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J Appl Physiol 95: 969-976, 2003. First published May 2, 2003; doi:10.1152/japplphysiol.00005.2003
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M1/70 attenuates blood-borne neutrophil oxidants, activation, and myofiber damage following stretch injury

S. Brickson,1 L. L. Ji,1 K. Schell,2 R. Olabisi,5 B. St. Pierre Schneider,3 and T. M. Best1,4,5,6

1Department of Kinesiology and 3School of Nursing, University of Wisconsin; Departments of 4Orthopedic Surgery, 5Biomedical Engineering, and 6Family Medicine, University of Wisconsin Medical School, Madison 53706; and 2Flow Cytometry Facility, University of Wisconsin Comprehensive Cancer Center, Madison, Wisconsin 53792

Submitted 6 January 2003 ; accepted in final form 30 April 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 ACKNOWLEDGMENTS
 REFERENCES
 
The purpose of this study was to determine the role of the CD11b-dependent respiratory burst in neutrophil oxidant generation and activation, interleukin-8 (IL-8) production, and myofiber damage after muscle stretch injury by using the monoclonal antibody M1/70 to block this pathway. Twelve male New Zealand White rabbits were randomly assigned to a treatment group: M1/70 (n = 6), IgG isotype control (n = 3), or saline control (n = 3). After intravenous injection of the assigned agent under gas anesthesia, a standardized single-stretch injury was created in the right tibialis anterior, whereas the left tibialis anterior underwent a sham surgery. Blood-borne neutrophil oxidant generation and CD11b receptor density and plasma IL-8 levels were measured pre- and 24 h postinjury. Damage was assessed histologically at the hematoma site by counting torn myofibers. M1/70 group demonstrated decreased blood-borne neutrophil oxidant generation (P < 0.05) and CD11b receptor density (P < 0.05), an increase in plasma IL-8 concentration (P < 0.01), and less torn myofibers (P < 0.01) compared with IgG isotype or saline control groups. These data indicate that 1) CD11b-dependent respiratory burst is a major source of oxidants produced by the neutrophil, and that treatment with M1/70 2) attenuates neutrophil activation status, 3) increases plasma IL-8 concentration, and 4) minimizes myofiber damage 24 h postmuscle stretch injury.

eccentric contraction; chemokine; respiratory burst; CD11b

NEUTROPHILS ARE THE FIRST line of defense after acute muscle injury. Acute muscle injury encompasses exposure to extreme temperatures, ischemia, or trauma, such as stretch injury that results in degeneration, inflammation, regeneration, or fibrosis (23, 32). Neutrophils may elicit unwanted tissue destruction through the release of oxidants and proteolytic enzymes (17, 27, 37). Whereas it has been established that neutrophils cause damage in cardiac and skeletal muscle after ischemia-reperfusion (16, 25, 34), this has not been confirmed after single-stretch injury. Stretch injury occurs when the load placed on the muscle is greater than the tension generated by the cross bridges, thereby causing the muscle to lengthen, despite contact between the myosin and actin filaments. One obstacle in evaluating myofiber damage after stretch injury is the lack of a gold standard for measuring damage. Histological verification is one of the most common markers, as it allows visualization of morphological changes that can be appreciated quantitatively (6). The role that neutrophils and oxidants play after muscle stretch injury is a clinically relevant question, as stretch injuries have been identified as the most common injury sustained in competitive athletics (54) and are associated with a high rate of morbidity (8).

Recently, the role of neutrophil-derived oxidants as immunoregulatory substances has also received considerable attention (2, 39, 42). Oxidants are signaling intermediates capable of modulating inflammation by promoting chemotaxis, facilitating migration by upregulating endothelial adhesion molecules (46), enhancing neutrophil activation by upregulating CD11b receptor density (10, 26, 33), and inducing the production of IL-8 (28, 41). Accordingly, oxidants may play a role in myofiber damage, both directly and indirectly.

