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Neutrophil Depletion Attenuates Muscle Injury after Exhaustive Exercise

KAWANISHI, NORIAKI; MIZOKAMI, TSUBASA; NIIHARA, HIROYUKI; YADA, KOICHI; SUZUKI, KATSUHIKO

Author Information
Medicine & Science in Sports & Exercise: October 2016 - Volume 48 - Issue 10 - p 1917-1924
doi: 10.1249/MSS.0000000000000980

Abstract

Prolonged exercise, such as running a marathon and eccentric muscle contraction, can induce skeletal muscle injury and decrease exercise performance due to muscle soreness and fatigue (2). Exercise-induced muscle injury can be divided into two phases (27). The initial phase of muscle damage occurs during exercise and is caused by mechanical factors such as eccentric contraction (33). The subsequent phase of muscle damage occurs after exercise and is suggested to be caused by the inflammatory response (7). Mechanically induced muscle injury may be the primary cause of the muscle damage with eccentric contraction exercise and downhill running, but it rarely occurs with prolonged endurance exercise. Nonmechanically related factors are likely to play important roles in causing muscle injury during prolonged endurance exercise. For example, both human and animal studies showed that prolonged endurance exercise increased the serum activities of creatine kinase and lactate dehydrogenase, which are biomarkers of muscle tissue damage (14,17). We and other researchers have shown histological evidence that exhaustive exercise causes myofiber lesions, including membrane damage (11,17,18). These studies strongly suggest that exhaustive exercise can promote muscle injury and lesions in the myofiber structure. However, the mechanisms underlying muscle injury induced by exhaustive exercise remain poorly understood.

Various experimental models of muscle damage concur that the infiltration of inflammatory cells, including neutrophils and macrophages, occurs in the injured skeletal muscle (24). Inflammatory cells release proinflammatory cytokines (e.g., tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6) and reactive oxygen species, which are key drivers of inflammation and the downstream effects of myofiber membrane lysis (34). Proinflammatory cytokines can also promote the activation of muscle satellite cells (1,37). Conversely, treatment with nonsteroidal anti-inflammatory drugs after exercise-induced muscle injury reduced the activation of muscle satellite cells (16,19). The damaging effects of inflammation on skeletal muscle structures suggest that inflammatory cells may play important roles in the mechanisms underlying exercise-related muscle injury and muscle regeneration. For example, several studies have reported increased presence of macrophages in skeletal muscle after exhaustive and downhill exercise (11,35,39). We have also shown that depletion of macrophages reduced the inflammatory responses and exercise-induced myofiber injury including membrane lesions (11). These results suggest that exercise-induced infiltration of macrophages in skeletal muscle is potentially an important factor in the development of muscle injury during endurance exercise.

Besides macrophages, recent evidence also suggests that neutrophils may play important roles in the development of muscle injury through the inflammatory pathway. For example, depleting muscle neutrophil content with the anti-Ly-6G/Ly-6C antibody before passive lengthening contraction (15) and reloading of atrophied soleus muscles (6) significantly reduced myofiber injury and membrane lesions in mice. In contrast, increased neutrophil infiltration into mouse skeletal muscle was observed 3 h after exhaustive exercise (22), which was also observed in several muscle injury mouse models (21). This evidence suggests that neutrophil infiltration may play important roles in mediating the inflammatory pathway that leads to the development of muscle injury after exhaustive exercise. However, the specific mechanisms of neutrophil-mediated muscle injury during exercise are currently unknown and warrant further investigation. In the present study, we tested the hypothesis that increased neutrophil infiltration after exhaustive exercise induces muscle injury and inflammation. The hypothesis was tested by investigating the effects of neutrophil depletion with antineutrophil antibody injection on muscle injury, macrophage infiltration, and inflammation after exhaustive exercise.

