Complement and myoblast transfer therapy: donor myoblast survival is enhanced following depletion of host complement C3 using cobra venom factor, but not in the absence of C5.
ABSTRACT Myoblast transfer therapy (MTT) is a potential cell therapy for myopathies such as Duchenne Muscular Dystrophy and involves the injection of cultured muscle precursor cells ('myoblasts') isolated from normal donor skeletal muscles into dystrophic host muscle. The failure of donor myoblast survival following MTT is widely accepted as being due to the immune response of the host. The role of complement as one possible mechanism for the initial, very rapid death of myoblasts following MTT was investigated. Donor male myoblasts were injected into the tibialis anterior (TA) muscles of female host mice that were: (i) untreated; (ii) depleted of C3 complement (24 h prior to MTT) using cobra venom factor (CVF); and/or (iii) deficient in C5 complement. Quantification of surviving male donor myoblast DNA was performed using the Y-chromosome specific (Y1) probe on slot blots for samples taken at 0 h, 1 h, 24 h, 1 week and 3 weeks after MTT. Peripheral depletion of C3 was confirmed using double immunodiffusion, and local depletion of C3 in host TA muscles was confirmed by immunostaining of muscle samples. Cobra venom factor treatment significantly increased the initial survival of donor myoblasts, but there was a marked decline in myoblast numbers after 1 h and little long-term benefit by 3 weeks. Strain specific variation in the immediate survival of donor male myoblasts following MTT in untreated C57BL/10Sn, DBA-1 and DBA-2 (C5-deficient) female hosts was observed. Cobra venom factor depletion of C3 increased initial donor male myoblast survival (approximately twofold at 0 h) in C57BL/10Sn and DBA-1 host mice and approximately threefold in DBA-2 hosts at 0 h and 1 h after MTT. The rapid and extensive number (approximately 90%) of donor male myoblasts in untreated DBA-2 mice (that lack C5) indicates that activation of the membrane attack complex (MAC) plays no role in this massive initial cell death. The observation that myoblast survival was increased in all mice treated with CVF suggests that CVF may indirectly enhance donor myoblast survival by a mechanism possibly involving activated C3 fragments.
- SourceAvailable from: PubMed Central[show abstract] [hide abstract]
ABSTRACT: C5-deficient mice differed from C5-sufficient mice both quantitatively and qualitatively in C5 protein, C5 mRNA, and the C5 gene. C5-deficient protein was present as decreased amounts of an unprocessed, single-chain precursor. C5-deficient mRNA was decreased in amount and present in two forms, the smaller of which was the same as the single form in normal cells. Nuclei from both normal and deficient cells contained the larger form of C5 mRNA, and C5-deficient DNA demonstrated differences from the normal pattern on Southern analysis for two restriction enzymes. These data suggest that the primary transcript of the C5-deficient gene is abnormal, retarding the processing of the C5 mRNA, and that the C5-deficient mRNA codes for an abnormal protein.Journal of Experimental Medicine 06/1987; 165(5):1442-7. · 13.21 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Identification of sex is often necessary to evaluate genetic or teratogenetic effects on embryonic development. A simple molecular technique to identify the sex of mouse embryos was studied using a Y chromosomal repetitive sequence (designated 145SC5). Since this technique does not require purification of DNA, it is particularly suitable for processing many embryos. Furthermore, 145SC5 detects 1% contamination of male DNA in a male-female DNA mixture. These results suggest that 145SC5 is a powerful molecular tool in a variety of studies on mouse development.Teratology 09/1988; 38(2):181-5.
