1Department of Physical Therapy, Georgia State University, Atlanta, Georgia 30303; 2Toxicology and Molecular Biology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, West Virginia 26505; and 3Autoimmune and Inflammatory Diseases, Protein Design Labs, Fremont, California 94555
Submitted 24 October 2003 ; accepted in final form 12 January 2004
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
inflammation; regeneration; chemokine receptors
Many inflammatory diseases with a marked mononuclear cell involvement such as rheumatoid arthritis, asthma, and atherosclerosis have been associated with elevated CC chemokine expression, and the progression of these diseases has been related to the functions of the CC chemokines (4, 7, 23). CC chemokines have also been demonstrated to play a role in traumatic injuries to the central and peripheral nervous systems as well as in skin wound healing, mainly through regulation of monocyte/macrophage recruitment or activation (5, 17, 18, 24). Recently, gene expression profiling demonstrated that enhanced expression of the CC chemokines is also a characteristic of injured skeletal muscle (9, 25). Migration and activation of cells including monocytes/macrophages as well as satellite cells is an obligatory component of the repair process after injury (11, 26). Although the role of chemokines in skeletal muscle injury and repair is not known, it is possible that these mediators may affect cells that express the appropriate receptors and thus influence the repair mechanisms after injury. In this respect, we hypothesized that 1) MCP-1, MIP-1, and MIP-1
expression is accompanied by expression of their major receptors including CCR2 and CCR5 in the injured skeletal muscle and 2) signaling through these receptors may influence the functional recovery of muscle after traumatic injury. Because these three chemokines share redundant physiological activities (1, 19), we first monitored the functional recovery of freeze-injured tibialis anterior (TA) muscles from mice that were genetically deficient for the CCR5 receptor (a major receptor for MIP-1
and MIP-1
) and also rendered MCP-1 deficient with neutralizing antibodies. To dissect the role of these chemokines, additional studies were conducted in CCR5- and CCR2-deficient mice. The results demonstrate that although both CCR2 and CCR5 receptors are expressed in injured muscle, the MCP-1/CCR2 axis, but not signaling through CCR5, plays a role in restoration of skeletal muscle function after traumatic injury.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
CCR2-/- mice on a mixed C57BL/6 x strain 129 genetic background were generated as described previously (15, 16). The CCR2-/- were then backcrossed to C57BL/G mice for 10 generations. CCR5-/- mice (B6 and C57BL/6J;129P2-Ccr5tm1Kuz) as well as wild-type controls (B6;129PF2/J) were obtained from the Jackson Laboratory (Bar Harbor, ME). Mean (±SD) weights for the CCR5-/- mice (n = 37) and their controls (n = 47) were 21.5 ± 2.1 and 21.3 ± 2.4 g, respectively; ages for all mice were between 13 and 16 wk. Mean weights for the CCR2-/- mice (n = 9) and their controls (n = 8) were 24.4 ± 2.1 and 21.5 ± 0.6 g, respectively; ages for these mice were between 18 and 24 wk. The mice were provided food and water ad libitum and were maintained on a 12:12-h light-dark cycle. Animal care and use procedures, including death by CO2 asphyxiation, were conducted in accordance with criteria outlined in the "PHS Policy on Humane Care and Use of Laboratory Animals" and the Guide for the Care and Use of Laboratory Animals (NIH pub. no. 8623, 1996). These procedures were approved by the Georgia State University and National Institute for Occupational Safety and Health institutional animal care and use committees.
Polyclonal antiserum to murine MCP-1 was prepared (Biosource International, Camarillo, CA) by injecting New Zealand White rabbits with recombinant murine MCP-1 (PeproTech, Rocky Hill, NJ) as described previously (13). The antiserum completely neutralizes the activity of 500 pg/ml of mouse MCP-1 and does not react with mouse MIP-1 or MIP-1
, as measured by a competitive ELISA (data not shown). CCR5-/- mice received intraperitoneal injections of MCP-1 antiserum (10 µl/g body wt) 2 h before injury and every 12 h for the first 3 days after injury. Some CCR5-/- mice received MCP-1 antiserum injections for 14 days (injected as described above for the first 3 days plus once daily from 4 to 14 days after injury).
