Journal of Leukocyte Biology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published online as doi:10.1189/jlb.0506356 on November 29, 2006

Published online before print November 29, 2006
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0506356v1
81/3/775    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Shireman, P. K.
Right arrow Articles by McManus, L. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shireman, P. K.
Right arrow Articles by McManus, L. M.
(Journal of Leukocyte Biology. 2007;81:775-785.)
© 2007 by Society for Leukocyte Biology

MCP-1 deficiency causes altered inflammation with impaired skeletal muscle regeneration

Paula K. Shireman*,{dagger},{ddagger},§,1, Verónica Contreras-Shannon||, Oscar Ochoa{dagger}, Bijal P. Karia{dagger}, Joel E. Michalek and Linda M. McManus§,#,**

* South Texas Veterans Health Care System, San Antonio, Texas, USA; and Departments of
{dagger} Surgery,
{ddagger} Medicine,
|| Cellular and Structural Biology,
Epidemiology and Biostatistics,
# Periodontics, and
** Pathology and
§ Sam and Ann Barshop Institute for Longevity and Aging Studies, University of Texas Health Science Center, San Antonio, Texas, USA

1 Correspondence: Departments of Surgery and Medicine and Sam and Ann Barshop Institute for Longevity and Aging Studies, University of Texas Health Science Center, MC 7741, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA. E-mail: shireman{at}uthscsa.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We examined the role of MCP-1, a potent chemotactic and activating factor for macrophages, in perfusion, inflammation, and skeletal muscle regeneration post-ischemic injury. MCP-1–/– or C57Bl/6J control mice [wild-type (WT)] underwent femoral artery excision (FAE). Muscles were collected for histology, assessment of tissue chemokines, and activity measurements of lactate dehydrogenase (LDH) and myeloperoxidase. In MCP-1–/– mice, restoration of perfusion was delayed, and LDH and fiber size, indicators of muscle regeneration, were decreased. Altered inflammation was observed with increased neutrophil accumulation in MCP-1–/– versus WT mice at Days 1 and 3 (P≤0.003), whereas fewer macrophages were present in MCP-1–/– mice at Day 3. As necrotic tissue was removed in WT mice, macrophages decreased (Day 7). In contrast, macrophage accumulation in MCP-1–/– was increased in association with residual necrotic tissue and impaired muscle regeneration. Consistent with altered inflammation, neutrophil chemotactic factors (keratinocyte-derived chemokine and macrophage inflammatory protein-2) were increased at Day 1 post-FAE. The macrophage chemotactic factor MCP-5 was increased significantly in WT mice at Day 3 compared with MCP-1–/– mice. However, at post-FAE Day 7, MCP-5 was significantly elevated in MCP-1–/– mice versus WT mice. Addition of exogenous MCP-1 did not induce proliferation in murine myoblasts (C2C12 cells) in vitro. MCP-1 is essential for reperfusion and the successful completion of normal skeletal muscle regeneration after ischemic tissue injury. Impaired muscle regeneration in MCP-1–/– mice suggests an important role for macrophages and MCP-1 in tissue reparative processes.

Key Words: CCL2 • macrophage • neutrophils • chemokines • ischemia


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Acute and chronic tissue ischemia is a common complication of atherosclerosis, embolic disease, and traumatic injury. Despite treatment, these conditions often result in lower extremity amputations. New and adjuvant therapies for limb salvage will probably derive from a better understanding of the mechanisms involved in postischemic collateral artery development and skeletal muscle regeneration.

Macrophages and inflammation are important components in collateral artery formation [1 ] and skeletal muscle regeneration [2 3 4 ] via biological responses that are incompletely understood. MCP-1 (also known as CCL2), through its receptor CCR2, is a chemokine with potent macrophage recruitment and activating functions [5 , 6 ]. In wild-type (WT) mice, within 3 days after femoral artery excision (FAE), MCP-1 protein levels were elevated maximally in ischemic calf muscle in association with a robust macrophage infiltrate; however, in the thigh, where arteriogenesis occurs, MCP-1 did not increase [7 ]. Thus, MCP-1 is probably involved in the modulation of macrophage responses following ischemia.

Previous studies have documented MCP-1 involvement in the restoration of perfusion after the induction of hind limb ischemia [8 9 10 11 ]; however, little is known about the role of MCP-1 in skeletal muscle regeneration following ischemic injury. Injured muscle secretes factors that are chemotactic for macrophages and other inflammatory cells [12 ], essential cells in the removal of damaged or necrotic tissue [13 ]. Further, macrophages enhance proliferation and delay differentiation [2 ] of satellite cells, the multipotent myogenic stem cells that are integral to regenerating damaged muscle [14 ].

Myogenic precursor cells can differentiate into skeletal muscle as well as transdifferentiate into adipocytes [15 ], osteoblasts [16 ], and endothelial cells [17 ]. The role that inflammation and perhaps MCP-1 plays in modulating the plasticity of myogenic stem cell growth and differentiation remains largely unknown but may be an important modifier in the skeletal muscle response to ischemia.

Given the essential roles of macrophages in muscle regeneration [2 , 4 ] and collateral artery formation [1 ], we hypothesized that MCP-1 may have a vital function in skeletal muscle responses to ischemia via recruitment and activation of macrophages. The present study was conducted to explore the relationship between the restoration of perfusion, inflammation, and skeletal muscle regeneration post-FAE in mice lacking MCP-1. Novel findings in this report include impairments in skeletal muscle regeneration and dysregulated inflammation in MCP-1–/– mice.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Experimental animals
MCP-1–/– mice, on a C57Bl/6J background, were derived as described previously [5 ] (original mice for the MCP-1–/– breeding colony were a kind gift from Barrett Rollins, Dana-Farber Cancer Institute, Harvard University, Boston, MA, USA) and backcrossed to C57Bl/6J mice from Jackson Labs (Bar Harbor, ME, USA) for six generations. MCP-1–/– mice, on the C57Bl/6J background, were bred at the Audie Murphy Veterans Hospital (San Antonio, TX, USA), and C57Bl/6J WT control mice were purchased from Jackson Labs. Male mice, 10–20 weeks old, were used in this study. All procedures complied with the National Institutes of Health (NIH) Animal Care and Use Guidelines and were approved by the Institutional Animal Care and Use Committee of the University of Texas Health Science Center at San Antonio and at the South Texas Veterans Health Care System (San Antonio, TX, USA).

Mouse FAE model and laser doppler imaging (LDI)
Male mice [MCP-1–/– or C57Bl/6J (WT)] underwent FAE. Mice were anesthetized with an i.p. injection of pentobarbital (2 mg; Abbott Laboratories, Chicago, IL, USA) or 2–3% isoflurane with oxygen for FAE and LDI. The femoral artery was excised from the inguinal ligament to just proximal to the bifurcation of the popliteal and saphena arteries as described previously [18 ]. In all animals, the femoral vein and nerve were preserved, and a sham surgery was performed on the left leg. LDI (Moor LDI, Moor Instruments, Wilmington, DE, USA) was performed sequentially on 20 mice/strain; each animal was imaged at all time points, as described previously [18 ].

Preparation of tissue lysates
After right FAE and left sham surgery (Days 1, 3, 7, and 14; n=5/time point), mice were killed humanely, and all of the muscles of the hind limb from the knee to the ankle (designated as "calf") or from the inguinal ligament to the knee (designated as thigh) were removed, weighed, and used immediately in preparation of tissue lysates, as described previously [7 ]. Similar samples were also collected from animals not subjected to surgery and served as negative controls (baseline). Briefly, on ice, muscles were minced and mixed, and a weighed portion was homogenized (Tissumizer, Tekmar, Cincinnati, OH, USA) on ice for 15 s in 1.5 ml lysate buffer, i.e., 50 mM Tris buffer (pH 7.4) containing 250 mM NaCl, 1% Nonidet P-40 (Roche Applied Science, Indianapolis, IN, USA), 50 mM NaF, 2 mM Na3VO4, 5.5 mM EDTA, 1 mM 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, 65 µM bestatin hydrochloride, 7 µM transepoxysuccinyl-L-leucylamido-(4-guanidino) butane, 11 µM leupeptin, 0.15 µM aprotinin, and 1 mM PMSF (from Sigma-Aldrich, St. Louis, MO, USA, unless otherwise specified). The tissue homogenate was then centrifuged immediately (4400 g, 5 min, 4°C), and the supernatant was removed and centrifuged further (2300 g, 5 min, 4°C). Aliquots of the final supernatant were stored at –80°C.

