Download Neutrophils injure cultured skeletal myotubes

Am J Physiol Cell Physiol
281: C335–C341, 2001.
Neutrophils injure cultured skeletal myotubes
FRANCIS X. PIZZA,1 THOMAS J. MCLOUGHLIN,1 STEPHEN J. MCGREGOR,1
EDWARD P. CALOMENI,2 AND WILLIAM T. GUNNING2
1
Department of Kinesiology, The University of Toledo, Toledo 43606 and
2
Department of Pathology, The Medical College of Ohio, Toledo, Ohio 43614
Received 1 December 2000; accepted in final form 28 February 2001
chromium assay; electron microscopy; muscle injury; muscle
regeneration
SKELETAL MUSCLE INJURY,
induced by eccentric contractions (15), muscle trauma (22, 23), or the loading of
atrophic muscle (7, 25), is associated with increased
muscle neutrophil concentrations. The biological function of neutrophils in muscle injury and subsequent
regeneration, however, is unclear. Previous investigators have reported that muscle neutrophil concentrations are increased within 2 h postinjury and remain
above control concentrations for at least 48 h of recovery (7, 22, 25). During this time, the muscle undergoes
further degeneration (secondary injury) (5, 23, 29).
Because of the temporal relationship between muscle
neutrophil concentrations and secondary injury and
Address for reprint requests and other correspondence: F. X. Pizza,
Dept of Kinesiology, The Univ. of Toledo, 2801 W. Bancroft St.,
Toledo, OH 43606 (E mail: [email protected]).
http://www.ajpcell.org
because neutrophils can release potentially injurious
reactive oxygen and nitrogen intermediates (ROIs and
RNIs, respectively) and lysosomal enzymes (reviewed
in Refs. 4 and 28), neutrophils have been suggested to
exacerbate skeletal muscle damage in the hours to
days following the injury. Direct evidence supporting
this contention, however, is lacking.
Ascertaining the biological function of neutrophils in
vivo is difficult because neutrophils are present when
injured muscle shows signs of both injury and regeneration (7, 10, 22, 26, 27, 29). Tissue-cultured skeletal
myotubes offer a distinct advantage over in vivo experimentation because the ability of neutrophils to damage previously uninjured myotubes can be evaluated.
We reasoned that if neutrophils exacerbate skeletal
muscle injury, then they should be capable of damaging previously uninjured myotubes.
To determine whether neutrophils injure skeletal
muscle myotubes, we cultured human neutrophils in a
non-in vitro-stimulated and an in vitro-stimulated
state with human myotubes. Myotube injury was
quantitatively and qualitatively determined using a
cytotoxicity (51Cr) assay and electron microscopy
(transmission and scanning), respectively. For transmission electron microscopy, lanthanum was used as
an extracellular tracer to qualitatively determine
whether neutrophils caused myotube membrane rupture and/or increased myotube membrane permeability. We expected that neutrophils would cause myotube
membrane rupture and thus hypothesized that lanthanum would be predominantly found diffusely in the
cytoplasm of myotubes. This hypothesis was based on
the fact that neutrophils can release ROIs, RNIs, and
lysosomal enzymes that may cause myotube membrane rupture and necrosis (reviewed in Refs. 4 and
28). Because previous investigators have reported that
traumatic injury to cultured cells (e.g., neurons and
fibroblasts) increases the rate of endocytosis (3, 30), we
also postulated that neutrophils would increase myotube endocytosis. Support for this hypothesis would be
revealed if lanthanum was found localized to cytoplasmic vacuoles of myotubes when cultured with neutrophils.
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.
0363-6143/01 $5.00 Copyright © 2001 the American Physiological Society
C335
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.2 on June 16, 2017
Pizza, Francis X., Thomas J. McLoughlin, Stephen J.
McGregor, Edward P. Calomeni, and William T. Gunning. Neutrophils injure cultured skeletal myotubes. Am J
Physiol Cell Physiol 281: C335–C341, 2001.—The purpose of
the study was to test the hypothesis that neutrophils can
injure cultured skeletal myotubes. Human myotubes were
grown and then cultured with human blood neutrophils.