Studies thus far have noted the abundance of neutrophils 24 h postinjury to correspond with peak oxidant production (12) and further morphological damage in stretch-injured skeletal muscle (52), but there is no direct evidence to indicate causation. Other investigators have administered antioxidants before repetitive eccentric contractions to alter redox status and determine the downstream effects on myofiber damage (57), but the role of oxidants as signaling intermediates was not evaluated (62). As antioxidant supplementation is not specific for leukocyte-derived oxidants, we chose to use the monoclonal antibody M1/70 to prevent oxidant production through the respiratory burst in neutrophils and macrophages. Although neutrophils and macrophages are capable of undergoing respiratory burst, superoxide contribution by macrophages is negligible compared with that by neutrophils, which possess a much greater protein content of the NADPH oxidase enzyme (61). M1/70 blocks the iC3b domain of the {beta}2-integrin CD11b, which is responsible for phagocytosis, the respiratory burst, and degranulation and does not directly interfere with endothelial adhesion and migration in vitro (18, 21, 50).

One way in which neutrophil-derived oxidants may participate in autocrine feed-forward signaling is by enhancing CD11b expression, a well-established marker of neutrophil activation (35). CD11b is one of three {beta}2-integrin cell surface receptors expressed on inflammatory cells (4). In the resting state, only 10% of CD11b molecules are constitutively expressed on neutrophils and exist in a low-avidity state (18). Translocation of CD11b from intracellular granules to the cell surface requires activation by signals, such as oxidants (3). Increased expression of CD11b receptors can markedly enhance the ability of neutrophils to adhere to endothelium and to initiate tissue injury through the release of oxidants (15, 53). Thus neutrophils may intensify their own recruitment, oxidant-generating capacity, and potential for damage through feed-forward signaling by oxidants (2, 56).

In addition to activating CD11b-receptor expression, oxidants may augment the neutrophilic response by signaling transcription of interleukin (IL)-8 through the redox-sensitive transcription factor NF-{kappa}B (28, 41). IL-8 is a chemoattractant cytokine secreted by a wide variety of cells, including neutrophils and endothelial cells, although its actions are highly selective for neutrophils (5). Specifically, IL-8 promotes a cascade of proinflammatory events, including neutrophil chemotaxis, priming for superoxide release, upregulation of CD11b adhesion molecules, and {beta}2-independent migration (20, 38, 60). IL-8 may also promote oxidant production by activating second messengers that trigger the respiratory burst and degranulation (28, 30). Neutralizing antibodies against IL-8 in vivo has been successful in reducing oxidant production and has proven beneficial in attenuating neutrophil-mediated tissue injury in several inflammatory conditions (11, 13, 14, 24).

Oxidants may be important regulators of the inflammatory response and, therefore, a target for treatment interventions. Presently, there are no published investigations that have directly explored the role of oxidants in regulating neutrophil activation status, IL-8 secretion, or myofiber damage after muscle stretch injury. Therefore, this study was designed to determine the contribution of oxidants generated through the CD11b-dependent respiratory burst to 1) the overall level of blood-borne neutrophil oxidant generation, the downstream effect of M1/70 on 2) neutrophil activation, as measured by CD11b receptor density, 3) plasma IL-8 concentrations, and 4)myofiber damage in response to stretch injury by using the anti-CD11b monoclonal antibody M1/70.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 DISCLOSURES
 ACKNOWLEDGMENTS
 REFERENCES
 
Experimental Procedure

Animal care. Twelve male New Zealand White rabbits (Covance Laboratories, Madison, WI; weight 2.7-3.0 kg) were housed individually and fed food and water ad libitum in a temperature-controlled room set at 22°C on a 12:12-h light-dark cycle. All experiments were conducted after Institutional Animal Care and Use Committee approval. Experiments were conducted after a 48-h acclimation period to the housing conditions for the animals.