- [show abstract] [hide abstract]
ABSTRACT: We have reassessed the effects of CVF administration on the humoral responses to two T-independent immunogens: DNP-Ficoll and DNP-Polyacrylamide. With high immunogen doses, little or no evidence of suppression was found. However, when the immunizing dose was reduced, suppression of both IgG and IgM responses became apparent. As indicated in a previous report, the immunosuppressive effect of CVF on T-dependent responses may result not only from C depletion but also from the generation of C cleavage products that may impair the auxiliary contribution of macrophages to the generation of these humoral responses. A similar mechanism may be applicable to the suppression of antibody production to DNP-Ficoll and DNP-PAA in view of recent reports showing a macrophage requirement for the response to these immunogens.The Journal of Immunology 12/1978; 121(5):2048-51. · 5.52 Impact Factor
Injection of cultured muscle precursor cells (‘myoblasts’)
isolated from normal donor muscles into dystrophic host
muscle is the basis of myoblast transfer therapy (MTT),
designed as a potential cell therapy for myopathies such as
Duchenne Muscular Dystrophy. The aim of this cell trans-
plantation strategy is to introduce many normal donor muscle
nuclei into dystrophic host muscle, thereby replacing the
missing gene for dystrophin and restoring muscle function
and integrity. Ideally, transplantation is followed by donor
myoblast replication, migration from the injection site, fusion
with dystrophic host myofibres and successful long-term
expression of a functional dystrophin molecule.
The major obstacle to the success of MTT is the extremely
rapid and massive death of donor myoblasts following
injection into the host muscle.1–6Quantification of DNA from
the nuclei of surviving male donor myoblasts using a mouse
Y-chromosome specific (Y1) probe4,7,8has shown that
approximately 85% of the injected donor male cells are killed
within minutes, irrespective of whether the female muscle
host environment is normal (C57BL/10Sn) or dystrophic
(mdx).9This is in marked contrast to the excellent long-term
survival (up to one year) of donor myoblasts using intact or
sliced muscle grafts from equivalent male donor mice
implanted into dystrophic female hosts.10–13
Recent studies in our laboratory14strongly support the
idea that tissue culture conditions per se may be largely
responsible for the adverse host immune response to isolated
injected donor myoblasts,15as cell isolation procedures
and/or subsequent culture conditions can alter the antigenic-
ity of the isolated donor myoblasts.16,17While host effector
cells, such as CD4/CD8 T cells and NK cells, are associated
with the host response that rapidly kills the injected donor
myoblasts,9it seems likely that a ‘cocktail’ of factors partic-
ipates in this immune response (reviewed in Smythe et al.15
and Skuk and Tremblay18). It is crucial that the immune
response of the host responsible for the extremely rapid death
Immunology and Cell Biology (2001) 79, 231–239
Complement and myoblast transfer therapy: Donor myoblast
survival is enhanced following depletion of host complement C3
using cobra venom factor, but not in the absence of C5
STUART I HODGETTS and MIRANDA D GROUNDS
Department of Anatomy and Human Biology, University of Western Australia, Crawley, Western Australia, Australia
Muscular Dystrophy and involves the injection of cultured muscle precursor cells (‘myoblasts’) isolated from
normal donor skeletal muscles into dystrophic host muscle. The failure of donor myoblast survival following MTT
is widely accepted as being due to the immune response of the host. The role of complement as one possible
mechanism for the initial, very rapid death of myoblasts following MTT was investigated. Donor male myoblasts
were injected into the tibialis anterior (TA) muscles of female host mice that were: (i) untreated; (ii) depleted of
C3 complement (24 h prior to MTT) using cobra venom factor (CVF); and/or (iii) deficient in C5 complement.