Myotube injury was quantitatively and qualitatively determined using a cytotoxicity (51Cr) assay and electron microscopy, respectively. For the 51Cr assay, neutrophils, under
non-in vitro-stimulated and N-formylmethionyl-leucyl-phenylalanine (FMLP)-stimulated conditions, were cultured
with myotubes at effector-to-target cell (E:T) ratios of 10, 30,
and 50 for 6 h. Statistical analyses revealed that myotube
injury was proportional to the E:T ratio and was greater in
FMLP-stimulated conditions relative to non-in vitro-stimulated conditions. Transmission electron microscopy, using
lanthanum as an extracellular tracer, revealed in cocultures
a diffuse appearance of lanthanum in the cytoplasm of myotubes and a localized appearance within cytoplasmic vacuoles of myotubes. These observations and their absence in
control cultures (myotubes only) suggest that neutrophils
caused membrane rupture and increased myotube endocytosis, respectively. Myotube membrane blebs were prevalent in
scanning and transmission electron micrographs of cultures
consisting of neutrophils and myotubes (E:T ratio of 5) and
were absent in control cultures. These data support the
hypothesis that neutrophils can injure skeletal myotubes in
vitro and may indicate that neutrophils exacerbate muscle
injury and/or delay muscle regeneration in vivo.
C336
NEUTROPHILS AND MUSCLE INJURY
MATERIALS AND METHODS
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.2 on June 16, 2017
Myoblasts. Human myoblasts were obtained from a 19-yrold female donor and were negative for mycoplasma, hepatitis B virus, hepatitis C virus, and human immunodeficiency
virus (Clonetics, San Diego, CA). Myoblasts were seeded at a
density of 10,000 cells/cm2 in either gelatin-coated microtiter
plate wells (24 well; Becton Dickinson, Lincoln Park, NJ) or
on gelatin-coated Thermanox coverslips (Fisher Scientific,
Pittsburgh, PA). Myoblast proliferation occurred in growth
medium (Clonetics) that was supplemented with 10 ng/ml
epidermal growth factor (EGF), 10 ␮g/ml insulin, 50 ␮g/ml
fetuin, 50 ␮g/ml bovine serum albumin, 375 ng/ml dexamethasone, 50 ␮g/ml gentamicin, and 50 ng/ml amphotericin-B.
Myoblasts were maintained in a humidified, 37°C, and 5%
CO2 atmosphere. At ⬃90% confluence, the growth medium
was exchanged for a differentiation medium that consisted of
Dulbecco’s modified Eagle’s medium (Sigma Chemical, St.
Louis, MO), 2 ng/ml EGF, and 2% heat-inactivated fetal
bovine serum (FBS). The differentiation medium was
changed every 2 days for 4 days. On the 5th day, myotubes
were cultured with neutrophils. Light microscopy observations of hematoxylin-and-eosin-stained cultures revealed numerous multinucleated myotubes.
Neutrophils. Human neutrophils were obtained from heparinized venous blood (50–70 ml) of healthy male volunteers
(n ⫽ 6) after obtaining verbal and written consent in accordance with institutional guidelines. Each subject’s neutrophils were used for both conditions (non-in vitro-stimulated
and FMLP-stimulated) and for all effector-to-target cell (E:T)
ratios.
Blood neutrophils were isolated from other cells using
density gradient centrifugation [neutrophil isolation medium
(NIM); Cardinal Assoc. Sante Fe, NM]. Briefly, blood was
layered on the NIM, centrifuged, and the polymorphonuclear
(PMN) cell layer was aspirated. Cells were then washed with
calcium- and magnesium-free Hanks’ balanced salt solution
(HBSS) and centrifuged. The remaining red blood cells were
lysed with an ammonium chloride solution and centrifuged,
and the PMN cells were washed again with HBSS. The PMN
cells were resuspended in Earle’s balanced salt solution
(EBSS) supplemented with 2% FBS and 400 ␮M of L-arginine
(coculture medium) to yield the desired E:T ratio. L-Arginine
was included in the coculture medium to provide a substrate
for nitric oxide synthesis and to enhance neutrophil degranulation (33). The final neutrophil preparation routinely
yielded ⬎98% neutrophils with cell viability ⬎98% as determined by trypan blue exclusion.