Antibody administration. Monoclonal rat anti-mouse antibody M1/70 was purified (Harlan Bioproducts, Madison, WI) from the M1/70.15.1.HL subcloned line (American Type Culture Collection) with permission from Dr. Daniel I. Simon (Department of Medicine, Harvard Medical School). M1/70 was sterile filtered through a 0.2-µm filter and obtained at a concentration of 2.15 mg/ml and endotoxin level of 4.2 endotoxin units/mg. The antibody was stored at -70°C in 1-ml aliquots. M1/70 was thawed and administered (1 mg/kg) via inner ear vein injection 2 h before injury (n = 6). The dose and frequency of administration were chosen based on previous reports (47, 49), and pilot data were collected on four animals in this laboratory. In the same fashion, the isotype control group (n = 3) received purified no-azide, low-endotoxin rat IgG2b,{kappa} monoclonal immunoglobulin (PharMingen, San Diego, CA) (1 mg/kg) 2 h before injury to exclude nonspecific antibody effects. The saline control group (n = 3) received 3.0 ml NaCl (0.9%) 2 h before injury to exclude the effects of protein injection. Rabbits were randomly assigned to one of the three treatment groups.

Isokinetic test system and injury protocol. Our laboratory's isokinetic injury and joint torque evaluation system has been previously described and validated (7, 9). All muscle injuries, blood sampling, and tissue harvesting were carried out under gas anesthesia by using 3-5% isoflurane (Isoflo, Abbott Laboratories, North Chicago, IL) and oxygen at 1.5-2 l/min. No analgesia was given pre- or postoperatively. A 6-mm incision was made over the dorsum of the right foot just distal to the ankle joint to isolate the tibialis anterior (TA) tendon. Normal saline solution (0.9%, room temperature) was used to maintain tissue hydration. To stimulate the TA, two Kendall Q-Trace Gold 5500 tin self-adhesive Ag-AgCl electrodes (Ludlow LP, Chicopee, MA) were placed above the thigh proximal to the knee and laterally over the fibular head. The animal was placed supine in the test apparatus, and the right foot was secured with a Velcro strap to the footplate. The right TA muscle-tendon unit was shortened 1.4 cm by using a stainless steel roller-clamp system (9). With the muscle-tendon unit in the shortened position, the peroneal nerve was stimulated by using an arbitrary waveform generator (model 75, Wavetek, San Diego, CA), which provides a 50-Hz pulse rate, 0.5-ms pulse width, and 2.66-mA current output. Once TA tetany was achieved, the ankle was plantarflexed through a 90° arc at 450°/s. The torque-angular displacement-time behavior was recorded. A sham operation consisting of a skin incision was performed in the left foot. Skin incisions in the feet were closed with 4-0 Ethilon suture. The animals were returned to their individual cages after recovery from anesthesia and fed food and water ad libitum with unrestricted activity.

Blood sampling. Venous blood samples were taken from the inner ear vein of the anesthetized animals and collected in sterile sodium citrate or EDTA-treated vacuum tubes (Becton-Dickinson, San Jose, CA) immediately preceding injury (internal control). Samples were collected again 24-h postinjury immediately before tissue harvest. Sodium-citrate-treated whole blood aliquots for flow cytometry were kept at room temperature, and the procedure of cell preparation was started within 30 min of collection (43). EDTA-treated aliquots for IL-8 ELISA were maintained on ice and immediately centrifuged at 2,750 rpm for 10 min at 4°C to avoid cytokine synthesis or degradation in vitro. Samples were stored at -70°C.

Muscle dissection. After the blood draw 24-h postinjury, the entire TA muscle-tendon unit from both legs was surgically removed, freeze-clamped immediately in liquid nitrogen, and then stored at -70°C. Animals were euthanized with intravenous Beusthanasia (0.4 ml/kg) injected into an inner ear vein.