Induction of Freeze-Induced Muscle Injury
The procedure used was identical to that previously described (27). In brief, a 1.5-cm-long incision was made through aseptically prepared skin overlying the left TA muscle belly. Injury was induced by applying a steel probe cooled to the temperature of dry ice to the TA muscle belly for 10 s. The skin incision was then closed with silk suture. In preparation for muscle injury induction and isometric strength testing, mice were anesthetized with 0.33 mg/kg fentanyl, 16.7 mg/kg droperidol, and 5.0 mg/kg diazepam administered intraperitoneally. We previously determined (12) that this anesthetic regimen is optimal for in vivo muscle strength testing in mice.
Measurement of In Vivo Isometric Strength
Maximal isometric tetanic torque of the left anterior crural muscles was measured before and after freeze injury with a miniature dynamometer as described previously (27, 28). In brief, the lateral surface of the lower left hindlimb was shaved and aseptically prepared. Two 36-gauge needle electrodes were inserted percutaneously to positions adjacent to the common peroneal nerve. These electrodes were connected to a stimulator, and the muscle was stimulated with 200-ms trains of 0.1-ms biphasic pulses at 300 Hz. Voltage and position of the electrodes were adjusted to yield the maximal isometric tetanic torque of the anterior crural muscle group. The TA muscle was then injured as described in Induction of Freeze-Induced Muscle Injury. Two minutes after injury, the maximal isometric tetanic torque measurement was repeated. In all mice, maximal isometric tetanic torque was measured at one additional time point after injury induction (i.e., 7, 14, or 28 days after injury). These time points were selected on the basis of previous work with this injury model in which we found minimal recovery of strength in the first days after injury, rapid recovery during the second week, and near-complete recovery after 4 wk (Ref. 27; unpublished observations).
Real Time RT-PCR
TA muscles were collected in RNAlater (Qiagen, Valencia, CA) and homogenized, and total RNA was extracted with a commercial kit (RNeasy, Qiagen) following the manufacturer's protocol. cDNA was synthesized from 1 µg of RNA with Superscript II (Life Technologies, Gaithersburg, MD). Real-time PCR for CCR2, CCR5, MIP-1, MIP-1
, MCP-1, and 18S/rRNA was performed with predeveloped primers and probes (TaqMan assay reagents, PE Applied Biosystems, Foster City, CA) on an ABI Prism 7700 Sequence detector (PE Applied Biosystems). The comparative threshold cycle (CT) method was used to calculate the relative concentrations as described previously (25). This method involved obtaining CT values for the transcript of interest, normalizing to the housekeeping gene 18S/rRNA (TaqMan assays), and comparing relative increases between controls and experimental samples.
Histopathology
Muscles processed for paraffin-embedded histology were removed and fixed by immersion in 10% neutral-buffered formalin. The tissues were then embedded in paraffin, cut into 6-µm-thick longitudinal sections, and stained with hematoxylin and eosin for blinded histopathological assessment. Histopathological findings were graded from 1 to 5 depending on severity (1 = minimal, 2 = slight/mild, 3 = moderate, 4 = moderately severe, 5 = severe/high) in six mice per group. Masson's trichrome stain to visualize collagen was applied as described originally (20).
Representative muscle tissue was embedded in Tissue Tek OCT (Miles Scientific), frozen in melting isopentane, and stored at -80°C. Ten cross sections (10 µm thick) were cut at each of six levels equally spaced along the length of the TA muscle in a microtome cryostat at -20°C. Immunostaining for Mac-3 was conducted as described previously (27). Immunostaining for MIP-1, MCP-1, CCR2, and CCR5 was conducted on formalin-fixed cryosections with specific polyclonal antibodies prepared in goats (Santa Cruz Biotechnology, Santa Cruz, CA). The antibodies were diluted 1:300 in phosphate-buffered saline (PBS) and applied to the sections for 18 h at 4°C. The slides were visualized with the ImmunoCruz staining system (Santa Cruz Biotechnology) and Tyramide Signal amplification kit (Molecular Probes, Eugene, OR) according to the manufacturers' instructions.