For the cytotoxicity assay, neutrophils in a non-in vitrostimulated and an in vitro-stimulated state were cultured
with myotubes at E:T ratios of 10, 30, and 50. In vitro
stimulation was accomplished by adding N-formyl-methionyl-leucyl-phenylalanine (FMLP; 2.0 ⫻ 10⫺6 M final concentration 0.02% vol/vol DMSO) to neutrophil suspensions just
before culturing them with myotubes. FMLP was used because it is a physiologically relevant stimulus that activates
neutrophils by receptor-mediated G protein signal transduction (33).
Cytotoxicity assay. Cytotoxicity against allogeneic myotubes was measured in a 6-h 51Cr release assay. Myotubes
were rinsed twice with EBSS and then labeled with 51Cr in
EBSS (Na251CrO4; 36 ␮Ci/well) for 1 h. After the wells were
washed twice with EBSS, neutrophils suspended in coculture
medium were added to the appropriate wells. To facilitate
neutrophil-myotube adhesion and to minimize neutrophil
aggregation, the plate was then centrifuged (50 g) for 1 min.
After centrifugation, the plate was incubated for 6 h in a
humidified, 37°C, 5% CO2 atmosphere.
Each plate contained wells for maximal 51Cr release, background 51Cr release, non-in vitro-stimulated neutrophils,
and FMLP-stimulated neutrophils. Maximal 51Cr release
was induced with a 4% Triton X-100 solution. Because preliminary experiments demonstrated that FMLP did not influence 51Cr release, background release wells contained only
coculture medium (data not reported). After the 6-h incubation, an aliquot was collected from each well and the radioactivity was counted using a gamma counter. An injury index
was calculated using the mean of triplicates using the following equation: injury index ⫽ [(e ⫺ b)/(m ⫺ b)], where e is
mean experimental release, b is mean background release,
and m is maximal release.
Electron microscopy. Transmission and scanning electron
microscopy were performed on several control cultures (myotubes only) and on cultures containing both neutrophils (nonin vitro-stimulated) and myotubes (E:T ratio of 5). Because of
the severity of the neutrophil-mediated myotube injury at
E:T ratios greater than five, an E:T ratio of five was used for
electron microscopy experiments to ensure that an adequate
quality and quantity of monolayers was obtained for analysis.
Using transmission electron microscopy, myotube membrane rupture and/or increased membrane permeability were
qualitatively determined utilizing lanthanum as an extracellular tracer. For transmission electron microscopy, cultures
were rinsed once with 0.1 M cacodylate buffer (pH 7.4) and
then fixed using a 3% glutaraldehyde solution that contained
1% lanthanum chloride. Cultures were kept at room temperature for 2 min and then transferred to 4°C for 2 h. After
primary fixation, cultures were washed with cacodylate. Secondary fixation took place at 4°C in S-collidine-buffered 2%
osmium tetroxide/1% lanthanum for 1 h followed by three
changes with S-collidine. To enhance the contrast of lanthanum against cellular structures, samples were not stained
with uranyl acetate and lead citrate. This practice facilitated
the determination of lanthanum within myotubes. After dehydration, monolayers were transferred to vertical molds
and infiltrated with 100% epoxy (Embed 812/Araldite). Following polymerization, thin silver-to-gold (70–90 nm) sections were cut on a Reichart OM-U3 ultramicrotome using a
diamond knife. Sections were viewed at 80 kV on a Phillips
CM10 transmission electron microscope.
For scanning electron microscopy, coverslips were washed
five times with EBSS without FBS (37°C) to remove soluble
proteins and detritus. Fixation was performed for 30 min
using sodium cacodylate-buffered 3% glutaraldehyde at 4°C.
Cultures were then washed with cacodylate buffer three
times before dehydration with graded changes of ethanol.
Following changes of absolute ethanol, coverslips were removed from culture dishes and critically point dried in a
Polaron E3000 (Watford) bomb. Coverslips were mounted to
stubs using silver paste and coated with gold using a Polaron
E5100 sputter coater. Samples were viewed at 5 kV on a
Cambridge Stereoscan 180 (Cambridge) scanning electron
microscope. All pictures were taken at a 65° tilt.
Statistical analyses. The injury index was statistically
analyzed using a repeated measures analysis of variance to
analyze the main effects and the interaction effect. The
Huynh-Feldt Epsilon was applied to degrees of freedom to
account for violation of the sphericity assumption. The Newman-Keuls post hoc test was used to locate the differences
between means when the observed F ratio was statistically
significant (P ⬍ 0.05).