Flow Cytometry and ELISA

Neutrophil viability, circulating count, CD11b receptor density, and oxidant production. CD11b density was measured by using flow cytometry, as described by Serotec (Serotec, Raleigh, NC), with modifications taken from other protocols (36, 48). The respiratory burst was measured as described by Taieb et al. (53), with modifications according to Vuorte et al. (55). Sodium-citrate whole blood samples (600 µl) were washed with 10 ml of phosphate-buffered saline (125 mM NaCl, 8 mM Na2HPO2, 2 mM NaH2PO2, and 5 mM KCl) containing 20 mM glucose and 1% BSA (PGB) at pH 7.4 and centrifuged at 1,000 rpm for 5 min at 24°C, and the supernatant was discarded. The cells were resuspended in 1 ml PGB, and three 300-µl aliquots (samples A, B, and C) were prepared. Samples A and C served as controls. Undiluted primary antibody (20 µl), mouse anti-rabbit Mab.198 (Serotec), was added to cell suspensions A and B and incubated for 20 min at room temperature. Samples A, B, and C were then washed with 3 ml PGB and centrifuged at 1,000 rpm for 5 min at 24°C, and the supernatant was discarded. The secondary antibody (1 µl), goat F(ab')2 anti-mouse IgG (heavy and light chain)-R-phycoerythrin (PE) (undiluted; Serotec), was added to the cells in A and B and incubated at room temperature for 20 min. Stock 10x NH4Cl red blood cell lysing solution (1.5 M NH4CL, 100 mM NaHCO3, 1 mM EDTA) was diluted 1:10, and 3 ml were added to A, B, and C. After a 10-min incubation at room temperature, the samples were centrifuged at 1,000 rpm for 5 min at 24°C, and the supernatant was discarded. The cells from all three samples were washed with 3 ml PGB and centrifuged at 1,000 rpm for 5 min at 24°C, and the supernatant was discarded. Cells from A were resuspended in 300 µl of PGB with propidium iodide (PI; Sigma Chemical, St. Louis, MO) diluted 1:200 in PGB. The cells in B and C were resuspended in 700 µl of PGB and loaded with 10 µM 2'-7'-dichlorofluorescein (DCF)-diacetate (Molecular Probes, Eugene, OR) and incubated at 37°C for 15 min in a water-jacketed incubator (Forma Scientific). Samples B and C were centrifuged at 1,000 rpm for 5 min at 24°C, the supernatant was discarded, and the cells were resuspended in 300 µl of PGB with PI diluted 1:200 in PGB. Data were acquired by using a FACSCalibur flow cytometer (Beckton Dickinson). All fluorophores were excited at 488 nm laser and light. The mean channel fluorescence intensity (MFI) of PE-stained cells in A and B was measured in the fluorescence detector equipped with a 585/42-nm band-pass filter. The MFI of DCF in B and C was detected through a 530/30-nm band-pass filter on the basis of a minimum number of 100,000 cells collected and analyzed by using the software CellQuest (Beckton Dickinson) (Figs. 1). Viability was determined by staining with PI, which was collected through a 660-nm long-pass filter. Cells were analyzed by using CellQuest acquisition and analysis software (Becton Dickinson). Neutrophils were selected based on physical properties of size and complexity, and the percentage of circulating neutrophils per 105 events in the blood was measured. Only viable neutrophils were analyzed for CD11b receptor density and oxidant production. The CD11b receptor density of CD11b+ cells was calculated as the increase in PE MFI over background fluorescence (A/C) and expressed as the relative change from preinjury to 24 h postinjury. Oxidant production in CD11b+ cells was calculated as the increase in DCF MFI over background fluorescence (A/B) and expressed as the relative change from preinjury to 24 h postinjury.



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  		Fig. 1. A: representative flow cytometry data from an IgG-treated animal 24 h postinjury. Oxidant production was detected by the fluorescence of dichlorofluorescin (DCF)-diacetate (DCFDA) through fluorescence channel 1 (FL1). CD11b receptor density was detected by the fluorescence of phycoerythrin (PE) through fluorescence channel 2 (FL2). The mean fluorescent intensities (MFI) of DCF and PE in this animal were 58.37 and 136.86, respectively. B: representative flow cytometry data from a M1/70-treated animal 24 h postinjury. Oxidant production was detected by the fluorescence of DCFDA through FL1. CD11b receptor density was detected by the fluorescence of PE through FL2. The MFI values of DCF and PE in this animal were 37.89 and 30.40, respectively.