MATERIALS AND METHODS

Animals

Male C57BL/6J mice (n = 40) were purchased from Kiwa Laboratory Animals (Wakayama, Japan) at 9 wk of age and were housed in groups of four mice per cage in a controlled environment, under a light/dark cycle (lights on at 9:00 and off at 21:00). The experimental procedures complied with the Guiding Principles for the Care and Use of Animals in Waseda University and were approved by the Institutional Animal Care and Use Committee in the university (2013-A110). The mice were randomly assigned to four groups, namely, sedentary with control antibody (S, n = 10), sedentary with antineutrophil antibody (SA, n = 10), exhaustive exercise with control antibody (E, n = 10), and exhaustive exercise with antineutrophil antibody (EA, n = 10). All the mice had access to standard chow ad libitum.

Injection of antineutrophil antibody

A neutrophil-specific antibody, anti-Ly-6G (clone 1A8), and an isotype control antibody (clone 2A3) were purchased from Bio X cell (Sunnyvale, CA). The 1A8 (0.5 μg) and 2A3 (0.5 μg) antibodies were each diluted in phosphate-buffered saline (PBS), and the mice were administered with 150 μL of either antibody solution intraperitoneally, according to their respective experimental groups. Mice in both the sedentary groups remained in resting conditions in the cage, whereas mice in both the exercise groups were subjected to a bout of exhaustive exercise 48 h after the antibody injection.

Exercise protocol

One week before undertaking the exhaustive exercise, mice in the exercise groups were familiarized with running on a motorized treadmill (Natsume, Kyoto, Japan) at 20 m·min−1, 0% grade, for 20 min·d−1. On the day of the experiment, the mice were placed on a treadmill that had a 7% gradient. The treadmill speed was then increased to 10 m·min−1 for 15 min, followed by 15 and 20 m·min−1 for 15 min each, and finally at 24 m·min−1 until exhaustion. Exhaustion was defined as the point when the mouse refused to run despite being given the shock grid five times. The mean running time to exhaustion was 320.3 ± 11.2 min for the E group and 331.3 ± 13.2 min for the EA group. No significant difference in running time to exhaustion was observed between the E and the EA groups. Twenty-four hours after the exhaustive exercise, mice in the exercise groups were sacrificed under light anesthesia with isoflurane inhalation (Abbott, Tokyo, Japan) and the gastrocnemius muscle was promptly removed, frozen in liquid nitrogen, and stored at −80°C until analysis.

Histological analysis

A portion of the gastrocnemius muscle sample was positioned on pieces of cork that were secured with gum tragacanth and snap frozen by immersing the samples in precooled isopentane at −80°C. Immunofluorescence staining was performed on the frozen gastrocnemius sections to examine the expression of Ly-6G and F4/80. Serial sections of 6-μm thickness were fixed in 4% paraformaldehyde. Anti-Ly-6G (BioLegend, San Diego, CA) and antidystrophin (Abcam, Cambridge, MA) primary antibodies or anti-F4/80 (Abcam) and antidystrophin primary antibodies were diluted in 1% bovine serum albumin solution and were incubated with the sections. Alexa Fluor 555 goat anti-rat IgG and Alexa Fluor 488 goat anti-rabbit IgG (Life Technologies, Carlsbad, CA) secondary antibodies were diluted in PBS and incubated with the sections. The concentrations of the antibodies were 20 μg·mL−1 for F4/80 and dystrophin and 10 μg·mL−1 for Ly-6G, Alexa Fluor 555 goat anti-rat IgG, and Alexa Fluor 488 goat anti-rabbit IgG. The stained sections of the muscle tissue were visualized by fluorescence microscopy (KEYENCE, Osaka, Japan). Ly-6G-positive and F4/80-positive cells were counted in four random high-magnification fields (200×) per slide using BZ-2 software (KEYENCE) to derive the average value for each section. F4/80-positive cells were detected with visual judgment of the observer.