Measurement of tissue levels of MCP-5, macrophage inflammatory protein (MIP)-2, MIP-1{alpha}, and keratinocyte-derived chemokine (KC) and enzymatic activity of lactate dehydrogenase (LDH) and myeloperoxidase (MPO)
Tissue lysates were thawed on ice for 15 min and centrifuged immediately (3300–16,100 g, 4°C, 10 min). Protein was determined by the Pierce BCATM protein assay (Pierce Biotechnology, Inc., Rockford, IL, USA) using a microtiter plate format; BSA from ICN Biomedicals Inc. (Costa Mesa, CA, USA) in lysate buffer was used as the standard. Absorption in all microtiter plate assays was monitored in a SpectraMax Plus plate reader (Molecular Devices, Sunnyvale, CA, USA), and results were analyzed with SOFTmax PRO software (Molecular Devices).

MCP-5, MIP-2, MIP-1{alpha}, and KC levels in the tissue lysates were assessed by ELISA by R&D Systems (Minneapolis, MN, USA), according to the manufacturer’s protocol with slight modifications. Standards and unknowns were diluted in lysate buffer and results were expressed as pg/mg protein.

Reaction rates (Vmax) for LDH and MPO were determined as described previously [7 ]. LDH and MPO activities were normalized to mg protein measured in each lysate. Data for LDH, MPO, muscle weight, and protein/weight for WT mice have been reported previously [7 ] and were included here for purposes of comparison with results from MCP-1–/– mice. Data for WT and MCP-1–/– mice were obtained in parallel experiments.

Histologic studies
After right FAE and left sham surgery, mice were sacrificed humanely after 1, 3, 7, 14, 21, or 28 days, and hind limb tissues were collected for histology; n = 4–12 mice/time point/strain. After the i.p. injection of heparin (250 U) and pentobarbital (3 mg), the abdominal aorta was cannulated and pressure-perfused as described previously [7 ]. Hind limbs were removed at the inguinal ligament and placed in 10% neutral-buffered formalin (NBF) for 1–2 h. Complete cross-sections of each leg at the calf or thigh (including bone, muscle, and skin) were then obtained and decalcified prior to routine paraffin embedding. All paraffin-embedded specimens were sectioned (6 µm) and stained with H&E prior to light microscopic examination. In a separate group of animals not pressure-perfused, tissues from the anterior and posterior compartments of the lower leg were removed en bloc, transected axially through the mid-portion of the muscle belly, fixed in 10% NBF, and paraffin-embedded.

Immunohistochemical studies
Routine, indirect immunohistochemical procedures were used to localize monocytes/macrophages (F4/80 and mac3) or neutrophils (Ly-6) in deparaffinized sections. Nonspecific binding of antibodies was blocked by treatment of sections (30 min) with 1% BSA in PBS. Primary antibodies (diluted in 1% BSA in PBS) were rat monoclonal antimouse F4/80 (Serotec, Inc., Raleigh, NC, USA), antimouse mac3 (BD Biosciences PharMingen, San Diego, CA, USA), and antimouse Ly-6 (BD Biosciences PharMingen). Biotinylated secondary antibodies (mouse-absorbed rabbit antirat IgG) and streptavidin-HRP were obtained from Vector Laboratories (Burlingame, CA, USA). After enzymatic development in diaminobenzidine and hydrogen peroxide, sections were counterstained with hematoxylin.

Histology and immunohistochemistry images were captured with a Nikon E600 Eclipse microscope (Nikon, Melville, NY, USA), equipped with a high-resolution digital camera (Nikon DXM 1200), interfaced to a personal computer equipped with Act-1 (Nikon) networking software for image-capture and archiving.

Histomorphometric measurement of intermuscular fat and muscle fiber area
Morphometric analyses were performed on images captured with a Nikon Eclipse TE2000-U inverted microscope, equipped with a high-resolution digital camera (Nikon DXM 1200F), interfaced with a personal computer, equipped with Metamorph (Nikon) software. Using the anterior compartment specimen removed en bloc at 21 days post-FAE, 2–6 µm cross-sections were obtained through the mid-portion of the muscle and stained with H&E or trichrome. Preliminary studies demonstrated no significant difference in fiber cross-sectional area between H&E and trichrome-stained sections (data not shown). The 21-day time point was chosen for morphometric analysis, as only small foci of necrotic muscle were present occasionally post-FAE in both mouse strains. The tibialis anterior (TA), the largest and most superficial muscle in the anterior compartment, was identified and used for all histomorphometric measurements. The TA was chosen, as it consistently underwent necrosis and regeneration, and muscles in the posterior compartment, such as the gastrocnemious muscle, exhibited various amounts of necrosis and regeneration [7 ]. For TA specimen following FAE, three different microscopic sections separated by at least 100 µm were used for morphometric analysis, and one microscopic section was used for sham surgery. Within each TA section, eight nonoverlapping areas (20x magnification) were selected randomly and digitally captured; areas containing large blood vessels or fibrous bands between muscle bundles were excluded.

In each of the digitally captured images, intermuscular adipocytes were outlined manually and divided by the total image area to calculate the percent fat. The average percent fat area of eight images for each TA specimen was then determined. The average percent fat among replicate TA sections for each animal was calculated for subsequent use in comparisons of results for each group of animals.

The average cross-sectional area (µm2) of individual muscle fibers for a given animal was determined after manual outlining of individual muscle fibers in each of the digitally captured images for a given TA; fibers, which were included only partially within the images, were excluded. In the TA of FAE limbs, only regenerated fibers (identified by the presence of a centrally located nucleus [19 ]) were measured, whereas fibers with peripherally located nuclei (i.e., mature, nonregenerated fibers) were measured in the TA of the sham surgery limbs.

Cell culture
Mouse C2C12 myoblasts [American Type Culture Collection (ATCC), Manassas, VA] were cultured in growth media (GM) containing DMEM (ATCC), supplemented with 10% FBS (Hyclone, Logan, UT) and 1% penicillin-streptomycin (Mediatech, Inc., Herndon, VA, USA) at 37°C in a humidified atmosphere of 5% CO2 in air. Cells were allowed to reach 60% confluency before passage.

Cell proliferation
Cell proliferation was measured by the incorporation of BrdU using the cell proliferation ELISA BrdU colorimetric kit (Roche, Mannheim, Germany). C2C12 cells were seeded in 96-well plates (Falcon, Becton Dickinson, Franklin Lakes, NJ, USA) in GM. After 24 h, the GM was removed, cells were washed with PBS, and the proliferation assay was performed under two different culture conditions: 4000 cells/well were grown in serum-free media (SFM); DMEM, supplemented with 5 µg/ml transferrin (Sigma-Aldrich) and 0.2 µg/ml selenium (Sigma-Aldrich) or 2000 cells/well, were grown in DMEM supplemented with 2% FBS. Cytokines or FBS were added after 2 h for SFM and after 24 h for 2% FBS. The cytokines and FBS additions were as follows: as the negative control, 1% BSA in PBS, the murine cytokine fibroblast growth factor 2 (FGF2) or MCP-1 (both from R&D Systems) in 1% BSA in PBS at 1, 10, or 100 ng/ml and 20% FBS as the positive control. Cells were incubated with cytokines or FBS for 24 h or 48 h, followed by 2 h incubation with BrdU. After removing the culture media, cells were fixed, and the plates were processed per manufacturer protocol. After a 10-min incubation, absorbance was measured at 370 nm using a SpectraMax Plus plate reader. Results for each experiment were expressed as fold change compared with the negative control (1% BSA in PBS), and the fold changes for three independent experiments were then averaged.