 

Plasma IL-8. Plasma samples were prepared according to methods described by Taieb et al. (53). Samples were stored at -70°C for no more than 1 mo before ELISA. Samples were centrifuged at 3,000 g at 4°C for 30 min. The plasma supernatants were diluted 1:2 and used for the determination of the IL-8 content, according to the manufacturer's instructions (PeliKine compact human IL-8 ELISA kit, Accurate Chemical and Scientific, Westbury, NY). Sensitivity of the assay was 0.09 ± 0.06 pg/ml. IL-8 concentration was determined by using a SPECTRAmax 340 microplate spectrophotometer (Molecular Devices, Sunnyvale, CA). Plates were washed by using a multiwash microplate washer (Tri-Continent Scientific, Grass Valley, CA). Data were analyzed by using SOFTmax Pro software (Molecular Devices), and plasma IL-8 concentration was expressed in picograms per milliliter.

Tissue Analyses

Histochemistry. Cross sections from the hematoma or corresponding muscle-tendon region of injured and uninjured TA muscles, respectively, were cut 10 µm in thickness at -20°C by using a cryostat (Leica Microsystems, Deerfield, IL). Sections were adhered to poly-L-lysine-coated slides (Sigma Chemical) and maintained in slide boxes at -70°C. Frozen muscle sections from injured muscles were air dried and then stained with hematoxylin and eosin stain. Quantification of torn fibers at the muscle-tendon junction was limited to the medial edge, as evidence of structural damage, and the hematoma was restricted to the medial 20% of the cross section, based on an average width of 1 cm (52). With the use of a x10 objective, two computer images of the medialmost edge, each ~0.5 mm2, were captured by using a Nikon light microscope (Fryer, Huntley, IL), Photometrics black-and-white videocamera (Tucson, AZ), the National Institutes of Health Image program (Bethesda, MD), and Macintosh G3 computer (Apple Computer, Cupertino, CA). These sections were quantified for the number of torn fibers per millimeter squared. Fibers that lacked distinct borders, were located on the outermost edge, or were not completely confined within the image were excluded from quantification.

Immunohistochemistry. Immunohistochemistry was employed to ensure that M1/70 did not block the binding site for Mab.198, the monoclonal antibody used to detect CD11b-receptor density through flow cytometry. Frozen muscle sections were air dried, fixed with acetone for 10 min, and air dried for 15 min. After washing with PBS, pH 7.6, for 8 min, sections were incubated in a humidified chamber with 2% BSA in PBS for 20 min. After rinsing in PBS, the primary antibodies were applied on separate sections and incubated for 2 h in a humidified chamber. The primary monoclonal antibodies included mouse anti-rabbit Mab.198 (undiluted; Serotec) and RPN3/57 (diluted 1:20; Serotec). Mab.198 recognizes the CD11b receptor present on neutrophils and other granulocytes, blood monocytes, macrophages, a subset of lymphocytes, and bone marrow cells (51). RPN3/57 recognizes uncharacterized antigens on neutrophils, T lymphocytes, thymocytes, and platelets (59). Of these cells, neutrophils are the most likely to be present in stretch-injured muscle (52). Our laboratory has previously demonstrated a lack of macrophages infiltrating stretch-injured muscle (52), indicating that infiltrating inflammatory cells should be both Mab.198+ and RPN3/57+. After a PBS rinse, biotinylated goat anti-mouse IgG F(ab')2 secondary antibody (diluted 1:200; Caltag Laboratories, Burlingame, CA) was applied for 30 min in a humidified chamber. The sections were washed in PBS, quenched with 0.3% hydrogen peroxide in methanol for 20 min, and washed again in PBS. The sections were incubated with Vectastain ABC peroxidase reagent kit (Vector Laboratories, Burlingame, CA) for 30 min and washed in PBS. The primary antibody was visualized by applying diaminobenzidine substrate solution with nickel chloride (Vector Laboratories) for 7 min. The substrate reaction was stopped by washing in distilled water for 10 min. Sections were dehydrated by 95 and 100% ethanol rinses and cleared by Citrisolv clearing agent (Fisher Scientific) for 3 min each. Coverslips were applied to sections by using the nonaqueous medium Cytoseal (VWR, Batavia, IL) and left to air-dry overnight. Each slide was coded to ensure that the identity of the cross section was not known during analysis. Sections were viewed with a Nikon light microscope (Fryer) and examined for dark purple-black structures, indicating primary antibody staining. Nonspecific binding of the primary antibody was tested with additional sections by omitting the primary antibodies.