IgG staining was applied to frozen muscle sections to evaluate muscle fiber membrane lesions using previously described methods with some modifications (5). The presence of IgG in the muscle fiber cytosol indicates the presence of muscle membrane lesions and increased cell membrane permeability. The 6-μm serial sections of the gastrocnemius were incubated in 1% bovine serum albumin solution with IgG fluorescein isothiocyanate-conjugated mouse anti-IgG (Vector, Burlingame, CA) and antidystrophin primary antibody (Abcam). Alexa Fluor 488 goat anti-rabbit IgG (Life Technologies) secondary antibody was diluted in PBS and incubated with the sections. The antibodies were diluted to achieve a concentration of 15 μg·mL−1 for IgG fluorescein isothiocyanate-conjugated mouse anti-IgG, 20 μg·mL−1 for dystrophin, and 10 μg·mL−1 for Alexa Fluor 488 goat anti-rabbit IgG. The stained sections of the muscle tissue were visualized by fluorescence microscopy (KEYENCE). The number of injured fibers showing cytosolic fluorescence and the total number of fibers were counted in four random high-magnification fields (200×) per slide using BZ-2 software (KEYENCE). IgG-positive muscle fibers were detected with visual judgment of the observer.

Real-time quantitative PCR

Total RNA was extracted from the gastrocnemius homogenate using the RNeasy Fibrosis Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. The purity of total RNA was assessed using the NanoDrop system (NanoDrop Technologies, Wilmington, DE). Total RNA was reverse transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA) according to the manufacturer’s instructions. PCR was performed with the Fast 7500 real-time PCR system (Applied Biosystems) using Fast SYBR Green PCR Master Mix (Applied Biosystems). The thermal profiles consisted of denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 3 s, and annealing at 60°C for 15 s. The 18S ribosomal RNA was used as the housekeeping control, and all the data were normalized to 18S ribosomal RNA expression. The data were expressed as the number of fold changed, relative to the values of the S group. The specific PCR primer pairs for each gene are shown in Supplemental Table 1 (see Table, Supplemental Digital Content 1, sequences of the primer pairs used in this study, https://links.lww.com/MSS/A696).

Statistical analyses

All statistical analyses were performed using version 19.0 of the Statistical Package for Social Sciences software (IBM, Chicago, IL). To calculate statistical significance, differences in the number of Ly-6G-, IgG-, and F4/80-positive cells and the mRNA expression levels between the groups were determined using two-way ANOVA. If significant interactions were observed, further comparisons were performed using the Tukey’s HSD post hoc test. The level of significance was set at P < 0.05.

RESULTS

Effects of neutrophil depletion and exhaustive exercise on neutrophil infiltration into the skeletal muscle

Ly-6G immunofluorescence staining revealed that the exhaustive exercise induced a substantial increase in neutrophil infiltration into the gastrocnemius muscle, but this infiltration was markedly reduced by preexercise injection of the 1A8 antibody, which depleted the neutrophils (Fig. 1A). The number of Ly-6G-positive cells was significantly higher by 3.06-fold in the E group compared with the S group (P < 0.01). However, the Ly-6G-positive cells in the EA group were significantly fewer in number than those observed in the E group by 0.42-fold (P < 0.01, Fig. 1B).

F1-8
FIGURE 1:
The effects of exhaustive exercise and 1A8 antibody treatment on neutrophil infiltration in skeletal muscle. A, Ly-6G immunofluorescence staining (red, Ly-6G-positive cells (arrows); green, dystrophin) of gastrocnemius muscle sections. Scale bar is 50 μm. B, Number of Ly-6G-positive cells in the gastrocnemius muscle. Values shown are mean ± SEM. **P < 0.01.

Effects of neutrophil depletion on exhaustive exercise-induced muscle injury

Immunostaining of IgG was used to detect the presence of extracellular proteins in muscle fiber cytoplasm as an indicator of muscle fiber injury and fiber membrane lesions. No cytosolic IgG was detected in the muscle fibers in the S group (Fig. 2A), but the E group had 7.3% IgG-positive muscle fibers (Fig. 2B). The percentage of IgG-positive muscle fibers in the EA group was significantly lower than that observed in the E group by 0.18-fold (P < 0.01, Fig. 2B).

F2-8
FIGURE 2:
The effects of exhaustive exercise and 1A8 antibody treatment on muscle injury. A, IgG immunofluorescence staining (green, IgG-positive muscle fibers (arrows); red, dystrophin) of gastrocnemius muscle sections. Scale bar is 50 μm. B, Number of IgG-positive cells. Values shown are mean ± SEM. *P < 0.01.