Data analysis
SAS software, Version 9.1 (SAS, Cary, NC), was used for all statistical analyses. Results were expressed as mean ± SEM, and statistical significance was set at the level of 5%.

Sequentially derived LDI ratios were analyzed by two different methods. First, a Bonferroni-corrected multiple comparisons procedure was used to determine if LDI ratios within a given group had returned to preoperative (baseline) levels. Data collected preoperatively were subtracted from each of the other time points to produce a dataset of comparable LDI ratio differences. Second, an ANOVA incorporating repeated measures across time was used with a Bonferroni-correction for multiple comparisons to determine if there were differences between MCP-1–/– and WT mice at each time point. Within each animal, time correlations and heterogeneity of the variances across time were modeled using the autoregressive order one-covariance structure. Animal was considered a random effect.

Weight, protein/weight, chemokine levels, and enzyme activity were analyzed by two different methods. First, a Dunnett-corrected multiple comparisons procedure, using a two-way ANOVA of least-square means to determine whether significant differences existed, was used to compare each time point to baseline values for each mouse strain. Second, a Hochberg or Bonferroni-corrected two-way ANOVA was used to determine if there were differences between MCP-1–/– and WT mice at each time point. The MPO and chemokine data were analyzed in a similar manner, except a log transformation was applied to the data prior to analysis to adjust for unequal variances. For lysate samples with chemokines below the level of detection in the ELISA (MIP-2, 7.8; MIP-1{alpha}, 4.7 and 15.625 pg/ml for MCP-5 and KC), a value of lowest detectable level/Formula pg/ml was assigned to these samples [20 ], and this value was corrected for the protein in each specimen.

Histomorphometry data for percent fat and fiber cross-sectional area were analyzed as follows. First, a repeated measures linear model was used to determine that there were no significant interactions among the three different levels of the TA muscle. Replicate data for all images and levels were then averaged to generate a single number for each limb for each mouse—FAE or sham limb for fiber cross-sectional area and FAE-only for percent fat. Paired t-tests were used to assess the significance of mean differences in fiber cross-sectional area between FAE versus sham limbs within each strain. One-way analyses of variance were used to assess the significance of mean differences between MCP-1–/– and WT mice for FAE (fiber cross-sectional area and percent fat) or sham (fiber cross-sectional area) limbs.

Prior to analysis of C2C12 proliferation data, the raw OD was background-corrected by subtracting the mean OD from blank wells and then divided by the mean OD from the negative control wells. ANOVA was used to contrast treatment and negative control OD means. The experiment-wide significance level was fixed at 5% using Dunnett’s method for repeated two-sided comparisons with the control.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Restoration of perfusion post-FAE
Before FAE, perfusion was equivalent in the right and left legs of WT and MCP-1–/– mice. Immediately after FAE, perfusion decreased to ~15% of that in the contralateral leg in both groups and remained unchanged through Day 3 (Fig. 1 ). In WT mice, perfusion increased by Day 7 and improved steadily thereafter. In contrast, there was a delay in the restoration of perfusion in the MCP-1–/– mice and a significant decrease in the perfusion ratio at Day 7 (P=0.01) as compared with WT animals. In MCP-1–/– and WT mice, there was a significant difference at all post-FAE time points (P≤0.0001) as compared with pre-excision levels. Thus, an extended period of resting ischemia was present post-FAE in both mouse strains.


Figure 1
View larger version (13K):
[in this window]
[in a new window]

  		Figure 1. Restoration of perfusion in MCP-1–/– and WT mice post-FAE. LDI perfusion ratios for MCP-1–/– and WT mice were measured sequentially for each mouse at all time points. For both groups, there were significant differences at all time points compared with pre-excision LDI measurements (P≤0.001). Results represent the mean ± SEM; n = 20 mice/strain. #, P = 0.01, between MCP-1–/– and WT groups.

 
FAE-induced changes in tissue weight, protein, and MPO activity
Thigh and calf muscle weights post-FAE were normalized to the contralateral leg for WT and MCP-1–/– animals to correct for small differences in total body weight between individual mice. With thigh muscles, there were no significant changes compared with baseline in the relationship between right (FAE) and left (sham surgery) thigh muscle weights at any postsurgical time point (1, 3, 7, or 14 days). Furthermore, there were no differences between thigh muscle weight ratios of WT and MCP-1–/– mice at corresponding time points (data not shown).

The relative weight of ischemic calf muscle in WT mice was increased significantly only at 3 days post-FAE (P≤0.001 vs. baseline) before returning to baseline values (Fig. 2A ). In contrast with MCP-1–/– mice, the calf muscle weight ratio was elevated significantly within 1 day and remained increased for an extended interval (i.e., at Days 1, 3, and 7, P≤0.001 vs. baseline, Fig. 2A ). In comparison with WT mice, the calf muscle weight ratio of MCP-1–/– animals was significantly greater than WT at 7 days post-FAE (P≤0.001). Thus, MCP-1–/– mice had elevated calf muscle weight ratios for an extended period of time as compared with WT animals. These observations were consistent with the time course of muscle regeneration and initial tissue edema (see below).


Figure 2
View larger version (18K):
[in this window]
[in a new window]

  		Figure 2. Calf muscle weight ratios, protein, and MPO activity in MCP-1–/– and WT mice post-FAE. (A) Calf muscle weight ratio. (B) Protein in FAE calf muscle. (C) MPO activity in calf muscle post-FAE or at baseline (samples collected in the absence of surgery). Results represent the mean ± SEM; n = 5 mice/time point/strain. *, P ≤ 0.02, compared with baseline; #, P ≤ 0.007, between MCP-1–/– and WT groups.

 
Calf tissue protein levels were determined in lysates and normalized to the weight of the muscle tissue. In parallel, the soluble protein/g tissue (protein/weight) followed an inverse pattern of the calf muscle weight; in the FAE calf muscle of WT mice, protein/weight was decreased significantly only at Day 3 (P=0.003) as compared with baseline (Fig. 2B) . In contrast, MCP-1–/– animals were decreased significantly from baseline at all time points (Days 1, 3, 7, and 14, P≤0.02 vs. baseline). In addition, there were significant differences between WT and MCP-1–/– mice at Day 1 (P=0.007) and Day 7 (P=0.002). In contralateral calf muscles, there were no significant differences from baseline for either mouse strain (data not shown). Thus, protein content in ischemic tissues was depressed for an extended period post-FAE in MCP-1–/– mice. The combined alterations in weight and protein/weight were consistent with increased edema and the prolonged presence of macrophages associated with residual necrotic tissue, as observed in histology (see below) of ischemic tissues in MCP-1–/– mice.

Edema is often associated with inflammation [21 ]. Therefore, MPO activity was used to measure neutrophil accumulation in ischemic calf muscle [22 ]. There were no significant differences from baseline in MPO activity in contralateral calf muscles for either mouse strain (data not shown). In calf muscle post-FAE, MPO was elevated significantly in WT mice at Day 3 (P≤0.001) and Day 7 (P=0.002; Fig. 2C ) and in MCP-1–/– mice, at Days 1, 3, and 7 (P≤0.008). Furthermore, MPO activity was significantly higher in MCP-1–/– mice than in WT mice at Days 1 and 3 (P≤0.003). Thus, MCP-1–/– mice exhibited increased neutrophil accumulation at early time points post-FAE compared with WT mice.