The medial edge of all sections was evaluated for the presence of Mab.198+ and RPN3/57+ cells. With the use of a x10 objective, two computer images of the medial 20% of the cross section were selected that contained the greatest amount of Mab.198+ or RPN3/57+ inflammatory cells. The images were of similar light intensity (76-78). Each image was set at a threshold of 125 so that only cells that exhibited the darkest intensity were counted. The automatic particle counting function was employed to count the number of distinct Mab.198+ and RPN3/57+ cells with a minimum and maximum pixel size of 3 and 9,999, respectively. Distinct cells within fibers and interstitial spaces were included, but cells within blood vessels or nerve bundles were not counted. Diffuse staining was excluded from quantification. The total number of cells per section was expressed as number per millimeter squared. The correlation between the Mab.198+ and RPN3/57+ cells was calculated.

Statistical Analysis

Means ± SEs were calculated for all data sets. Data were analyzed with a one-way ANOVA with Fisher's protected least significant difference post hoc tests to determine the significance of differences among means. The number of circulating neutrophils, blood-borne neutrophil oxidant production, and CD11b receptor density were expressed as the relative change from preinjury to 24-h postinjury values. Preinjury and postinjury plasma IL-8 concentrations were compared among treatment groups. Also, the difference in the plasma IL-8 concentrations between the preinjury and the postinjury time points was compared among the three treatment groups. Means for torn fibers, Mab.198+, and RPN3/57+ cellular concentrations represent the difference between injured and uninjured torn fibers and cellular concentrations obtained from one section per muscles. The significance of differences among means for P < 0.05 was considered significant. All analyses were performed by using Minitab Statistical Software Release 12 (Mintab, State College, PA).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 DISCLOSURES
 ACKNOWLEDGMENTS
 REFERENCES
 
Neutrophil Viability and Circulating Count

Viability was at least 90%, as determined by staining with PI. The percentage of circulating neutrophils in response to injury was not impaired by the administration of the M1/70 antibody (P = 0.21) (Fig. 2).



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  		Fig. 2. The mean relative change in the no. of live circulating neutrophils measured in blood drawn preinjury and 24 h postinjury was calculated for the M1/70-, IgG-, and saline-treated groups. No difference was detected among the 3 treatment groups (P = 0.21). Values are means ± SE.

 

Oxidant Production

Blood-borne neutrophils from M1/70-treated animals demonstrated a 20.1% decrease in oxidant production between preinjury and 24 h postinjury compared with a 96.3 and 102% increase in oxidant production in the IgG- and saline-treated groups, respectively (P < 0.05) (Fig. 3).



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  		Fig. 3. The mean relative change in oxidant generation from live neutrophils measured in blood drawn preinjury and 24 h postinjury was calculated for the M1/70-, IgG-, and saline-treated groups. The M1/70 group demonstrated a relative decrease in oxidant generation (*P < 0.05). Values are means ± SE.

 

CD11b Receptor Density

Blood-borne neutrophils from M1/70-treated animals demonstrated a 30.6% decrease in CD11b receptor density between preinjury and 24 h postinjury compared with a 12.3 and 11.0% increase in CD11b receptor density in the IgG- and saline-treated groups, respectively (P < 0.05) (Fig. 4).



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  		Fig. 4. The mean relative change in CD11b receptor density from live neutrophils measured in blood drawn preinjury and 24 h postinjury was calculated for the M1/70-, IgG-, and saline-treated groups. The M1/70 group demonstrated a relative decrease in CD11b receptor density (*P < 0.05). Values represent means ± SE.