NEUTROPHILS AND MUSCLE INJURY
C337
RESULTS
Fig. 1. Injury index (means ⫾ SE) determined by 51Cr release assay.
*Significant (P ⬍ 0.05) difference between effector-to-target cell (E:T)
ratios of 10 and 30 and between 10 and 50. #Significant difference
between E:T ratios of 30 and 50. $Significantly greater myotube
injury for N-formylmethionyl-leucyl-phenylalanine (FMLP)-stimulated conditions relative to non-in vitro-stimulated conditions across
all E:T ratios.
Fig. 2. Control culture (myotubes only). For all micrographs, the
electron-dense areas represent lanthanum, whereas visualization of
cellular structures is attributable to osmium tetroxide staining.
Samples were not stained with uranyl acetate or lead citrate. Lanthanum was present on the extracellular surface of myotube membranes (arrows) and was not found diffusely or localized in the
cytoplasm of myotubes. Magnification, ⫻7,500.
with lanthanum. In most cases, the lumina of the
larger vacuoles contained no internal structures visible
by electron microscopy. Small isolated lanthanum-containing vacuoles were also found near myotube membranes (Fig. 3). The second observed pattern of lanthanum’s appearance was one in which it was found
diffusely within myotubes (Figs. 4 and 5).
Myotube membrane blebs (bubble-like protrusions
on the plasma membrane) were frequently observed on
myotubes cultured with neutrophils (see Figs. 5, 6, and
8) and were absent in control cultures (Fig. 2). Most of
the membrane blebs were ruptured as indicated by the
diffuse appearance of lanthanum within membrane
blebs and the cytoplasm of myotubes (Figs. 4 and 5). At
some of the sites of membrane rupture (Fig. 4), we
observed neutrophils in close proximity to vacuoles
that presumably were contained within membrane
blebs before their rupture. Whether neutrophils caused
the membrane bleb rupture or the membrane rupture
resulted in the chemotaxis of neutrophils cannot be
determined from our observations. We also observed
intact membrane blebs and blebs that were presumably shed from myotubes (Fig. 6). Transmission electron micrographs revealed that membrane blebs (ruptured, intact, and shed blebs) contained vacuoles that
either were rimmed with or contained lanthanum
(Figs. 4–6). The presence of lanthanum within vacuoles of intact and shed membrane blebs suggests that
lanthanum was incorporated into membrane bleb
vacuoles before bleb rupture. The vacuoles in membrane blebs contained no visible cellular structures.
DISCUSSION
Neutrophils, the first inflammatory cell type to appear in injured muscle (7, 22, 23), have been suggested
to contribute to the exacerbation in skeletal muscle
injury in the hours to days following the injury. Neu-
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.2 on June 16, 2017
Cytotoxicity assay. Statistical analysis revealed a
significant main effect for E:T ratio and condition (nonin vitro-stimulated and FMLP-stimulated neutrophils)
with no interaction detected (Fig. 1). Post hoc analysis
revealed significant differences between E:T ratios of
10 and 30, 10 and 50, and 30 and 50. The injury index
was also significantly greater for the FMLP-stimulated
conditions relative to the non-in vitro-stimulated conditions across all E:T ratios.
To demonstrate that FMLP activated neutrophils,
we quantified superoxide anion production (O2⫺䡠) using
the cytochrome c assay (24) in a separate set of experiments. These experiments (n ⫽ 15) demonstrated that
FMLP resulted in a fourfold increase (P ⬍ 0.05) in
neutrophil-derived O2⫺䡠 (means ⫾ SE; 16.0 nmol 䡠 10
min⫺1 䡠 2 ⫻ 106 neutrophils⫺1 ⫾ 0.7) relative to non-in
vitro-stimulated neutrophils (3.7 nmol 䡠 10 min⫺1 䡠 2 ⫻
106 neutrophils⫺1 ⫾ 2.0).
Electron microscopy observations. In control cultures
(myotubes only), lanthanum was found on the extracellular surface of myotube membranes and was not
found in the cytoplasm of myotubes (Fig. 2). In contrast, cultures consisting of both neutrophils and myotubes showed two different patterns of lanthanum’s
appearance within myotubes.
The most frequently observed pattern was a localization of lanthanum to cytoplasmic vacuoles of myotubes
(Fig. 3). In most cases, these vacuoles consisted of a
larger vacuole with smaller vacuoles surrounding and
appearing to be associated with the larger vacuoles.