Kinetics of the inflammatory infiltrate and skeletal muscle regeneration post-FAE
In the bilateral thigh specimen, an inflammatory infiltrate was located near the skin incision where focal areas of skeletal muscle necrosis were identified occasionally in WT and MCP-1–/– mice. The histologic appearance of the calf muscle of WT and MCP-1–/– mice at 1-day post-FAE was similar; a comprehensive description of changes in WT mice has been published previously [7 ]. In brief, at the Day 1 time point, ischemic injury of skeletal muscle was widespread, and an inflammatory infiltrate, consisting mainly of neutrophils, was present; injured muscle was easily identified as swollen, intensely eosinophilic segments of muscle fibers that lacked peripherally located nuclei. At 3 days, a prominent mononuclear inflammatory infiltrate, primarily macrophages, was evident in association with muscle injury in WT, but not MCP-1–/– mice (Figs. 3 A-D and 4 ). Neutrophils were also present in both strains of mice at Day 3 but were more prevalent in the MCP-1 –/– mice. By 7 days post-FAE, the inflammatory infiltrate was greatly diminished in WT mice and replaced by regenerated muscle fibers containing multiple, centrally located nuclei (Fig. 3F) , whereas large regions of necrotic muscle remained in MCP-1–/– animals (Fig. 3E) . It is interesting that macrophage infiltration around necrotic muscle fibers was more prevalent by this time in MCP-1–/– animals. Muscle regeneration was essentially complete in WT mice within 14–21 days post-FAE, and the histologic appearance in WT animals was similar at both of these time points. In contrast, residual necrotic myofibers with associated muscle regeneration, macrophage infiltration, and adipocyte accumulation were widespread in MCP-1–/– animals at Day 14 (Fig. 3G and 3H) . Thus, in MCP-1–/– mice, macrophage accumulation was decreased at Day 3, when WT macrophage accumulation in WT mice was maximal, but the macrophage infiltrate persisted longer in association with residual necrotic tissue. At 21 days post-FAE, muscle regeneration was largely complete in the MCP-1–/– mice, with adipocyte accumulation between regenerated fibers (Fig. 3I) , and fewer adipocytes were present in WT mice (Fig. 3J) . Thus, impaired skeletal muscle regeneration in MCP-1–/– mice was associated with dysregulated inflammation in the regenerating muscle and a tendency to form adipocytes.


Figure 3
View larger version (132K):
[in this window]
[in a new window]

  		Figure 3. Macrophage (F4/80) infiltration, skeletal muscle regeneration, and fat accumulation in the ischemic hind limb in the absence of MCP-1. (A) MCP-1–/–, 3 days post-FAE, neutrophilic inflammatory infiltrate with few mononuclear cells, gastrocnemius muscle. (B) WT, 3 days post-FAE, robust mononuclear cell infiltrate, gastrocnemius muscle. (C) MCP-1–/–, 3 days post-FAE, F4/80 immunolocalization, a few positive cells are present adjacent to the injured muscle fibers, gastrocnemius muscle. (D) WT, 3 days post-FAE, F4/80 immunolocalization with robust infiltration of positive cells, gastrocnemius muscle. (E) MCP-1–/–, 7 days post-FAE, TA muscle with small, regenerated fibers with one or more centrally located nuclei, a few normal fibers at the periphery, necrotic muscle fibers. (F) WT, 7 days post-FAE, TA muscle with regenerated fibers, a few normal fibers at the periphery. (G) MCP-1–/–, 14 days post-FAE, regenerated TA muscle with intermuscular adipose tissue, necrotic fibers, a few normal fibers at the periphery. (H) MCP-1–/–, 14 days post-FAE, F4/80 immunolocalization, positive mononuclear cells interspersed among regenerated muscle fibers (arrows). (I) MCP-1–/–, 21 days post-FAE, regenerated TA muscle with intermuscular adipose tissue (arrows). (J) WT, 21 days post-FAE, regenerated TA muscle with focal intermuscular adipocytes (arrows). H&E stain except for C, D, and H, which were counterstained with hematoxylin; n, Normal skeletal muscle fiber; *, necrotic skeletal muscle fiber.

 

Figure 4
View larger version (157K):
[in this window]
[in a new window]

  		Figure 4. Inflammatory cell infiltration at 3 days post-FAE. The triangular, soleus muscle (arrow). (A) WT with intense intramuscular mononuclear cell infiltrate. (B) MCP-1–/– with reduced intramuscular inflammatory cell infiltrate. Inset, edge of soleus muscle (arrow) at higher magnification; H&E stain.

 
Elevated tissue chemokine levels in MCP-1–/– mice post-FAE
Given the alterations in neutrophil recruitment in MCP-1–/– mice, as described above, tissue levels of the neutrophil chemoattractants MIP-2 [23 ] and KC [24 ] were measured, corrected for protein, and expressed as pg/mg protein. Baseline levels of both chemokines were below the limit of detection in MCP-1–/– and WT mice. In both mouse strains, MIP-2 was elevated significantly (P≤0.002) over baseline at post-FAE Days 1, 3, and 7 (Fig. 5A ). KC levels (Fig. 5B) were elevated significantly (P≤0.003) over baseline at Days 1 and 3 in WT mice and at Days 1, 3, and 7 in MCP-1–/– mice. Although there were no significant differences between MCP-1–/– and WT mice at any single time point for MIP-2 or KC, the means of MIP-2 and KC were increased at Day 1 in MCP-1–/– mice compared with WT mice.


Figure 5
View larger version (17K):
[in this window]
[in a new window]

  		Figure 5. Neutrophil chemoattractant tissue levels in MCP-1–/– and WT mice post-FAE. Tissue levels of the potent neutrophil chemoattractant agents (A) MIP-2 and (B) KC post-FAE. Baseline results were derived from samples collected in the absence of surgery, and chemokine levels were not detectable (ND). Results represent the mean ± SEM of only those samples with detectable chemokine levels; n = 4–5 mice/time point/strain. *, P ≤ 0.003, compared with baseline.

 
As macrophage recruitment was also altered in MCP-1–/– mice, tissue levels of the macrophage chemoattractants MIP-1{alpha} [25 ] and MCP-5 [26 ] were measured. Baseline levels of both chemokines were below the limit of detection in MCP-1–/– and WT mice. Although MIP-1{alpha} was elevated significantly (P≤0.05) over baseline at post-FAE Days 1, 3, and 7, levels were similar between MCP-1–/– mice and WT mice at all time points (Fig. 6A ). In contrast to MIP-1{alpha}, MCP-5 (Fig. 6B) was not only elevated significantly (P≤0.001) over baseline in both mouse strains at post-FAE Days 1, 3, and 7 but was increased (P=0.05) in WT compared with MCP-1–/– mice at Day 3. However, at Day 7, MCP-5 was elevated significantly (P=0.05) in MCP-1–/– mice compared with WT mice. These differences between MCP-5 levels in the two mouse strains may account, in part, for the robust macrophage accumulation in WT mice at Day 3 post-FAE and the persistence of macrophages in MCP-1–/– mice at later time points.


Figure 6
View larger version (18K):
[in this window]
[in a new window]

  		Figure 6. Macrophage chemoattractant tissue levels in MCP-1–/– and WT mice post-FAE. Tissue levels of the potent macrophage chemoattractant agents (A) MIP-1{alpha} and (B) MCP-5 post-FAE. Baseline results were derived from samples collected in the absence of surgery, and chemokine levels were not detectable. Results represent the mean ± SEM of only those samples with detectable chemokine levels; n = 4–5 mice/time point/strain. *, P ≤ 0.05, compared with baseline; #, P = 0.05, between MCP-1–/– and WT groups.

 
Impaired muscle regeneration in MCP-1–/– mice post-FAE
LDH is an enzyme that is present in skeletal muscle; its activity decreases with injury and is restored with regeneration [7 , 27 ]. It is not surprising that contralateral calf muscles (sham surgery side) from either mouse strain demonstrated no significant differences from baseline in LDH activity (data not shown). In WT mice post-FAE (Fig. 7A ), LDH activity was lowest at Day 3 and returned to near baseline by Day 14 with significant differences from baseline at Days 3 and 7 (P≤0.002). In contrast, in MCP-1–/– mice, LDH activity was decreased significantly from baseline at 3, 7, and 14 days post-FAE (P≤0.006). Finally, LDH activity was decreased significantly in MCP-1–/– mice at Day 14 (P≤0.001) compared with WT animals.


Figure 7
View larger version (27K):
[in this window]
[in a new window]

  		Figure 7. Kinetics of hind limb skeletal muscle injury and regeneration, fiber size, and percent fat in MCP-1–/– and WT mice post-FAE. (A) LDH activity in calf muscle post-FAE or at baseline (samples collected in the absence of surgery). (B) Muscle fiber cross-sectional area (µm2); TA muscle at 21 days post-FAE. (C) Fat area (%) of the TA at 21 days post-FAE. Mean ± SEM; number of mice/group indicated in parentheses. *, P ≤ 0.006, compared with baseline or sham levels; #, P ≤ 0.05, between MCP-1–/– and WT groups.