Effects of neutrophil depletion on muscle inflammation after exhaustive exercise

We next evaluated the expression of cytokines (TNF-α, IL-1β, and IL-6) as biomarkers for muscle inflammation (Fig. 3). The levels of mRNA expression for TNF-α, IL-1β, and IL-6 were significantly higher in the E group compared with the S group (TNF-α, P < 0.01; IL-1β, P < 0.05; IL-6, P < 0.01). However, the mRNA expression of TNF-α and IL-6 decreased significantly after preexercise injection with the 1A8 antibody in the EA group (P < 0.05).

F3-8
FIGURE 3:
The effects of exhaustive exercise and 1A8 antibody treatment on proinflammatory cytokine responses in skeletal muscle. The mRNA expression levels of TNF-α, IL-1β, and IL-6 in the gastrocnemius muscle. Values shown are mean ± SEM. *P < 0.05, **P < 0.01.

Effects of neutrophil depletion and exhaustive exercise on macrophage infiltration into the skeletal muscle

Immunofluorescence staining of F4/80 was performed to determine the presence of macrophages in the gastrocnemius muscle (Fig. 4A). Macrophage content increased substantially (3.91-fold) 24 h after exhaustive exercise (P < 0.01), but the macrophage content in the EA group decreased significantly by 0.44-fold after preexercise injection with 1A8 antibody (P < 0.01, Fig. 4B). These results were further supported by the gene expression of the macrophage marker using reverse transcription PCR. Consistent with the increased macrophage content, the levels of mRNA expression for F4/80 and MCP-1 increased significantly in the E group compared with the S group (P < 0.01). However, the mRNA levels were significantly lower in the EA group than those in the E group (P < 0.01, Fig. 4C).

F4-8
FIGURE 4:
The effects of exhaustive exercise and 1A8 antibody treatment on macrophage infiltration in skeletal muscle. A, F4/80 immunofluorescence staining (red, F4/80-positive cells (arrows); green, dystrophin) of gastrocnemius muscle sections. Scale bar is 50 μm. B, Number of F4/80-positive cells. C, mRNA expression levels of F4/80 and MCP-1 in the gastrocnemius muscle. Values shown means ± SEM. *P < 0.05, **P < 0.01.

Immunofluorescence staining for MCP-1 was performed on muscle sections to visualize the localization of MCP-1 (see Figure, Supplemental Digital Content 2, immunofluorescence staining of Ly-6G and MCP-1 in skeletal muscle, https://links.lww.com/MSS/A697). MCP-1 colocalized with the neutrophil marker Ly-6G. Exhaustive exercise significantly increased the number of MCP-1-positive neutrophils in muscle by 3.99-fold (P < 0.01), but the neutrophil content was markedly reduced by 0.24-fold when treated with preexercise injection of 1A8 antibody (P < 0.01).

DISCUSSION

Although it is well established that exhaustive exercise induces muscle injury and inflammation, the underlying mechanisms and etiology of the muscle injury are not as well understood. This study was an attempt to further understand the mechanisms of muscle injury during exhaustive exercise by investigating the effects of suppressing neutrophil infiltration on muscle fiber injury and inflammation, 24 h after exhaustive exercise.

Both eccentric and prolonged exercises increase cell concentration and migration activity of blood neutrophils (9,31,32). The activated neutrophils, in turn, produce inflammatory mediators, reactive oxygen species, and proteases, which promote tissue injury. Notably, neutrophil infiltration into the muscle was elevated after exhaustive running in mice (22). In our study, immunofluorescence staining showed a 3.1-fold increase in neutrophil infiltration into the muscle 24 h after exhaustive exercise. We used clone 1A8 of an anti-Ly-6G antibody to deplete neutrophils from the tissue with inflammatory cell infiltration (4) and injection of the 1A8 antibody before exercise reduced neutrophil infiltration in the skeletal muscle after exhaustive exercise. These results demonstrated the efficacy of our protocol with antineutrophil antibody injection for reducing neutrophil infiltration into the skeletal muscle after exhaustive exercise.