Some of the larger and smaller vacuoles contained
lanthanum in their lumen and others were rimmed
C338
NEUTROPHILS AND MUSCLE INJURY
trophils could contribute to muscle injury by damaging
uninjured areas of damaged fibers, damaging adjacent
uninjured fibers, and/or delaying muscle regeneration
by injuring muscle precursor cells and myotubes. Our
results provide support for this hypothesis by demonstrating that neutrophils injure cultured skeletal muscle myotubes. The myotube injury was proportional to
neutrophil number (E:T ratio) and to their state of
activation (non-in vitro-stimulated vs. FMLP-stimulated; Fig. 1). The neutrophil-mediated injury was con-
Fig. 4. Neutrophil and myotube culture. The diffuse presence of lanthanum in the cytoplasm of
myotubes (solid arrows) and remnants of a ruptured membrane bleb (broken arrows) indicate
myotube membrane rupture. Magnification,
⫻22,400.
firmed via transmission and scanning electron microscopy. The presence of lanthanum in the cytoplasm of
myotubes (Figs. 3–5) and myotube membrane blebs
(Figs. 4–6 and 8) in cultures consisting of both neutrophils and myotubes support our quantitative data. In
addition, lanthanum’s localized and diffuse appearance in the cytoplasm of myotubes when cultured with
neutrophils suggests that neutrophils are capable of
increasing myotube endocytosis and rupturing myotube membranes, respectively.
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.2 on June 16, 2017
Fig. 3. Neutrophil and myotube culture. The majority of lanthanum-containing cytoplasmic vacuoles consisted
of a larger vacuole that was surrounded by smaller vacuoles (solid arrows). The less frequently observed cytoplasmic vacuoles were small isolated
vacuoles near myotube membranes
(broken arrows). These vacuoles were
either rimmed with or contained lanthanum, and morphologically, they resemble endocytotic vesicles. Magnification, ⫻18,000.
NEUTROPHILS AND MUSCLE INJURY
C339
Our results represent the first report to provide
quantitative evidence supporting the contention that
neutrophils are capable of damaging previously uninjured myotubes (Fig. 1). The greater myotube injury in
the FMLP-stimulated condition relative to the non-in
vitro-stimulated condition is attributable to a greater
state of neutrophil activation. In separate experiments, we demonstrated that FMLP resulted in a fourfold increase in neutrophil-derived O2⫺䡠 relative to
non-in vitro-stimulated neutrophils. Previous investigators have reported similar effects of FMLP on neutrophil ROIs (reviewed in Ref. 4) and have also reported FMLP-mediated release of lysosomal enzymes
from neutrophil granules (33).
Myotube injury in the non-in vitro-stimulated condition is most likely the result of a basal state of neutrophil activation and/or an adhesion-dependent increase
in neutrophil activation. We and others have demon-
Fig. 6. Neutrophil-myotube culture. The shed membrane bleb (broken arrow) is intact and contains vacuoles that are either rimmed or
filled with lanthanum (solid arrow). Magnification, ⫻30,000.
Fig. 7. Neutrophil-myotube culture. The clathrin-coated pit on the
myotube membrane (arrow) is filled with lanthanum. Using a lower
magnification (⫻22,400), the structure adjacent to the clathrincoated pit is a membrane bleb (asterisk). Magnification, ⫻120,000.
strated that non-in vitro-stimulated blood neutrophils
produce low concentrations of ROIs in vitro (reviewed
in Ref. 4). Thus, in the non-in vitro-stimulated conditions, neutrophils were in a state of activation before
culturing them with myotubes. Because neutrophil adhesion to extracellular matrix proteins is known to
activate neutrophils (20), neutrophils likely became
further activated when they adhered to myotubes.
Our quantitative data are consistent with other in
vitro studies that have demonstrated that neutrophils
Fig. 8. Neutrophil-myotube culture. Myotube membrane bleb (broken arrow) and myotube membrane pits (solid arrows). The membrane pits are suggestive of endocytosis. Magnification, ⫻15,000.
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.2 on June 16, 2017
Fig. 5. Neutrophil-myotube culture. The myotube membrane bleb
(broken arrow) contains vacuoles that are either rimmed or filled
with lanthanum (solid arrow). Myotube membrane rupture is indicated by the diffuse presence of lanthanum in cytoplasm (arrowheads). An apparent endocytotic vesicle (asterisk) is shown near the
membrane bleb. Magnification, ⫻52,000.