 
In addition, at 21 days post-FAE, TA muscle fiber cross-sectional area (µm2) of regenerated fibers was decreased significantly in MCP-1–/– (789±3) compared with WT mice (1154±79; P=0.005; Fig. 7B ). It was interesting that the mature fiber cross-sectional area in the sham surgery TA was significantly increased in MCP-1–/– mice compared to WT mice (1943±61 and 1529±150, respectively; P=0.05). Furthermore, the muscle fiber cross-sectional area was significantly smaller in the FAE compared with the sham surgery TA in MCP-1–/– (P=0.004) but not in WT mice. Thus, regenerated muscle fibers in MCP-1–/– mice were significantly smaller than mature muscle fibers, as well as smaller than regenerated fibers in WT mice at 21 days post-surgery.

Adipocyte deposition in regenerated muscle of MCP-1–/– and WT mice
Intermuscular adipocytes were not identified in the TA of sham surgery limbs in either strain of mice. However, in the TA of FAE limbs, intermuscular adipocytes were present in the areas of regenerated tissue (Fig. 3I and 3J) . Although MCP-1–/– mice had an increased area of intermuscular fat within the TA (5.4±1.6% vs. 3.4±0.2%, for MCP-1–/– and WT mice, respectively), this difference did not achieve significance (Fig. 7C) .

MCP-1 does not induce C2C12 proliferation
C2C12 cells are a murine myoblast cell line, commonly used to study myoblast proliferation and differentiation in vitro [28 , 29 ]. FGF2, a known myoblast mitogen [14 ], was used as a positive control in addition to 20% FBS. In SFM, 48 h after cytokine addition, proliferation in response to FGF2 increased in a dose-dependent manner, and similar proliferation occurred at 10 and 100 ng/ml (1.73±0.14-fold increase over control for 10 ng/ml; P≤0.001 compared with control). Maximal proliferation was observed in cells supplemented with 20% FBS (2.65±0.26-fold increase over control; P≤0.001 compared with control; Fig. 8 ). In contrast, the addition of MCP-1 did not alter proliferation of C2C12 cells. Similar results were obtained in SFM, when proliferation was measured at 24 h as well as at 24 h and 48 h in cells in 2% FBS. MCP-1 activity was confirmed in an in vivo assay with inflammatory cell attraction in response to MCP-1 injected s.c., as described previously [30 ]. Thus, MCP-1 did not affect C2C12 proliferation in low-serum or serum-free conditions.


Figure 8
View larger version (15K):
[in this window]
[in a new window]

  		Figure 8. No change in C2C12 proliferation in response to MCP-1. C2C12 cells, a murine myoblast cell line, had increased proliferation to FGF2 and 20% FBS, but not MCP-1. SFM were used, and proliferation was measured 48 h after the addition of cytokines or FBS. Results represent the mean ± SEM of three independent experiments. *, P ≤ 0.001, compared with negative control.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The tissue response to ischemia is a critical component of limb salvage and the maintenance of function. The present study documented that regeneration of ischemia-damaged skeletal muscle is contingent on macrophage recruitment via MCP-1. In the absence of MCP-1, alterations in neutrophil recruitment and tissue chemotactic factors occurred in parallel with impaired skeletal muscle regeneration.

Restoration of perfusion post-FAE is a complex process that involves the development of collateral arteries, and MCP-1, as a result of its macrophage recruitment activities, is beneficial to this process [9 10 11 ]. Previous studies using MCP-1–/– mice have documented decreased restoration of perfusion after induction of hind limb ischemia [9 , 10 ], observations that have been confirmed in the present study. Curiously, restoration of perfusion using CCR2–/– mice, the main receptor for MCP-1 [6 ], has been variable [31 , 32 ], and in our lab, there was no significant difference in the restoration of perfusion between CCR2–/– and WT mice [33 ]. Several possibilities may explain these seemingly contradictory results. First, MCP-1 may function through receptors other than CCR2 [34 ], and the loss of MCP-1 signaling through these undefined receptors may be responsible for the delayed restoration of perfusion in MCP-1–/– mice. Alternatively, CCR2–/– mice exhibit increased tissue MCP-1 as compared with WT mice in response to hind limb ischemia [33 ]. This increased MCP-1 concentration may allow MCP-1 to bind to alternate receptors, which are not activated at physiological levels of MCP-1 and may compensate for the loss of CCR2 receptors. Nevertheless, the post-ischemic restoration of perfusion has been delayed consistently in MCP-1–/– mice.

The role of MCP-1 in inflammation and the regeneration of ischemic muscle have not been defined previously. Although perfusion was similar in WT and MCP-1–/– mice at early time points, the accumulation of neutrophils, as measured by MPO activity, was elevated in ischemic calf muscle at Days 1 and 3, and edema was present earlier and remained for an extended period in MCP-1–/– mice (Fig. 2) . In previous studies with MCP-1–/– mice, similar numbers of neutrophils, as compared with WT, were recruited to various inflammatory lesions and counted in peritoneal [5 ] or bronchiolar lavage [35 ] fluid or in histological specimen after tissue immunolocalization [36 , 37 ]. Explanations for divergent results in neutrophil recruitment between the current and previous studies are unknown. However, as compared with the present study, alternative inflammatory insults as well as neutrophil enumeration methods were used previously; those histological approaches are prone to sampling error and difficulties in cell identification. Tissue MPO activity used herein reflects a larger, more representative sampling of tissue neutrophil content.

In the present study, the mechanisms of increased neutrophil accumulation in MCP-1–/– mice are consistent with elevations of neutrophil chemoattractant agents. Of note and consistent with the current findings, an increase and/or persistence of neutrophils in various inflammatory states have been observed previously after blocking MCP-1 [36 ] as well as in CCR2–/– mice [39 , 40 ]. A possible explanation for enhanced neutrophil recruitment in MCP-1–/– mice derives from a recent study that demonstrated increased mRNA for MIP-2 in excisional wounds and stimulated macrophages from MCP-1–/– mice [40 ]. Consistent with this observation, the mean MIP-2 and KC tissue levels herein were increased in MCP-1–/– mice compared with WT mice at Day 1 but did not attain statistical significance, most likely secondary to small sample size and large variance between animals. These chemokines may account for the significant increases in MPO activity at Days 1 and 3 in MCP-1–/– mice versus WT mice. Of note, MIP-2 and MPO elevations in WT mice followed the same pattern (i.e., maximal at Day 3), and the increased MIP-2 at Day 1 in MCP-1–/– mice may account for the exaggerated Day 3 MPO activity. Finally, the clearance of neutrophils may have been decreased secondary to reduced macrophage accumulation [42 ]. Thus, although the physiological basis remains to be established, the present study demonstrates that the absence of MCP-1 appears to alter/increase early inflammatory events in ischemic tissues.

Neutrophils are key players in early inflammation associated with muscle injury, but these cells can also exaggerate muscle damage (c.f., Tidball [3 ] and Toumi and Best [43 ]). Although some reports suggest a beneficial or neutral role of neutrophils in muscle regeneration [44 , 45 ], most suggest that neutrophils increase muscle injury via generation of oxidative stress [3 , 43 , 46 ]. In fact, MPO may be a major factor in neutrophil-mediated damage; i.e., MPO–/– mice had less muscle membrane injury induced by mechanical loading than WT mice despite similar levels of inflammation [46 ]. However, in the current study, a slight but not significant decrease in LDH activity at Day 3 in MCP-1–/– compared with WT mice does not support the likelihood that there was increased tissue damage as a result of increased neutrophil accumulation. Nevertheless, altered skeletal muscle regeneration in the absence of MCP-1 may, in part, be attributable to increased MPO accumulation at early time points and is the basis for ongoing studies.