Inflammatory cells, such as neutrophils and macrophages, trigger the inflammatory response in the skeletal muscle by activating the production of proinflammatory cytokines, which can further exacerbate muscle injury by causing myofiber lesions (34). We and other researchers have reported that the protein and gene expression levels of TNF-α were elevated in the muscle after exhaustive exercise (8,11,25). In contrast, preexercise treatment with an anti-inflammatory substance lowered plasma concentrations of creatine kinase and lactate dehydrogenase after exhaustive exercise (29). These results supported the hypothesis that inflammatory mediators released from activated inflammatory cells, such as neutrophils, may play important roles in exercise-induced muscle injury. In the present study, the increase in myofiber injury 24 h after exhaustive exercise was reversed by blocking neutrophil infiltration into the muscle with a preexercise injection of anti-Ly-6G antibody. Our results agree with a previous study that significantly reduced myofiber injury after the passive lengthening contraction in neutrophil-depleted mice (15). We also observed that neutrophil depletion with preexercise injection of the anti-Ly-6G antibody lowered mRNA expression of proinflammatory cytokines (e.g., TNF-α and IL-6) in the skeletal muscle. Taken together, the results from our study support the notion that inflammatory cells play important roles in causing muscle injury and that neutrophil infiltration into the skeletal muscle after exhaustive exercise regulates muscle injury by activating the inflammatory pathway. These results also suggest that exercise-induced muscle injury can be attenuated by blocking neutrophil infiltration.

A limitation of this study was the single-point evaluation of mRNA expression for proinflammatory cytokines at 24 h after exercise. This approach did not take into account reports from Rosa Neto et al. (25) who found that mRNA levels and protein contents of proinflammatory cytokines increased immediately after exhaustive exercise and was maintained at the same level for 6 h after exercise. However, the time-course response of mRNA expression for proinflammatory cytokines at 24 h after exhaustive exercise has not been investigated. It is possible that mRNA expression of proinflammatory cytokines is decreased after 24 h and the activation of inflammatory pathways occurring in the initial 6 h after exercise may be more important in the pathology for muscle injury. Future study should investigate whether the activation of inflammatory pathways that occurs early after exercise can be decreased by blocking neutrophil infiltration.

Previous studies have shown that macrophage depletion markedly reduced myofiber injury and inflammation in several muscle injury mouse models (30,38). We also reported that macrophage depletion reduced muscle injury and inflammation after exhaustive exercise in mice (11). This evidence suggests that macrophage infiltration is the likely primary cause of muscle injury after exhaustive exercise. In the present study, neutrophil depletion correlated with the decrease in macrophage content (r = 0.83, P < 0.01) and resulted in decreased F4/80 mRNA expression in the skeletal muscle after exhaustive exercise. These results are consistent with an earlier observation that neutrophil depletion decreased muscle injury and macrophage infiltration after hindlimb unloading and reloading (30). Taken together, these results suggest that neutrophil depletion may attenuate the severity of exercised-induced muscle injury by suppressing macrophage infiltration. However, the mechanisms by which neutrophils regulate infiltration of macrophages into muscles remain unclear.

Other studies have shown that macrophages are recruited to various tissues through chemotactic factors, such as chemokines and adhesion molecules, and that prolonged exercise can rapidly increase the expression of macrophage-specific chemokines in human skeletal muscle (20,28). These findings are consistent with our observation that exhaustive exercise upregulated MCP-1 mRNA expression and macrophage infiltration in mice. Collectively, the evidence presented indicates that elevated MCP-1 production may be associated with enhanced macrophage infiltration after exhaustive exercise. However, the cellular source underlying the production of chemokines in muscles is unknown.

