpha} and other cytokines play in skin, angiogenesis and connective tissue. For example, keratinocyte proliferation is decreased and differentiation increased by TNF-{alpha} (Vassalli 1992 ), and TNF-{alpha} induces expression of cell adhesion molecules, such as ICAM on the surface of keratinocytes (Matsuura et al. 1998 ). Therefore, the anomalies observed in the skin of TNF-{alpha} knockout mice could be due to deregulation of keratinocyte proliferation, differentiation, and adhesion. TNF-{alpha} also stimulates fibroblast proliferation in culture (Vilcek et al. 1986 ), so the lack of TNF-{alpha} might result in decreased fibroblast proliferation and thus reduced connective tissue density. Because matrix metalloproteinases (MMPs) are involved in matrix remodeling during wound healing, angiogenesis, and tumor metastasis (for review see Forget et al. 1999 ) and are increased in inflammatory states such as chronic wounds (Wysocki et al. 1999 ), and because MMP2 and pro-MMP2 production by cultured human skin and isolated dermal fibroblasts is activated by TNF-{alpha} (Han et al. 2000 ), this potentially introduces further changes to the regulation of the skin and connective tissue in these mice.

Stress, Glucocorticoids, and TNF-{alpha}
The hyperactive behavior and highly stressed appearance of the single-knockout mice may be accounted for by the complex cytokine interactions in vivo, and the strong interaction of TNF-{alpha} with pituitary secretion of glucocorticoid hormones and the negative feedback interaction existing between them (as previously discussed). Upregulation of IL-1 may further exacerbate this situation because IL-1 is an important player in a number of the physical manifestations of stress (Nguyen et al. 1998 ) and administration of IL-1 produces stress-related changes (Shintani et al. 1995 ).

"Bystander Effect" Damage to the Muscle of Knockout Mice
The unexpected presence of myotubes throughout the host muscle after graft implantation is not a feature of normal muscle. Although small areas of myotube formation are sometimes seen on the adjacent host muscle, this is characteristically restricted to the surface of the host TA muscle lying immediately beneath the graft and is considered to be the result of direct physical damage from the surgery. The presence of myotubes throughout the majority of host muscle sections beneath the graft in both single- and double-knockout mice, and their absence from both local and distant muscle at Day 1, strongly supports the idea that such myotubes are not normally present in TNF-{alpha} null mice but are a consequence of the presence of the overlying graft. Bystander host muscle damage has also been observed in mouse soleus muscles after implantation of MHC-matched myoblasts (Wernig and Irintchev 1995 ), and this was attributed to locally released cytokines such as TNF-{alpha}.

The conspicuous number of leukocytes seen between host myofibers in knockout mice suggests that potential alterations in the absolute numbers or subtypes of inflammatory cells present in the double-knockout muscle could result from loss of the normal Type 1 inflammatory response with an increased or abnormal Type 2 inflammatory response (Durbin et al. 2000 ). Alternatively (as discussed above), an altered cytokine profile within the knockout mice may increase the toxicity of these inflammatory cells, resulting in bystander damage to the muscle fibers in their immediate vicinity. Because cytokine levels and inflammatory pathways are linked by regulatory mechanisms, a combination of these two possibilities may be responsible. Further investigation is under way in our laboratory into the cause of the observed bystander effect in the knockout mice. This bystander effect in knockout mice causing muscle damage and subsequent myotube formation has potentially important clinical implications with respect to various muscle diseases (such as myositis). A recent study of the pathology of X-linked muscular dystrophy in the mdx mouse in the absence of TNF-{alpha} revealed a complicated picture, with increased pathology of diaphragm muscle at 4 weeks of age but no effect on diaphragm muscle and decreased pathology of quadriceps muscles at 8 weeks in TNF-{alpha}(-/-)/mdx mice (Spencer et al. 2000 ). Although it was predicted that the absence of TNF-{alpha} would ameliorate the dystrophic process, this was not supported by the complex results. The compensatory role of other cytokines in the absence of TNF-{alpha} probably accounted for this. In addition, there may be a sustained bystander effect in the TNF-{alpha}(-/-)/mdx mice. We propose that the alternative strategy of administering antibodies to block normal TNF-{alpha} activity (Ackerman et al. 1992 ; Illei and Lipsky 2000 ) might produce quite different results and reduce the pathology of the muscular dystrophy in the mdx mice (due to alterations to other cytokines as discussed above), and this approach has the major advantage of potential clinical application.


  Footnotes

1 Present address: Division of Clinical Sciences, TVW Telethon Institute for Child Health Research, Subiaco, Western Australia.


  Acknowledgments

Supported by funding from the National Health and Medical Research Council of Australia.

We are grateful to Hugh Reid (LaTrobe University, Victoria) for the generous donation of single- and double-knockout mice, to Jim Hopley for technical assistance, to Marilyn Davies for expert surgery, and to Gayle Smythe, Jason White, and Stuart Hodgetts for advice and helpful discussions. Thanks also to Nguyen Ly of the Image Analysis and Acquisition Facility, UWA, for assistance with micrographs.

Received for publication December 18, 2000; accepted March 14, 2001.


  Literature Cited
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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