MCP-1 may affect the interactions of neutrophils and macrophages. As MCP-1 has been immunolocalized to vascular endothelial cells and macrophages within ischemic hind limb muscles [7 ], the absence of endothelial MCP-1 likely alters the balance of neutrophils and monocytes transmigrating across the endothelium. In this regard, previous reports have noted an interdependence between monocyte and neutrophil trafficking [47 ] as well as a need for macrophages to replace neutrophils to facilitate the resolution of inflammation [48 ]. Furthermore, MCP-1 enhances the ability of macrophages to phagocytose apoptotic neutrophils [42 ], potentially removing harmful neutrophilic enzymes, which could increase muscle damage. Thus, the absence of MCP-1 could alter neutrophil interactions with endothelial cells and macrophages.

Macrophages are important in muscle regeneration [2 3 4 ], and MCP-1 is a potent chemotactic factor for macrophage recruitment [6 ]. In the current study, it was not surprising that macrophages were decreased in ischemic muscle tissue, as defects in macrophage recruitment have been reported previously in MCP-1–/– mice [5 , 37 , 49 ]. Normally, macrophages predominate during skeletal muscle regeneration and are essential for removal of necrotic tissue [13 ]. Macrophages also produce a vast array of growth factors and enzymes that influence many aspects of the muscle regenerative process, including angiogenesis and the chemotaxis, proliferation, and differentiation of myoblasts [50 ]. Unless the damaged muscle fiber becomes invaded by macrophages, it remains arrested in the stage of intrinsic degeneration [4 , 51 ]. Further support for a macrophage role in muscle regeneration derives from inhibition of P-selectin, an adhesion molecule important in the early events of leukocyte extravasation, which resulted in fewer inflammatory cells in injured tissue as well as impaired muscle regeneration [52 ] and angiogenesis [53 ]. Thus, defective macrophage recruitment and/or function likely contributed to impaired muscle regeneration in MCP-1–/– mice in the current study.

Macrophage recruitment was delayed in the MCP-1–/– mice, but macrophages persisted longer as compared with WT mice. Persistence of macrophages could potentially derive from the extended presence of necrotic tissue in the MCP-1–/– mice. Macrophages are critical to the removal of necrotic tissue [13 ] and injured muscle fibers release factors that are chemotactic for macrophages [12 ]. In fact, chronic muscle injury, as observed in muscular dystrophy, can result in chronic macrophage infiltration, which can actually increase muscle damage and fibrosis [54 ]. Other mechanisms that may contribute to the persistence of macrophages include alterations in the production of macrophage chemoattractants in the MCP-1–/– mice. Support for this possibility derives from the significantly elevated MCP-5 levels at Day 7 in MCP-1–/– mice and may provide a mechanism for the prolonged presence of macrophages in the absence of MCP-1. It is interesting that MIP-1{alpha}, another potent macrophage chemoattractant, was similarly elevated in both mouse strains, suggesting that the delayed and prolonged accumulation of macrophages in MCP-1–/– mice was regulated by specific chemokines rather than a generalized increase in chemotactic factors.

Another important aspect of skeletal muscle regeneration is the activation of satellite cells, the multipotent adult stem cells that reside in skeletal muscle [14 ]. Satellite cell proliferation is required to repair/replace injured myofibers. Normally, satellite cells are quiescent and are physically distinct from the myofiber, as they reside in indentations between the sarcolemma and the basal lamina. In response to muscle damage, satellite cells become activated, proliferate, and express myogenic markers (now termed myoblasts) [14 ]. Myoblasts ultimately fuse to existing muscle or fuse together to form new myofibers during regeneration of damaged skeletal muscle (reviewed in Hawke and Garry [14 ]). It is interesting that macrophage-conditioned media increase myoblast proliferation and chemotaxis [12 , 55 56 57 ].

Although the cellular mechanisms responsible for the delayed skeletal muscle regeneration in MCP-1 animals remain to be established, it is conceivable that MCP-1 has direct effects upon satellite cells and/or myoblasts to stimulate their proliferation and/or chemotaxis. Support for this possibility is provided by observations that CCR2 [58 ] and MCP-1 [59 ] are localized to satellite cells in vivo, and MCP-1 is synthesized by myoblasts in tissue culture [59 ]. MCP-1 is chemotactic for satellite cells [59 ] and could influence muscle regeneration by inducing satellite cells from neighboring regions to migrate into areas of muscle injury. Nevertheless, exogenous MCP-1 did not enhance in vitro proliferation of C2C12 myoblasts in the current study (Fig. 6) . Additional experiments to examine the mitogenic and chemotactic effect of MCP-1 on isolated satellite cells and/or myoblasts are necessary to address this important question.

Impaired muscle regeneration in MCP-1–/– mice was demonstrated in the current study by decreased tissue LDH and diminished muscle fiber size as compared with WT mice (Fig. 7) . Although the increased adipocyte accumulation in MCP-1–/– mice was not significant, it followed an interesting trend in comparison with CCR2–/– mice, which do exhibit increased adipocyte deposition in regenerated muscle following ischemic [33 ] and freeze [60 ] injury compared with WT mice. It is interesting that myogenic progenitor cells can transdifferentiate into adipocytes [15 , 61 ]. Thus, although MCP-1 deficiency may contribute to adipocyte accumulation in injured muscle, other ligands of CCR2 may play a more important role. Whether impaired muscle regeneration was a direct effect of MCP-1 on satellite cells, an indirect effect (i.e., altered inflammation) or a combination of both factors cannot be answered by the present study.

In conclusion, the absence of MCP-1 during ischemic injury of skeletal muscle was associated with a modest delay in the restoration of perfusion, altered inflammation, altered chemokine levels, and impaired muscle regeneration. In combination, these findings demonstrate that MCP-1 is essential for restoration of perfusion and the successful completion of normal skeletal muscle regeneration following ischemic injury, probably via macrophage recruitment and activation.


   ACKNOWLEDGEMENTS
 
This work is supported, in part, by the San Antonio Area Foundation, Veterans Administration Merit Review, the American Heart Association, Texas Affiliate (0365123Y), and NIH (HL070158, HL074236, HL04776, and AR052610). We acknowledge the technical assistance of Richard Castillo, Jefferey Jimenez, and Didier Nuno in the completion of these studies. A portion of the statistical analyses was graciously provided by John Cornell and Gary Chisholm.