Quantification of surviving male donor myoblast DNA was performed using the Y-chromosome specific (Y1) probe
on slot blots for samples taken at 0 h, 1 h, 24 h, 1 week and 3 weeks after MTT. Peripheral depletion of C3 was
confirmed using double immunodiffusion, and local depletion of C3 in host TA muscles was confirmed by
immunostaining of muscle samples. Cobra venom factor treatment significantly increased the initial survival of
donor myoblasts, but there was a marked decline in myoblast numbers after 1 h and little long-term benefit by
3 weeks. Strain specific variation in the immediate survival of donor male myoblasts following MTT in untreated
C57BL/10Sn, DBA-1 and DBA-2 (C5-deficient) female hosts was observed. Cobra venom factor depletion of C3
increased initial donor male myoblast survival (approximately twofold at 0 h) in C57BL/10Sn and DBA-1 host mice
and approximately threefold in DBA-2 hosts at 0 h and 1 h after MTT. The rapid and extensive number (approxi-
mately 90%) of donor male myoblasts in untreated DBA-2 mice (that lack C5) indicates that activation of the
membrane attack complex (MAC) plays no role in this massive initial cell death. The observation that myoblast
survival was increased in all mice treated with CVF suggests that CVF may indirectly enhance donor myoblast
survival by a mechanism possibly involving activated C3 fragments.
Myoblast transfer therapy (MTT) is a potential cell therapy for myopathies such as Duchenne
Key words: cobra venom factor, complement, depletion, DNA quantification, myoblast transfer therapy, survival,
Correspondence: Dr SI Hodgetts, Department of Anatomy and
Human Biology, University of Western Australia, 35 Stirling
Highway, Crawley, WA 6009, Australia.
Received 4 August 2000; accepted 8 January 2001.
of donor myoblasts is understood and overcome, in order for
MTT to be successful in potential clinical situations.
Activation of the complement cascade is one possible
mechanism that could account for the initial, very rapid death
of the donor myoblasts. This system consists of many
complex proteins that form a proteolytic cleavage cascade
(reviewed in Carroll19), that ultimately results in target cell
lysis via the generation of a membrane attack complex
(MAC) that can lyse target cells extremely rapidly and is
active for less than one second20(see Fig. 1). Complement
activation is involved in the initiation of an inflammatory
response to reperfusion of ischaemic skeletal muscle,21
occurs in damaged muscle fibres in certain myopathies,22–27
as well as after bupiviacaine-induced necrosis in vivo,28and
cultured myoblasts have been shown to fix complement in
vitro.29While one study has investigated the potential role of
complement in MTT,30the extent of donor myoblast death
was not quantified. The present study was designed to quan-
tify the survival of male donor myoblasts after injection into
host mice where the complement response was prevented
using two strategies; cobra venom factor (that depletes C3)
and the use of C5-deficient (DBA-2) host mice.31,32
Cobra venom factor (CVF) is a well-characterized anti-
complement glycoprotein (reviewed in Vogel and Muller-
Eberhard33,34), that has a C3/C5 convertase activity that
rapidly depletes the host’s C3 component by activation and
conversion,35making CVF an effective compound to deplete
animals of C3 (see Fig. 1). Cobra venom factor modifies
hyperacute xenograft rejection in the discordant guinea pig
to Lewis rat cardiac xenograft model36and delayed-type
hypersensitivity reactions in mice.37Quantitative analyses
(using the male-specific Y1 probe) of donor (male) myoblast
survival after injection into (female) normal C57BL/10Sn
and DBA-1 host mice (the parental strain of DBA-2) depleted
of complement component C3 using CVF, as well as in
C5-deficient (DBA-2) host mice are presented here.
Materials and Methods
C57BL/10Sn, DBA-1 and DBA-2 were obtained from the Animal
Resource Centre, Murdoch, Western Australia. All animal procedures
were carried out in strict accordance with National Health and
Medical Research Council of Australia guidelines and with approval
from the Animal Ethics Committee of the University of Western
Skeletal muscles were taken from the hind limbs and lower back of
4–6-week-old donor male C57BL/10Sn or DBA-1 mice. Myoblasts
were isolated from these muscles by enzymic digestion and filtration
through 100 µm nylon gauze as described previously.2,9The resulting
primary culture was maintained in Hams F10 medium (Trace, Castle
Hill, NSW, Australia) supplemented with 20% (v/v) FCS (Trace),
4 mmol/L L-Glutamine (Sigma, St Louis, MO, USA), 100 IU/mL
Penicillin, 100 µg/mL Streptomycin (Sigma) and 25 ng/mL basic
fibroblast growth factor (bFGF) (Sigma). Medium was replaced
every other day and cells were grown to 70–80% confluency before
harvesting by digestion with 0.1% (w/v) trypsin (ICN-Flow). Cells
were reseeded at approximately 1–2 × 105cells per 75 cm2flask (pre-
coated with 1% (w/v) gelatin) in fresh medium and passaged
successively in this way.