Statistics
The strength differences among groups of mice were analyzed with a two-way (group x time) repeated-measures ANOVA. When significant interactions were found, single degree of freedom contrasts were applied as post hoc tests. For comparison of mRNA transcript levels between control and injured muscles, unpaired Student t-tests were used. An -level of 0.05 was used for all analyses. All statistical testing was conducted with SPSS (version 10.0). Values presented are means ± SE.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
RNA, isolated from an injured or a contralateral uninjured (control) muscle, was examined for transcripts of MIP-1, MIP-1
, MCP-1, CCR5, and CCR2 by TaqMan real-time PCR after muscle freeze injury. The largest increases in MIP-1
, MIP-1
, and MCP-1 expression occurred within 24 h of injury. The rapid increases in the chemokine mRNA transcript levels were followed by gradual reductions over days 37 and a return to control levels by day 14. CCR2 and CCR5 mRNA expression in the injured TA muscle were increased only in the first 24 h after injury (Fig. 1B). The contralateral uninjured muscles expressed low constitutive levels of MIP-1
, MIP-1
, MCP-1, CCR2, and CCR5 mRNA that did not vary over time after injury. Immunostaining for CCR5, MIP-1
, CCR2, and MCP-1 revealed that mRNA changes were accompanied by increases in the corresponding proteins within the freeze-injured TA muscle (Fig. 2). Minimal immunostaining was observed in the first 24 h of injury (data not shown), but considerable staining was observed by postinjury day 3. The immunoreactive proteins were not detected in uninjured muscles but were readily observed in the damaged muscle region adjacent to the deep, uninjured region.
|
|
Evaluation of Effects of CCR5/MCP-1 Deficiency on TA Muscle Injury and Recovery
Muscle strength in vivo. Wild-type controls and chemokine-deficient mice (CCR5-/- mice injected with MCP-1 antiserum for the first 3 days after injury) experienced nearly identical losses of maximal isometric tetanic torque immediately after injury (i.e., 75 vs. 73%; Fig. 3). The torque deficits for the two groups after 7 days were also very similar (i.e., 55%). However, the chemokine-deficient group recovered more slowly during the second week, and at 14 days after injury the torque deficit for these mice was double that of the control mice (i.e., 33 vs. 17%; P = 0.002). By 28 days after injury, neither group showed significant deficits in maximal isometric tetanic torque. The difference in torque between the two groups at 14 days after injury was not exacerbated by a more prolonged administration of the MCP-1 antiserum. Compared with control mice, CCR5-/- mice injected with antiserum for 14 days exhibited a twofold greater torque deficit at 14 days after injury (i.e., 31.0 ± 5.1% vs. 16.1 ± 2.9%; P = 0.018). These findings were virtually identical to those obtained for the CCR5-/- mice receiving antiserum for only 3 days (Fig. 3).
|
Histopathology. Hematoxylin and eosin staining indicated that 60% of the TA muscle was affected by the freeze injury. Approximately three-quarters of the muscle cross section were damaged directly beneath where the steel probe had been applied, whereas the deepest one-quarter of the muscle cross section appeared histologically normal. By postinjury day 3, the levels of inflammatory cell infiltration had peaked in the injured muscles of both wild-type controls and chemokine-deficient mice including CCR5-/- mice injected with anti-MCP-1 for 3 days. Furthermore, there were no marked differences in the degree of tissue damage or inflammatory cell response between control mice and chemokine-deficient mice at this time point (Fig. 4A). The majority of the inflammatory cells in the injured TA muscles from both control and experimental groups were positive for Mac-3, a marker of activated monocytes/macrophages (Fig. 4B). However, by postinjury day 14, no sign of damage was detectable in the mice from the control group, indicating a complete regeneration, whereas the chemokine-deficient mice exhibited increased fat infiltration (severity grade of 2 for 4 mice or 3 for 2 mice) into the interstitium of the injured TA muscles (Fig. 4C). We next investigated the presence of collagen in the injured muscle with the Masson's trichrome procedure, which stains collagen blue. As shown in Fig. 4D, small amounts of collagen were seen in the interstitium of injured muscles from the chemokine-deficient mice (severity grade of 1) but not in muscles from control animals.