 

Plasma IL-8

Baseline preinjury plasma IL-8 concentrations were similar among the three treatment groups (P = 0.35). Plasma IL-8 concentrations 24 h postinjury were increased in the M1/70-treated animals compared with the IgG- and saline-treated groups (P < 0.01) (Fig. 5). The M1/70 group showed an increase of 5.33 ± 1.94 pg/ml from preinjury to 24 h postinjury (P < 0.05) compared with a decrease of 3.64 ± 2.41 and 3.10 ± 0.24 pg/ml in the IgG and saline control groups, respectively.



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  		Fig. 5. Plasma interleukin (IL)-8 concentrations were measured in animals injected with M1/70, IgG, or saline before injury. There were no differences in IL-8 levels among the 3 treatment groups before injury (P = 0.35). The M1/70 group demonstrated a greater increase in IL-8 concentrations compared with the IgG and saline control groups 24 h postinjury (+P < 0.01) and was greater than M1/70 preinjury levels (*P < 0.05). Values are means ± SE.

 

Histological Characteristics

A hematoma near the muscle-tendon junction was visible for all stretch-injured TA muscles. Hematoxylin-and-eosin-stained sections of injured TA from animals treated with M1/70 (Fig. 6A) or IgG (Fig. 6B) depict these damage characteristics. The number of torn myofibers per millimeter squared at the hematoma site for the M1/70, IgG, and saline treatment groups was 3.32 ± 1.49, 16.7 ± 2.71, and 10.7 ± 2.31, respectively. M1/70-treated animals demonstrated less torn myofibers 24 h postinjury compared with the other treatment groups (P < 0.01) (Fig. 7).



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  		Fig. 6. Medial aspect of the muscle-tendon junction from an injured TA taken from a M1/70-treated (A) and an IgG-treated (B) animal 24 h postinjury stained with hematoxylin and eosin. Nos. represent torn fibers counted in that section.

 


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  		Fig. 7. The means ± SE of torn myofibers per mm2 are presented for the 3 different treatment groups. Data represent torn myofibers counted on 1 section from each animal. *P < 0.01, M1/70 vs. IgG or saline control groups.

 

Immunohistochemistry

The Pearson correlation coefficient for Mab.198+ and RPN3/57+ cells was 0.901 (P < 0.01). A difference in Mab.198+ and RPN3/57+ cellular concentrations from injured minus uninjured TAs was detected among the three treatment groups. The M1/70 group exhibited 17.0 ± 11 Mab.198+ cells/mm2 compared with 129 ± 47 and 105 ± 41 cells/mm2 for the IgG and saline control groups, respectively (P < 0.05). Similarly, the M1/70 group exhibited 12.0 ± 7 RPN3/57+ cells/mm2 compared with 123 ± 22 and 123 ± 36 cells/mm2 for the IgG and saline control groups, respectively (P < 0.01).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
DISCLOSURES
 ACKNOWLEDGMENTS
 REFERENCES
 
The role of oxidants as potential immunoregulators of inflammation has been a topic of recent discussion (42). Oxidants can signal events that may augment the capacity of neutrophils to create excessive damage, specifically CD11b activation and increased IL-8 levels (45, 53). We selected the monoclonal antibody M1/70 to block oxidant production through the CD11b-dependent respiratory burst and demonstrated this pathway to be a major source of oxidants produced by blood-borne neutrophils. Treatment with M1/70 decreased neutrophil activation status, increased plasma IL-8 concentration, and minimized myofiber damage 24 h postmuscle stretch injury.

The decrease in blood-borne neutrophil oxidant production after M1/70 treatment in the present study indicates that the CD11b-dependent respiratory burst pathway accounts for a substantial part of total neutrophil oxidant production. Therefore, the CD11b-dependent respiratory burst may be a more significant source of oxidants in our paradigm of single-stretch muscle injury than other sources of oxidants not blocked by M1/70, such as the lipooxygenase and cyclooxygenase pathways (29), or the respiratory burst by stimulation of receptors other than CD11b (19, 22). Thus the CD11b receptor may be an effective target for minimizing oxidant production after stretch injury. Current treatment strategies include primarily NSAIDs, which interfere with the cyclooxygenase pathway (1, 40).