Male C57BL/10Sn and DBA-1 primary myoblast cultures were
harvested using trypsin/EDTA, adjusted to a concentration of
2.5 × 105/10 µL in endotoxin-tested PBS (pH 7.2) and the cells kept
on ice until injected. Ten microlitres of this cell suspension was
injected longitudinally into each tibialis anterior (TA) of 6–8-week-
old female host mice using a Hamilton syringe with a 29G needle.
The needle was retracted carefully as the cells were injected in order
to minimize the physical trauma of the procedure. Host mice used for
0 h time points were injected after surgically exposing the TA
muscle, so that the sample was ready for immediate removal. Control
mice were injected with 10 µL of endotoxin-tested sterile PBS
(Trace). Female host mice (C57BL/10Sn, DBA-1 and C5-deficient
DBA-2) were killed at 0 h, 1 h, 24 h, 1 week and 3 weeks after
myoblast injection and TA muscles were isolated. Samples were used
for DNA quantification, and separate representative TA samples
were used for the generation of frozen sections for immunohisto-
chemistry. Control (no PBS, no MTT, as well as PBS injected) TA
muscles, were also taken.
C3 depletion using cobra venom factor
Host mice were depleted of C3 with a single i.p. injection of 100 µL
(25 mg) of CVF (Venom Supplies, Tanunda, SA, Australia), 24 h
prior to MTT. This is the time required for systemic C3 depletion.
Host mice were then subjected to MTT as described. Serum samples
of control and depleted host mice were also taken at 0 h, 1 h, 24 h,
1 week and 3 weeks after MTT and stored at 4°C before assaying for
circulating C3 complement levels. Serum samples were taken via
cardiac bleed immediately after removal of the TA muscle.
Complement (C3) detection
Circulating C3 in serum samples were analysed using double
immunodiffusion. Briefly, the presence and/or absence of C3 com-
ponent in serum samples taken from control (PBS-injected), as well
as CVF-treated and untreated (no CVF) host mice at 0 h, 1 h, 24 h,
1 week and 3 weeks following MTT was detected by the formation
of precipitation arcs in 1% (w/v) agarose using a goat antiserum to
mouse complement C3 (ICN/Cappel, Costa Mesa, CA, USA). Equal
SI Hodgetts and MD Grounds
Outline of the complement cascade.
volumes of antigen (serum) and antibody were added to wells and
left overnight at 4°C. Precipitation arcs were clearly visible between
antigen and antibody wells and photographed using oblique light or
further visualized by staining with 1% (w/v) Coomassie Brilliant
Frozen sections of whole representative TA muscles taken at 0 h, 1 h,
24 h, 1 week and 3 weeks after MTT, as well as ‘PBS-injected’
controls and ‘no PBS, no MTT’ controls were immunostained for
the presence and location of C3 using a goat antiserum to mouse
complement C3 (ICN/Cappel, USA) at 1/200 dilution, followed by a
donkey antigoat Alexa 488 conjugated secondary antibody (Molec-
ular Probes, Eugene, OR, USA) at 1/1000 dilution. Briefly, sections
were rehydrated in a small volume of PBS, and blocked using 10%
(v/v)/FCS/1% (w/v) BSA in PBS for 30 min at room temperature.
Incubation with primary antibody was performed overnight at 4°C.