C340
NEUTROPHILS AND MUSCLE INJURY
pletion, elevations in free cytoplasmic calcium concentrations, activation of calpains, altered thiol status,
and ROIs cause the formation of membrane blebs (9).
Fidzianska and Kaminska (6) reported plasma membrane blebs in skeletal muscle of newborn rats 24 h
after a myotoxin (bupivacaine) injection. Their plasma
membrane blebs were filled with vacuoles, some of
which contained presumed remnants of skeletal muscle organelles, an observation that is consistent with
apoptosis (31, 34). Our myotube membrane blebs also
contained vacuoles; however, these vacuoles contained
no visible cellular structures (Figs. 6–8). The organelle-free vacuoles in our membrane blebs is consistent with changes in cell morphology associated with
oncosis, a type of prelethal injury in which injured cells
swell before necrosis (16, 31). Pyknotic nuclei and apoptotic bodies were also reported at 24 h in bupivacaineinjected rats (6). However, we observed no electron
microscopy signs of neutrophil-mediated apoptosis of
myotubes. Thus our observations may indicate that the
myotube membrane blebs were the result of neutrophil-mediated oncosis.
The results from the present study demonstrate that
neutrophils can injure cultured skeletal muscle myotubes. However, the results may not be applicable to
the in vivo events associated with muscle injury for
several reasons. First, in vitro conditions cannot mimic
the complex interactions between the various cell types
that are present in injured muscle or the vascular
response to muscle injury. Second, our experiments
were performed on developing muscle and not adult
myofibers. The effect of neutrophils on myotubes may
be different from their effect on adult myofibers, since
it is likely that differences in muscle defense mechanisms exist between myotubes and myofibers. However, previous investigators have reported proliferation of muscle precursor cells and the formation of
myotubes at times when neutrophil concentrations are
elevated (10, 26, 27). Thus, based on our results, it is
conceivable that neutrophils could exacerbate muscle
injury and delay muscle regeneration in vivo by injuring muscle precursor cells and/or myotubes. If this
scenario is true, then strategies to ameliorate neutrophil-mediated myotube injury may be important in
myoblast transplantation and to enhance muscle regeneration following skeletal muscle injury. Further
work is needed to determine the mechanism for the
neutrophil-mediated myotube injury and whether similar mechanisms are operating in vivo.
We gratefully acknowledge Eleni Mylona and Susan Tsivitse for
their assistance.
This project was supported by the University of Toledo deArce
Memorial Research Endowment Fund.
REFERENCES
1. Bursztajn S and Libby P. Morphological changes in cultured
myotubes treated with agents that interfere with lysosomal
function. Cell Tissue Res 220: 573–588, 1981.
2. Eddleman CS, Ballinger ML, Smyers ME, Godell CW, Fishman HM, and Bittner GD. Repair of plasmalemmal lesions by
vesicles. Proc Natl Acad Sci USA 94: 4745–4750, 1997.
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.2 on June 16, 2017
injure cultured endothelial cells (8, 11, 17, 18, 32) or
cardiac myoblasts (3). Neutrophil-derived superoxide
anion, hydrogen peroxide, hydroxyl radical, hypochlorous acid, and lysosomal proteases have all been demonstrated to cause varying degrees of injury to cultured
endothelial cells (8, 11, 17, 18, 32). In addition, blocking neutrophil adhesion and ROI production using
anti-CD18 and intracellular adhesion molecule-1 antibodies has been reported to ameliorate neutrophilmediated injury to endothelial cells and cardiac myoblasts (3, 21). Future experiments are needed to
investigate whether similar mechanisms are operating
in our neutrophil and myotube skeletal muscle cultures.
Electron micrographs of neutrophil-myotube cultures revealed unexpected and novel observations. We
hypothesized that neutrophils would cause myotube
membrane rupture, and thus we postulated that lanthanum would predominantly be found diffusely in the
cytoplasm of myotubes. Although diffuse appearance of
lanthanum was observed (Figs. 4 and 5), lanthanum
was more frequently localized to cytoplasmic vacuoles
of myotubes when cultured with neutrophils (Fig. 3).