Received May 23, 2006; revised October 31, 2006; accepted November 2, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Deindl, E., Schaper, W. (2005) The art of arteriogenesis Cell Biochem. Biophys. 43,1-16[CrossRef][Medline]
  2. Merly, F., Lescaudron, L., Rouaud, T., Crossin, F., Gardahaut, M. F. (1999) Macrophages enhance muscle satellite cell proliferation and delay their differentiation Muscle Nerve. 22,724-732[CrossRef][Medline]
  3. Tidball, J. G. (2005) Inflammatory processes in muscle injury and repair Am. J. Physiol. Regul. Integr. Comp. Physiol. 288,R345-R353[Abstract/Free Full Text]
  4. Lescaudron, L., Peltekian, E., Fontaine-Perus, J., Paulin, D., Zampieri, M., Garcia, L., Parrish, E. (1999) Blood borne macrophages are essential for the triggering of muscle regeneration following muscle transplant Neuromuscul. Disord. 9,72-80[CrossRef][Medline]
  5. Lu, B., Rutledge, B. J., Gu, L., Fiorillo, J., Lukacs, N. W., Kunkel, S. L., North, R., Gerard, C., Rollins, B. J. (1998) Abnormalities in monocyte recruitment and cytokine expression in monocyte chemoattractant protein 1-deficient mice J. Exp. Med. 187,601-608[Abstract/Free Full Text]
  6. Charo, I. F., Taubman, M. B. (2004) Chemokines in the pathogenesis of vascular disease Circ. Res. 95,858-866[Abstract/Free Full Text]
  7. Shireman, P. K., Contreras-Shannon, V., Reyes-Reyna, S. M., Robinson, S. C., McManus, L. M. (2006) MCP-1 parallels inflammatory and regenerative responses in ischemic muscle J. Surg. Res. 134,145-157[CrossRef][Medline]
  8. Niiyama, H., Kai, H., Yamamoto, T., Shimada, T., Sasaki, K., Murohara, T., Egashira, K., Imaizumi, T. (2004) Roles of endogenous monocyte chemoattractant protein-1 in ischemia-induced neovascularization J. Am. Coll. Cardiol. 44,661-666[Abstract/Free Full Text]
  9. Waeckel, L., Mallat, Z., Potteaux, S., Combadiere, C., Clergue, M., Duriez, M., Bao, L., Gerard, C., Rollins, B. J., Tedgui, A., Levy, B. I., Silvestre, J. S. (2005) Impairment in postischemic neovascularization in mice lacking the CXC chemokine receptor 3 Circ. Res. 96,576-582[Abstract/Free Full Text]
  10. Voskuil, M., Hoefer, I. E., van Royen, N., Hua, J., de Graaf, S., Bode, C., Buschmann, I. R., Piek, J. J. (2004) Abnormal monocyte recruitment and collateral artery formation in monocyte chemoattractant protein-1-deficient mice Vasc. Med. 9,287-292[Abstract/Free Full Text]
  11. Ito, W. D., Arras, M., Winkler, B., Scholz, D., Schaper, J., Schaper, W. (1997) Monocyte chemotactic protein-1 increases collateral and peripheral conductance after femoral artery occlusion Circ. Res. 80,829-837[Abstract/Free Full Text]
  12. Robertson, T. A., Maley, M. A., Grounds, M. D., Papadimitriou, J. M. (1993) The role of macrophages in skeletal muscle regeneration with particular reference to chemotaxis Exp. Cell Res. 207,321-331[CrossRef][Medline]
  13. Tidball, J. G. (1995) Inflammatory cell response to acute muscle injury Med. Sci. Sports Exerc. 27,1022-1032
  14. Hawke, T. J., Garry, D. J. (2001) Myogenic satellite cells: physiology to molecular biology J. Appl. Physiol. 91,534-551[Abstract/Free Full Text]
  15. Teboul, L., Gaillard, D., Staccini, L., Inadera, H., Amri, E. Z., Grimaldi, P. A. (1995) Thiazolidinediones and fatty acids convert myogenic cells into adipose-like cells J. Biol. Chem. 270,28183-28187[Abstract/Free Full Text]
  16. Sun, J. S., Wu, S. Y., Lin, F. H. (2005) The role of muscle-derived stem cells in bone tissue engineering Biomaterials 26,3953-3960[CrossRef][Medline]
  17. Steffel, J., Wernig, M., Knauf, U., Kumar, S., Wiestler, O. D., Wernig, A., Brustle, O. (2003) Migration and differentiation of myogenic precursors following transplantation into the developing rat brain Stem Cells 21,181-189[Abstract/Free Full Text]
  18. Shireman, P. K., Quinones, M. P. (2005) Differential necrosis despite similar perfusion in mouse strains after ischemia J. Surg. Res. 129,242-250[CrossRef][Medline]
  19. Charge, S. B., Rudnicki, M. A. (2004) Cellular and molecular regulation of muscle regeneration Physiol. Rev. 84,209-238[Abstract/Free Full Text]
  20. Hornung, R. W., Reed, L. D. (1990) Estimation of average concentration in the presence of nondetectable values Appl. Occup. Environ. Hyg. 5,46-51
  21. Ueno, A., Oh-ishi, S. (2002) Critical roles for bradykinin and prostanoids in acute inflammatory reactions: a search using experimental animal models Curr. Drug Targets Inflamm. Allergy 1,363-376[CrossRef][Medline]
  22. Bradley, P. P., Priebat, D. A., Christensen, R. D., Rothstein, G. (1982) Measurement of cutaneous inflammation: estimation of neutrophil content with an enzyme marker J. Invest. Dermatol. 78,206-209[CrossRef][Medline]
  23. Jerva, L. F., Sullivan, G., Lolis, E. (1997) Functional and receptor binding characterization of recombinant murine macrophage inflammatory protein 2: sequence analysis and mutagenesis identify receptor binding epitopes Protein Sci. 6,1643-1652[Abstract]
  24. Bozic, C. R., Kolakowski, L. F., Jr, Gerard, N. P., Garcia-Rodriguez, C., von Uexkull-Guldenband, C., Conklyn, M. J., Breslow, R., Showell, H. J., Gerard, C. (1995) Expression and biologic characterization of the murine chemokine KC J. Immunol. 154,6048-6057[Abstract]
  25. Menten, P., Wuyts, A., Van Damme, J. (2002) Macrophage inflammatory protein-1 Cytokine Growth Factor Rev 13,455-481[CrossRef][Medline]
  26. Sarafi, M. N., Garcia-Zepeda, E. A., MacLean, J. A., Charo, I. F., Luster, A. D. (1997) Murine monocyte chemoattractant protein (MCP)-5: a novel CC chemokine that is a structural and functional homologue of human MCP-1 J. Exp. Med. 185,99-109[Abstract/Free Full Text]
  27. Fink, E., Fortin, D., Serrurier, B., Ventura-Clapier, R., Bigard, A. X. (2003) Recovery of contractile and metabolic phenotypes in regenerating slow muscle after notexin-induced or crush injury J. Muscle Res. Cell Motil. 24,421-429[CrossRef][Medline]
  28. Koleva, M., Kappler, R., Vogler, M., Herwig, A., Fulda, S., Hahn, H. (2005) Pleiotropic effects of sonic hedgehog on muscle satellite cells Cell. Mol. Life Sci. 62,1863-1870[CrossRef][Medline]
  29. Epstein, J. A., Lam, P., Jepeal, L., Maas, R. L., Shapiro, D. N. (1995) Pax3 inhibits myogenic differentiation of cultured myoblast cells J. Biol. Chem. 270,11719-11722[Abstract/Free Full Text]
  30. Bleul, C. C., Fuhlbrigge, R. C., Casasnovas, J. M., Aiuti, A., Springer, T. A. (1996) A highly efficacious lymphocyte chemoattractant, stromal cell-derived factor 1 (SDF-1) J. Exp. Med. 184,1101-1109[Abstract/Free Full Text]
  31. Tang, G., Charo, D. N., Wang, R., Charo, I. F., Messina, L. (2004) CCR2–/– knockout mice revascularize normally in response to severe hindlimb ischemia J. Vas. Surg. 40,786-795
  32. Heil, M., Ziegelhoeffer, T., Wagner, S., Fernandez, B., Helisch, A., Martin, S., Tribulova, S., Kuziel, W. A., Bachmann, G., Schaper, W. (2004) Collateral artery growth (arteriogenesis) after experimental arterial occlusion is impaired in mice lacking CC-chemokine receptor-2 Circ. Res. 94,671-677[Abstract/Free Full Text]
  33. Contreras-Shannon, V., Ochoa, O., Reyes-Reyna, S. M., Sun, D., Michalek, J. M., Kuziel, W. A., McManus, L. M., Shireman, P. K. (2006) Fat accumulation with altered inflammation and regeneration in skeletal muscle of CCR2 –/– mice following ischemic injury Am. J. Physiol. Cell Physiol. Epub ahead of print