Sections were washed three times in PBS and incubated with sec-
ondary antibody overnight at 4°C. After washing briefly with PBS,
sections were mounted using DPX (BDH Laboratory Supplies,
Poole, UK) and examined.
Quantification of DNA from male donor myoblasts
Female host mice (C57BL/10Sn, DBA-1 and C5-deficient DBA-2)
were killed at 0 h, 1 h, 24 h, 1 week and 3 weeks after MTT and TA
muscles isolated. Each muscle was cut into 2–3 mm3pieces and then
homogenized in DNA isolation buffer (50 mmol/L Tris-HCl,
150 mmol/L NaCl, 2 mmol/L EDTA (pH 8)) using an Ultra-Turrax
homogeniser (Janke and Kunkel, Staufen, Germany). After adding
SDS to a final concentration of 0.2% (w/v) and incubating at 65°C
for 20 min, Proteinase K (Boehringer Mannheim, Mannheim,
Germany) was added to a final concentration of 200 µg/mL and the
samples incubated overnight at 37°C. Samples were extracted once
in phenol (Tris-HCl buffered, pH 7.4–7.9) (Gibco BRL, Rockville,
MD, USA), once in 25:24:1 phenol : chloroform : isoamyl alcohol,
once in 24:1 chloroform : isoamyl alcohol and then ethanol precipi-
tated overnight at –20°C. Deoxyribonucleic acid pellets were resus-
pended in double distilled water overnight at 4°C and quantified by
measuring the absorbance at 260/280 nm. Deoxyribonucleic acid
integrity was also checked visually under UV illumination after
electrophoresis in 1% (w/v) agarose in the presence of ethidium
bromide. The remainder of the DNA was applied to Hybond-N+
nylon membrane (Amersham, Little Chalfont, UK) using a slot-blot
apparatus (Bio Rad, Hercules, CA, USA) according to the manufac-
turer’s instructions. Donor male DNA was quantified by hybridiza-
tion with the Y-chromosome specific Y1 probe,7–9which was random
prime-labelled using α32P dCTP (DuPoint, Boston, MA, USA). Dilu-
tions of Y1 were applied to each slot blot as a standard positive
control, as well as control samples of 2.5 × 105male donor myoblasts
(in triplicate), designated as 100%. Quantification was performed by
densitometric analysis after exposure to phosphorimaging screens
(Fuji, Osaka, Japan) using a MacBas 2500 phosphorimaging system
(FujiFilm) and Image Reader 1.5E/Image Gauge V3.0 software.
Statistical analysis of quantitative DNA data was performed using
Minitab software (Minitab Inc., State College, PA, USA).
Levels of C3 complement
Serum samples collected from C57BL/10Sn, DBA-1 and
DBA-2 host mice that were either control (PBS-injected, no
MTT), untreated (no CVF prior to MTT) or CVF-treated (C3
depleted, 24 h prior to MTT) were subjected to double
immunodiffusion assays in order to determine the presence
and/or absence of circulating C3 (Fig. 2). Sera from control
C57BL/10Sn, DBA-1 and DBA-2 host mice contains circu-
lating C3 (well 1, Fig. 2a–c) as indicated by comparable pre-
cipitation arcs. The presence of C3, as indicated by
precipitation arcs, in all untreated (non-CVF, MTT) host mice
at 0 h, 1 h, 24 h, 1 week and 3 weeks following MTT, were
similar to that detected in control (PBS-injected, no MTT)
mice (Fig. 2a, wells 2–6, respectively). C3 was undetectable
in CVF-treated host mice at 0 h, 1 h and 24 h after MTT, as
indicated by the absence of precipitation arcs (Fig. 2b,c, wells
2–4, respectively). C3 was detectable in the serum of all
CVF-treated host mice at 1 week after MTT (Fig. 2b,c, well
5) and by 3 weeks (Fig. 2b,c, well 6) was comparable to C3
detected in control mice (Fig. 2a). Note that Fig. 2 shows
results for C57BL/10Sn and DBA-1 host mice only, although
detection of C3 for each treatment was the same for DBA-2
mice (data not shown).