|
Muscle strength recovery in CCR5-/- and CCR2-/- mice. To evaluate whether the delay in muscle function recovery was dependent more on the CCR5 signaling chemokines or on MCP-1, which signals through CCR2, maximal isometric tetanic torque at 14 days after injury was measured in CCR2-/- mice and CCR5-/- mice not injected with MCP-1 antiserum. The CCR5-/- mice not injected with MCP-1 antiserum exhibited a torque deficit at 14 days after injury similar to that of the wild-type controls (i.e., 20.3 vs. 19.6%, respectively; P = 0.90; Fig. 5A). In contrast, injured CCR2-/- mice retested at 14 days after injury exhibited a greater torque deficit compared with the wild-type controls (i.e., 38 vs. 25%, respectively; P = 0.03; Fig. 5B). The effect of the CCR2 deficiency on the strength deficit was comparable in magnitude to that of MCP-1 neutralization.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Strength loss is the most important functional consequence and a reliable indicator of muscle injury (29). Satellite cells, myogenic precursor cells, have been demonstrated to be necessary for the recovery of strength after injury, probably by assisting in the restoration of contractile protein content (22). Resident satellite cells migrate from surrounding uninjured areas to the injured site and fuse together to form myoblasts, which in turn fuse to form myotubes (8). In addition, it was demonstrated recently that bone marrow-derived cells can migrate to areas of induced degeneration to undergo myogenic differentiation and participate in the regeneration of damage fibers (6). Many growth factors, such as hepatocyte growth factor (HGF) and insulin-like growth factor (IGF), have been studied and demonstrated as potential activator and chemotactic factors for the muscle precursor cells (2, 8, 30). Other immune mediators, such as IL-4, have been shown to modulate the fusion process of the satellite cells through induction of calcium-dependent cell signals (10). Many of these mediators, like chemokines, are released by activated macrophages involved in the injury process. Although the role of chemokines in modulating satellite cell migration and activation has not been studied, our findings of a delay in the functional restoration of animals deficient for MCP-1/CCR2 suggest that the chemokines may modulate the satellite cell response after injury. This effect may be by stimulating migratory, proliferative, or fusion activities of the satellite cells or indirectly through macrophage activation. Although neutralization of MCP-1 did not influence the inflammatory cell accumulation after injury, a role of MCP-1 in macrophage activation is not excluded. Future studies should be directed toward identifying the mechanisms through which MCP-1 and/or CCR2 modulate skeletal muscle regeneration and how they affect the recruitment and activation of the satellite cells.
![]() |
FOOTNOTES |
---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Bischoff R. Chemotaxis of skeletal muscle satellite cells. Dev Dyn 208: 505-515, 1997.[CrossRef][ISI][Medline]
3. Boring L, Gosling J, Monteclaro FS, Lusis AJ, Tsou CL, and Charo IF. Molecular cloning and functional expression of murine JE (monocyte chemoattractant protein 1) and murine macrophage inflammatory protein 1alpha receptors: evidence for two closely linked C-C chemokine receptors on chromosome 9. J Biol Chem 271: 7551-7558, 1996.
4. Dawson J, Miltz W, Mir AK, and Wiessner C. Targeting monocyte chemoattractant protein-1 signalling in disease. Expert Opin Ther Targets 7: 35-48, 2003.[CrossRef][ISI][Medline]
5. DiPietro LA, Burdick M, Low QE, Kunkel SL, and Strieter RM. MIP-1alpha as a critical macrophage chemoattractant in murine wound repair. J Clin Invest 101: 1693-1698, 1998.
6. Ferrari G, Cusella-De Angelis G, Coletta M, Paolucci E, Stornaiuolo A, Cossu G, and Mavilio F. Muscle regeneration by bone marrow-derived myogenic progenitors. Science 279: 1528-1530, 1998.
7. Gu L, Okada Y, Clinton SK, Gerard C, Sukhova GK, Libby P, and Rollins BJ. Absence of monocyte chemoattractant protein-1 reduces atherosclerosis in low density lipoprotein receptor-deficient mice. Mol Cell 2: 275-281, 1998.[ISI][Medline]
8. Hawke TJ and Garry DJ. Myogenic satellite cells: physiology to molecular biology. J Appl Physiol 91: 534-551, 2001.
9. Hirata A, Masuda S, Tamura T, Kai K, Ojima K, Fukase A, Motoyoshi K, Kamakura K, Miyagoe-Suzuki Y, and Takeda S. Expression profiling of cytokines and related genes in regenerating skeletal muscle after cardiotoxin injection: a role for osteopontin. Am J Pathol 163: 203-215, 2003.
10. Horsley V, Jansen KM, Mills ST, and Pavlath GK. IL-4 acts as a myoblast recruitment factor during mammalian muscle growth. Cell 113: 483-494, 2003.[ISI][Medline]
11. Huard J, Li Y, and Fu FH. Muscle injuries and repair: current trends in research. J Bone Joint Surg Am 84-A: 822-832, 2002.[Medline]
12. Ingalls CP, Warren GL, Lowe DA, Boorstein DB, and Armstrong RB. Differential effects of anesthetics on in vivo skeletal muscle contractile function in the mouse. J Appl Physiol 80: 332-340, 1996.