We have demonstrated that the M1/70 antibody was effective in attenuating the increase in blood-borne neutrophil CD11b receptor density after muscle stretch injury. Subsequently, neutrophils may have less capability to adhere to the endothelium and infiltrate the injured muscle (15, 53). We postulate that the M1/70-induced decrease in CD11b receptor density was responsible for the significant reduction in Mab.198+ and RPN3/57+ cellular infiltration in stretch-injured muscle, and that lack of infiltration resulted in less myofiber damage. Our findings are similar to other in vivo studies in which M1/70 was administered to protect against vascular injury and was reported to decrease neutrophil adhesion (58) and macrophage infiltration (49). Despite in vitro data demonstrating non-anti-adhesive properties of M1/70 (18, 21, 50) and immunohistochemical findings in this study demonstrating that M1/70 does not interfere with binding of the anti-CD11b antibody Mab.198, we cannot definitively rule out the possibility of steric hindrance of M1/70 with the adhesive domain of CD11b as the cause for decreased neutrophil infiltration. Despite this limitation, this study is the first, to our knowledge, to demonstrate a direct link between neutrophils and myofiber damage.

Yet another way in which oxidants can modulate the inflammatory is through the induction of IL-8, which has been directly linked to promoting damage in other inflammatory models (11, 13, 24, 31). Neutrophils constitutively produce low levels of IL-8 mRNA and protein and require stimulation to produce detectable amounts of the protein (2). This led us to hypothesize that oxidants produced by neutrophils after muscle stretch injury would elevate plasma IL-8 levels similar to values after exercise (44) and that M1/70 treatment would result in comparably lower plasma IL-8 levels by blocking oxidants generated through the respiratory burst. However, we unexpectedly observed a decrease in plasma IL-8 levels in the IgG and saline control groups 24 h poststretch injury compared with preinjury levels. One possible explanation for this observation is that IL-8 produced in response to injury in the IgG and saline control groups had already bound to its receptor by 24 h, and, therefore, plasma IL-8 levels in the control groups were below preinjury constitutive levels.

The elevation of the plasma IL-8 level in the M1/70-treated group was also unanticipated. We speculate that the increase in the IL-8 level in the M1/70 group was a compensatory effort by other cells to increase IL-8-mediated intravascular neutrophil activation in the M1/70 group. It is also possible that the increase in plasma IL-8 in M1/70-treated animals was an attempt to elicit {beta}2-independent migration of neutrophils into injured tissue (38). Another possibility is that this antibody may have signaled the upregulation of IL-8 by binding to the CD11b receptor. There are in vitro data demonstrating that CD11b is the only one of the three related heterodimers able to upregulate IL-8 (56).

In summary, the CD11b-dependent respiratory burst is a major contributor to the overall level of oxidants produced by blood-borne neutrophils after muscle stretch injury. M1/70 diminished neutrophil activation, or CD11b receptor density, possibly by attenuating signaling by oxidants. However, other functions of the CD11b iC3b domain blocked by M1/70 may have played a role as well. Neutrophil infiltration and myofiber damage were attenuated in the M1/70 treatment group. Directions for future study would be to block NADPH oxidase enzyme and evaluate the impact on neutrophil activation status, infiltration, and myofiber damage. This would provide further evidence for the role of oxidants as immunoregulators and point to the respiratory burst as an effective target for treatment interventions.


   DISCLOSURES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
ACKNOWLEDGMENTS
 REFERENCES
 
The authors acknowledge the Virginia Horne Henry Distinguished Graduate Fellowship (to S. Brickson) and The Whitaker Foundation (to T. M. Best) for funding this study.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 ACKNOWLEDGMENTS
REFERENCES
 
We appreciate the statistical consultation provided by Dr. Murray Clayton, Professor of Statistics at the University of Wisconsin-Madison, and the generosity of Dr. Daniel I. Simon, Department of Medicine at the Harvard Medical School, for use of the M1/70 cell line.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. Brickson, Univ. of Wisconsin-Madison, 2000 Observatory Dr., Madison, WI 53706 (E-mail: sbrickson{at}education.wisc.edu).

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
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 ACKNOWLEDGMENTS
 REFERENCES
 

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