We interpret the localized appearance of lanthanum
to indicate neutrophil-mediated myotube endocytosis.
This interpretation is based on 1) the observation that
lanthanum was found in the lumen of cytoplasmic
vacuoles (Fig. 3) and in vacuoles of intact and shed
membrane blebs (Fig. 6), 2) the appearance of clathrincoated pits that contained lanthanum (Fig. 7), 3) pitted
regions on myotube membranes (Fig. 8), 4) an apparent
endocytotic lanthanum-containing vesicle near a membrane bleb (Fig. 5), and 5) the morphological similarities between our lanthanum-containing cytoplasmic
vacuoles and endocytotic vesicles previously reported
in cultured myotubes (1, 13).
Several possibilities exist to explain the postulated
neutrophil-mediated myotube endocytosis. One possibility is that, as a result of myotube injury, myotubes
were internalizing injured membrane components. Another possibility is that the increased endocytosis in
our injured myotubes may have been an attempt to
reseal the membrane. This possibility is based on the
work of previous investigators who have established
that cytoplasmic vesicles that fuse and reseal injured
membrane originate from endocytotic vesicles (2, 30).
Further investigation is needed to test these hypotheses.
Plasma membrane blebs have been reported in a
variety of cells and is considered a prominent feature of
cell injury regardless of its etiology (9, 12, 14, 31).
Membrane blebs have been reported in cells undergoing apoptosis or oncosis, and thus their mere formation
provides no insight on the type of cell injury (i.e.,
apoptosis or oncosis) (31). Membrane blebs can rupture
or be shed from the membrane and have been suggested to represent irreversible and reversible injury,
respectively (9, 12, 14). Membrane bleb formation appears to be the result of a disassociation of the plasma
membrane lipid bilayer from the cytoskeleton (19).
Previous investigators have postulated that ATP de-
NEUTROPHILS AND MUSCLE INJURY
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
activation in plasma membrane bleb formation during tert-butyl
hydroperoxide-induced rat hepatocyte injury. Gastroenterology
110: 1897–1904, 1996.
Nathan CF. Neutrophil activation on biological surfaces. J Clin
Invest 80: 1550–1560, 1987.
Ohno S and Malik AB. Polymorphonuclear leukocyte (PMN)
inhibitory factor prevents PMN-dependent endothelial cell injury by an anti-adhesive mechanism. J Cell Physiol 171: 212–
216, 1997.
Orimo S, Hiyamatu E, Arahata K, and Sugita H. Analysis of
inflammatory cells and complement C3 in bupivacaine-induced
myonecrosis. Muscle Nerve 14: 515–520, 1991.
Papadimitriou JM, Robertson TA, Mitchell CA, and
Grounds MD. The process of new plasmalemma formation in
focally injured skeletal muscle fibers. J Struct Biol 103: 124–
134, 1990.
Pizza FX, Cavender D, Stockard A, Baylies H, and Beighle
A. Anti-inflammatory doses of ibuprofen: effect on neutrophils
and exercise-induced muscle injury. Int J Sports Med 20: 98–
102, 1999.
Pizza FX, Hernandez IJ, and Tidball JG. Nitric oxide synthase inhibition reduces muscle inflammation and necrosis in
modified muscle use. J Leukoc Biol 64: 427–433, 1998.
Robertson TA, Grounds MD, Mitchell CA, and Papadimitriou JM. Fusion between myogenic cells in vivo: an ultrastructural study in regenerating murine skeletal muscle. J Struct Biol
105: 170–182, 1990.
Robertson TA, Papadimitriou JM, and Grounds MD. Fusion of myogenic cells to the newly sealed region of damaged
myofibres in skeletal muscle regeneration. Neuropathol Appl
Neurobiol 19: 350–358, 1993.
Rosen GM, Pou S, Ramos CL, Cohen MS, and Britigan BE.
Free radicals and phagocytic cells. FASEB J 9: 200–209, 1995.
Thompson JL, Balog EM, Fitts RH, and Riley DA. Five
myofibrillar lesion types in eccentrically challenged, unloaded
rat adductor lungus muscle—a test model. Anat Rec 254: 39–52,
1999.
Togo T, Alderton JM, Bi GQ, and Steinhardt RA. The
mechanism of facilitated cell membrane resealing. J Cell Sci
112: 719–731, 1999.