  34. Schecter, A. D., Berman, A. B., Yi, L., Ma, H., Daly, C. M., Soejima, K., Rollins, B. J., Charo, I. F., Taubman, M. B. (2004) MCP-1-dependent signaling in CCR2(–/–) aortic smooth muscle cells J. Leukoc. Biol. 75,1079-1085[Abstract/Free Full Text]
  35. Koth, L. L., Rodriguez, M. W., Bernstein, X. L., Chan, S., Huang, X., Charo, I. F., Rollins, B. J., Erle, D. J. (2004) Aspergillus antigen induces robust Th2 cytokine production, inflammation, airway hyperreactivity and fibrosis in the absence of MCP-1 or CCR2 Respir. Res. 5,12[CrossRef][Medline]
  36. Kim, W. J., Chereshnev, I., Gazdoiu, M., Fallon, J. T., Rollins, B. J., Taubman, M. B. (2003) MCP-1 deficiency is associated with reduced intimal hyperplasia after arterial injury Biochem. Biophys. Res. Commun. 310,936-942[CrossRef][Medline]
  37. Dewald, O., Zymek, P., Winkelmann, K., Koerting, A., Ren, G., Abou-Khamis, T., Michael, L. H., Rollins, B. J., Entman, M. L., Frangogiannis, N. G. (2005) CCL2/monocyte chemoattractant protein-1 regulates inflammatory responses critical to healing myocardial infarcts Circ. Res. 96,881-889[Abstract/Free Full Text]
  38. Li, P., Garcia, G. E., Xia, Y., Wu, W., Gersch, C., Park, P. W., Truong, L., Wilson, C. B., Johnson, R., Feng, L. (2005) Blocking of monocyte chemoattractant protein-1 during tubulointerstitial nephritis resulted in delayed neutrophil clearance Am. J. Pathol. 167,637-649[Abstract/Free Full Text]
  39. Jinnouchi, K., Terasaki, Y., Fujiyama, S., Tomita, K., Kuziel, W. A., Maeda, N., Takahashi, K., Takeya, M. (2003) Impaired hepatic granuloma formation in mice deficient in C-C chemokine receptor 2 J. Pathol. 200,406-416[CrossRef][Medline]
  40. Kuziel, W. A., Morgan, S. J., Dawson, T. C., Griffin, S., Smithies, O., Ley, K., Maeda, N. (1997) Severe reduction in leukocyte adhesion and monocyte extravasation in mice deficient in CC chemokine receptor 2 Proc. Natl. Acad. Sci. USA 94,12053-12058[Abstract/Free Full Text]
  41. Ferreira, A. M., Rollins, B. J., Faunce, D. E., Burns, A. L., Zhu, X., Dipietro, L. A. (2005) The effect of MCP-1 depletion on chemokine and chemokine-related gene expression: evidence for a complex network in acute inflammation Cytokine 30,64-71[CrossRef][Medline]
  42. Amano, H., Morimoto, K., Senba, M., Wang, H., Ishida, Y., Kumatori, A., Yoshimine, H., Oishi, K., Mukaida, N., Nagatake, T. (2004) Essential contribution of monocyte chemoattractant protein-1/C-C chemokine ligand-2 to resolution and repair processes in acute bacterial pneumonia J. Immunol. 172,398-409[Abstract/Free Full Text]
  43. Toumi, H., Best, T. M. (2003) The inflammatory response: friend or enemy for muscle injury? Br. J. Sports Med. 37,284-286[Free Full Text]
  44. Teixeira, C. F., Zamuner, S. R., Zuliani, J. P., Fernandes, C. M., Cruz-Hofling, M. A., Fernandes, I., Chaves, F., Gutierrez, J. M. (2003) Neutrophils do not contribute to local tissue damage, but play a key role in skeletal muscle regeneration, in mice injected with Bothrops asper snake venom Muscle Nerve 28,449-459[CrossRef][Medline]
  45. Teixeira, C. F., Chaves, F., Zamuner, S. R., Fernandes, C. M., Zuliani, J. P., Cruz-Hofling, M. A., Fernandes, I., Gutierrez, J. M. (2005) Effects of neutrophil depletion in the local pathological alterations and muscle regeneration in mice injected with Bothrops jararaca snake venom Int. J. Exp. Pathol. 86,107-115[CrossRef][Medline]
  46. Nguyen, H. X., Lusis, A. J., Tidball, J. G. (2005) Null mutation of myeloperoxidase in mice prevents mechanical activation of neutrophil lysis of muscle cell membranes in vitro and in vivo J. Physiol. 565,403-413[Abstract/Free Full Text]
  47. Maus, U., von Grote, K., Kuziel, W. A., Mack, M., Miller, E. J., Cihak, J., Stangassinger, M., Maus, R., Schlondorff, D., Seeger, W., Lohmeyer, J. (2002) The role of CC chemokine receptor 2 in alveolar monocyte and neutrophil immigration in intact mice Am. J. Respir. Crit. Care Med. 166,268-273[Abstract/Free Full Text]
  48. DiTirro, J., Rhoades, E. R., Roberts, A. D., Burke, J. M., Mukasa, A., Cooper, A. M., Frank, A. A., Born, W. K., Orme, I. M. (1998) Disruption of the cellular inflammatory response to Listeria monocytogenes infection in mice with disruptions in targeted genes Infect. Immun. 66,2284-2289[Abstract/Free Full Text]
  49. Huang, D. R., Wang, J., Kivisakk, P., Rollins, B. J., Ransohoff, R. M. (2001) Absence of monocyte chemoattractant protein 1 in mice leads to decreased local macrophage recruitment and antigen-specific T helper cell type 1 immune response in experimental autoimmune encephalomyelitis J. Exp. Med. 193,713-726[Abstract/Free Full Text]
  50. Grounds, M. D. (1998) Age-associated changes in the response of skeletal muscle cells to exercise and regeneration Ann. N. Y. Acad. Sci. 854,78-91[Abstract/Free Full Text]
  51. Carlson, B. M., Faulkner, J. A. (1983) The regeneration of skeletal muscle fibers following injury: a review Med. Sci. Sports Exerc. 15,187-198
  52. Baker, W., Schneider, B. A., Kulkarni, A., Sloan, G., Schaub, R., Sypek, J., Cannon, J. G. (2004) P-selectin inhibition suppresses muscle regeneration following injury J. Leukoc. Biol. 76,352-358[Abstract/Free Full Text]
  53. Egami, K., Murohara, T., Aoki, M., Matsuishi, T. (2006) Ischemia-induced angiogenesis: role of inflammatory response mediated by P-selectin J. Leukoc. Biol. 79,971-976[Abstract/Free Full Text]
  54. Gosselin, L. E., McCormick, K. M. (2004) Targeting the immune system to improve ventilatory function in muscular dystrophy Med. Sci. Sports Exerc. 36,44-51
  55. Cantini, M., Carraro, U. (1995) Macrophage-released factor stimulates selectively myogenic cells in primary muscle culture J. Neuropathol. Exp. Neurol. 54,121-128[Medline]
  56. Cantini, M., Massimino, M. L., Bruson, A., Catani, C., Dalla Libera, L., Carraro, U. (1994) Macrophages regulate proliferation and differentiation of satellite cells Biochem. Biophys. Res. Commun. 202,1688-1696[CrossRef][Medline]
  57. Massimino, M. L., Rapizzi, E., Cantini, M., Libera, L. D., Mazzoleni, F., Arslan, P., Carraro, U. (1997) ED2+ macrophages increase selectively myoblast proliferation in muscle cultures Biochem. Biophys. Res. Commun. 235,754-759[CrossRef][Medline]
  58. Bartoli, C., Civatte, M., Pellissier, J. F., Figarella-Branger, D. (2001) CCR2A and CCR2B, the two isoforms of the monocyte chemoattractant protein-1 receptor, are up-regulated and expressed by different cell subsets in idiopathic inflammatory myopathies Acta Neuropathol. (Berl.) 102,385-392[Medline]
  59. Chazaud, B., Sonnet, C., Lafuste, P., Bassez, G., Rimaniol, A. C., Poron, F., Authier, F. J., Dreyfus, P. A., Gherardi, R. K. (2003) Satellite cells attract monocytes and use macrophages as a support to escape apoptosis and enhance muscle growth J. Cell Biol. 163,1133-1143[Abstract/Free Full Text]
  60. Warren, G. L., Hulderman, T., Mishra, D., Gao, X., Millecchia, L., O’Farrell, L., Kuziel, W. A., Simeonova, P. P. (2005) Chemokine receptor CCR2 involvement in skeletal muscle regeneration FASEB J. 19,413-415[Abstract/Free Full Text]
  61. Li, W. C., Yu, W. Y., Quinlan, J. M., Burke, Z. D., Tosh, D. (2005) The molecular basis of transdifferentiation J. Cell. Mol. Med. 9,569-582[Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
S. C. Bryer, G. Fantuzzi, N. Van Rooijen, and T. J. Koh
Urokinase-Type Plasminogen Activator Plays Essential Roles in Macrophage Chemotaxis and Skeletal Muscle Regeneration
J. Immunol., January 15, 2008; 180(2): 117