A few studies have attempted to identify the cells that produce chemokines in the skeletal muscle. Our previous studies demonstrated that skeletal muscle cells and macrophages produce MCP-1 in response to stimulation by cytokines and lipopolysaccharide (12). Other scholars have found strong MCP-1 expression in inflammatory cells in human muscle, but not within the myofibers (23). Neutrophils are known to release macrophage chemoattractants, such as MCP-1, and also regulate the infiltration of macrophages in local tissue to induce inflammation (26). These findings suggest that neutrophils may be a source of muscle tissue-derived chemokines that contributes to the infiltration of macrophages in skeletal muscles after exhaustive exercise. To investigate the possibility of MCP-1 production in infiltrated muscle cells, we costained serial sections of the muscle tissue with MCP-1 and Ly-6G antibodies. We found that MCP-1 was expressed in close proximity to the Ly-6G-positive cells, suggesting that MCP-1 is produced by neutrophils in the skeletal muscle tissue. We also found an increase in the content of the MCP-1-positive neutrophils in the muscle after exhaustive exercise. In contrast, neutrophil depletion decreased both MCP-1 mRNA expression and content of MCP-1-positive neutrophils in the muscle. These results suggest that the induction of macrophage infiltration after exhaustive exercise might be caused by neutrophil-derived factors, which include MCP-1.

Acute exercise-induced muscle injury triggers the activation and proliferation of muscle satellite cells (3) and promote muscle repair and regeneration. The activation of satellite cells after muscle injury is regulated by the inflammatory response. For example, both TNF-α knockout and TNF receptor–deficient mice showed reduced satellite cell proliferation after cardiotoxin- and trauma-induced muscle injury (1,37). These results imply that inflammatory mediators may play important roles in satellite cell proliferation after muscle injury and support other findings that nonsteroidal anti-inflammatory drug infusion or ingestion suppressed exercise-induced satellite cell proliferation 8 d after resistance exercise (19) and prolonged running (16). Therefore, the inflammatory response that is regulated by neutrophil and macrophage infiltration after exhaustive exercise might be required for muscle repair and regeneration after muscle injury. A limitation in the present study is lack of insights into longer term responses in muscle recovery after injury, which requires up to 8 d of monitoring. The present study was designed to examine the influence of neutrophil depletion at the onset of muscle injury and the inflammatory response 24 h after exercise, which did not address the effects of neutrophil depletion on muscle repair and regeneration. The effect of neutrophil depletion on muscle repair and regeneration after exhaustive exercise-induced muscle injury should be further investigated by evaluating longer term responses in the proliferation of satellite cells after exercise.

There is more evidence in recent years suggesting the contribution of the extracellular matrix (ECM) to muscle regeneration after injury. ECM, such as metalloproteinase (MMP), plays important roles in muscle development through the activation of myoblast proliferation and fusion, and the migration of satellite cells into the site of muscle injury (10). Furthermore, MMP also promotes muscle regeneration through the induction of ECM remodeling (10). The isoform MMP9 is produced from neutrophils and macrophages (13), and the mRNA level of MMP9 increased markedly, together with increased inflammation and infiltration of macrophages, after 3 and 7 d of muscle damage by freezing (36). In addition, suppression of inflammation and macrophage infiltration by cryotherapy cause a decrease in the MMP9 mRNA level (36). Therefore, the proinflammatory mediators produced by neutrophils and macrophages might be associated with activation of satellite cells and the induction of MMP-mediated ECM remodeling after exhaustive exercise-induced muscle injury. Future studies can use neutrophil depletion as a model to investigate the interaction between inflammation and ECM remodeling in influencing muscle regeneration after exhaustive exercise-induced muscle injury.

In summary, we have demonstrated that neutrophil depletion substantially decreased the severity of myofiber injury, macrophage infiltration, and expression of proinflammatory cytokines in the muscle after exhaustive exercise. These findings are novel and provide evidence that neutrophils can contribute to the mechanisms that cause muscle injury by regulating inflammation through induction of macrophage infiltration.

We would like to thank Dr. Fabian C. L. Lim (Associate Professor, Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore) for editing the language in the manuscript.

This work was supported by a Grant-in-Aid for Challenging Exploratory Research (K. Suzuki, number 24650410) from the Japan Society for the Promotion of Science and the Strategic Research Foundation at Private Universities, 2015–2020 (S1511017), from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

There is no conflict of interest. The results of the present study do not constitute endorsement by the American College of Sports Medicine.

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Keywords:

RUNNING; INFLAMMATORY CELL; MUSCLE DAMAGE; CYTOKINE

Supplemental Digital Content

© 2016 American College of Sports Medicine