Trump BF, Berezesky IK, Chang SH, and Phelps PC. The
pathways of cell death: oncosis, apoptosis, and necrosis. Toxicol
Pathol 25: 82–88, 1997.
Weiss SJ, Yound J, LoBuglio AF, and Slivka A. Role of
hydrogen peroxide in neutrophil-mediated destruction of cultured endothelial cells. J Clin Invest 68: 714–721, 1981.
Wyatt TA, Lincoln M, and Pryzwansky KB. Regulation of
human neutrophil degranulation by LY-83583 and L-arginine:
role of cGMP-dependent protein kinase. Am J Physiol Cell
Physiol 265: C201–C211, 1993.
Wyllie AH, Kerr JF, and Currie AR. Cell death: the significance of apoptosis. Int Rev Cytol 68: 251–306, 1980.
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.2 on June 16, 2017
3. Entman ML, Youker K, Shoji T, Kukielka G, Shappell SB,
Taylor AA, and Smith CW. Neutrophil induced oxidative injury of cardiac myocytes. J Clin Invest 90: 1335–1345, 1992.
4. Fantone JC and Ward PA. Role of oxygen-derived free radicals
and metabolities in leukocytes-dependent inflammatory reactions. Am J Pathol 107: 397–418, 1982.
5. Faulkner JA and Brooks S. An in situ single skeletal muscle
model of contraction-induced injury: mechanistic interpretations. Basic Appl Myol 4: 17–23, 1994.
6. Fidzianska A and Kaminska A. Apoptosis: a basic pathological reaction of injured neonatal muscle. Pediatr Pathol 11: 421–
429, 1991.
7. Frenette J, Cai B, and Tidball JG. Complement activation
promotes muscle inflammation during modified muscle use.
Am J Pathol 156: 2103–2110, 2000.
8. Gannon DE, Varani J, Phan SH, Ward JH, Kaplan J, Till
GO, Simon RH, Ryan US, and Ward PA. Source of iron in
neutrophil-mediated killing of endothelial cells. Lab Invest 57:
37–44, 1987.
9. Gores GJ, Herman B, and Lemasters JJ. Plasma membrane
bleb formation and rupture: a common feature of hepatocellular
injury. Hepatology 11: 690–698, 1990.
10. Grounds MD, Garrett KL, Lai MC, Wright WE, and Beilharz MW. Identification of skeletal muscle precursor cells in
vivo by use of MyoD1 and myogenin probes. Cell Tissue Res 267:
99–104, 1992.
11. Hardy MM, Flickinger AG, Riley D, Weiss RH, and Ryan
US. Superoxide dismutase mimetics inhibit neutrophil-mediated human aortic endothelial cell injury in vitro. J Biol Chem
269: 18535–18540, 1994.
12. Herman B, Nieminen AL, Gores GJ, and Lemasters JJ.
Irreversible injury in anoxic hepatocytes precipitated by an
abrupt increase in plasma membrane permeability. FASEB J 2:
146–151, 1988.
13. Janeczko RA, Carriere RM, and Etlinger JD. Endocytosis,
proteolysis, and exocytosis of exogenous proteins by cultured
myotubes. J Biol Chem 260: 7051–7058, 1985.
14. Lemasters JJ, Ji S, Stemkowski CJ, and Thurman RG.
Hypoxic hepatocellular injury. Pharm Biochem Behav 18, Suppl
1: 455–459, 1983.
15. MacIntyre DL, Reid WD, Lyster DM, and McKenzie DC.
Different effects of strenuous exercise on the accumulation of
neutrophils in muscle in women and men. Eur J Appl Physiol 81:
47–53, 2000.
16. Majno G and Joris I. Apoptosis, oncosis, and necrosis. Am J
Pathol 146: 3–15, 1995.
17. Martin WJ. Neutrophils kill pulmonary endothelial cells by a
hydrogen-peroxide-dependent pathway. Am Rev Respir Dis 130:
209–213, 1984.
18. Miller RA and Britigan BE. Protease-cleaved iron-transferrin
augments oxidant-mediated endothelial cell injury via hydroxyl
radical formation. J Clin Invest 95: 2491–2500, 1995.
19. Miyoshi H, Umeshita K, Sakon M, Imajoh-Ohmi S, Fujitani
K, Gotoh M, Oiki E, Kambayashi J, and Monden M. Calpain
C341