13. Kayama F, Yoshida T, Elwell MR, and Luster MI. Role of tumor necrosis factor-alpha in cadmium-induced hepatotoxicity. Toxicol Appl Pharmacol 131: 224-234, 1995.[CrossRef][ISI][Medline]
14. Kunkel SL. Through the looking glass: the diverse in vivo activities of chemokines. J Clin Invest 104: 1333-1334, 1999.
15. Kuziel WA, Dawson TC, Quinones M, Garavito E, Chenaux G, Ahuja SS, Reddick RL, and Maeda N. CCR5 deficiency is not protective in the early stages of atherogenesis in apoE knockout mice. Atherosclerosis 167: 25-32, 2003.[CrossRef][ISI][Medline]
16. Kuziel WA, Morgan SJ, Dawson TC, Griffin S, Smithies O, Ley K, and Maeda N. Severe reduction in leukocyte adhesion and monocyte extravasation in mice deficient in CC chemokine receptor 2. Proc Natl Acad Sci USA 94: 12053-12058, 1997.
17. Low QE, Drugea IA, Duffner LA, Quinn DG, Cook DN, Rollins BJ, Kovacs EJ, and DiPietro LA. Wound healing in MIP-1-/- and MCP-1-/- mice. Am J Pathol 159: 457-463, 2001.
18. Ma M, Wei T, Boring L, Charo IF, Ransohoff RM, and Jakeman LB. Monocyte recruitment and myelin removal are delayed following spinal cord injury in mice with CCR2 chemokine receptor deletion. J Neurosci Res 68: 691-702, 2002.[CrossRef][ISI][Medline]
19. Mantovani A. The chemokine system: redundancy for robust outputs. Immunol Today 20: 254-257, 1999.[CrossRef][ISI][Medline]
20. Masson PJ. Trichrome staining and their preliminary techniques. J Tech Methods 12: 75, 1929.
21. Raport CJ, Gosling J, Schweickart VL, Gray PW, and Charo IF. Molecular cloning and functional characterization of a novel human CC chemokine receptor (CCR5) for RANTES, MIP-1beta, and MIP-1alpha. J Biol Chem 271: 17161-17166, 1996.
22. Rathbone CR, Wenke JC, Warren GL, and Armstrong RB. Importance of satellite cells in the strength recovery after eccentric contraction-induced muscle injury. Am J Physiol Regul Integr Comp Physiol 285: R1490-R1495, 2003.
23. Rose CE Jr, Sung SS, and Fu SM. Significant involvement of CCL2 (MCP-1) in inflammatory disorders of the lung. Microcirculation 10: 273-288, 2003.[CrossRef][ISI][Medline]
24. Siebert H, Sachse A, Kuziel WA, Maeda N, and Bruck W. The chemokine receptor CCR2 is involved in macrophage recruitment to the injured peripheral nervous system. J Neuroimmunol 110: 177-185, 2000.[CrossRef][ISI][Medline]
25. Summan M, McKinstry M, Warren GL, Hulderman T, Mishra D, Brumbaugh K, Luster MI, and Simeonova PP. Inflammatory mediators and skeletal muscle injury: a DNA microarray analysis. J Interferon Cytokine Res 23: 237-245, 2003.[CrossRef][ISI][Medline]
26. Tidball JG. Inflammatory cell response to acute muscle injury. Med Sci Sports Exerc 27: 1022-1032, 1995.[ISI][Medline]
27. Warren GL, Hulderman T, Jensen N, McKinstry M, Mishra M, Luster MI, and Simeonova PP. Physiological role of tumor necrosis factor in traumatic muscle injury. FASEB J 16: 1530-1632, 2002.
28. Warren GL, Ingalls CP, Shah SJ, and Armstrong RB. Uncoupling of in vivo torque production from EMG in mouse muscles injured by eccentric contractions. J Physiol 515: 609-619, 1999.
29. Warren GL, Lowe DA, and Armstrong RB. Measurement tools used in the study of eccentric contraction-induced injury. Sports Med 27: 43-59, 1999.[ISI][Medline]
30. Winn N, Paul A, Musaro A, and Rosenthal N. Insulin-like growth factor isoforms in skeletal muscle aging, regeneration, and disease. Cold Spring Harb Symp Quant Biol 67: 507-518, 2002.[ISI][Medline]
31. Zlotnik A, Morales J, and Hedrick JA. Recent advances in chemokines and chemokine receptors. Crit Rev Immunol 19: 1-47, 1999.[ISI][Medline]