DNA quantification of surviving donor male myoblasts
The amount of surviving donor male (Y1 positive) myoblast
DNA recovered from injected female host TA muscles was
quantified by calculating the percentage of radioactive 32P
signal compared to that obtained from Y1 hybridization to
Complement and myoblast transfer therapy
samples from untreated [no cobra venom factor (CVF)] (a)
C57BL/10Sn, (b) CVF-treated C57BL/10Sn and (c) CVF-treated
DBA-1 host mice following myoblast transfer therapy (MTT).
Cobra venom factor treatment was performed 24 h prior to MTT.
Precipitation arcs indicate the presence and relative amounts of
circulating C3 present in serum of host mice. Well 1 contains sera
from host mice from PBS-injected (no MTT) control, and sera
taken from the three groups of host mice at 0 h (well 2), 1 h (well
3), 24 h (well 4), 1 week (well 5) and 3 weeks (well 6) after MTT,
Double immunodiffusion agarose plates of serum
2.5 × 105cultured donor male myoblasts (designated as
Untreated and CVF-treated C57BL/10Sn host mice
Quantification of male DNA on slot blots using the Y1 probe
showed a characteristically rapid and massive loss of male
DNA after injection of normal C57BL/10Sn donor male
myoblasts into untreated C57BL/10Sn female host mice.9An
average of only approximately 10% of donor cells survived at
0 h (2–5 min after injection) and numbers of donor cells
declined over time such that by 3 weeks (504 h) only approx-
imately 2% of donor myoblasts were present (Fig. 3a). A
twofold increase at 0 h (to approximately 20%) was seen in
the average survival of C57BL/10Sn donor myoblasts after
myoblast injection into CVF-treated C57BL/10Sn hosts and
these donor myoblasts persisted for at least 24 h. Numbers of
donor myoblasts declined over 1 week (168 h), although at
3 weeks (504 h) there were still significantly more (threefold)
donor myoblasts than in untreated C57BL/10Sn hosts. ANOVA
showed statistically significant differences (P < 0.005) in the
average number of C57BL/10Sn donor myoblasts at each
time point after injection between untreated and CVF-treated
C57BL/10Sn host mice.
Untreated and CVF-treated DBA-1 host mice
A similar rapid and massive loss of male DNA was seen after
injection of normal DBA-1 donor male myoblasts into
untreated DBA-1 female host mice (Fig. 3b). However, there
was a marked strain specific difference as the initial number
of donor myoblasts surviving in DBA-1 hosts was more than
double that seen with C57BL/10Sn hosts. Approximately
40% of the injected donor myoblasts survived in DBA-1 mice
at 0 h (compared with approximately 20% in C57BL/10Sn
hosts – see also Hodgetts et al.9). This rapidly declined to
10% at 1 h and by 3 weeks only approximately 5% of donor
myoblasts were present. The average survival of donor
myoblasts was significantly increased in CVF-treated DBA-
1 hosts (compared to untreated DBA-1 hosts), over the first
week. At 1 h, approximately 85% of donor myoblasts had
survived (nearly eightfold over untreated DBA-1 host mice).
By 1 week there were still significantly more myoblasts in
CVF-treated hosts, although numbers gradually declined. By
3 weeks only approximately 5% of donor myoblasts remained
and this value was not different from untreated DBA-1 hosts.
ANOVA showed statistically significant differences (P < 0.005)
in the average numbers of donor myoblasts at 1 h, 24 h and
1 week after injection between untreated and CVF-treated
DBA-1 host mice.
Untreated and CVF-treated DBA-2 host mice
At 0 h after MTT only approximately 10% of injected DBA-1
donor myoblasts remained in untreated DBA-2 female hosts
(Fig. 3c). Over time this value declined so that by 3 weeks
only approximately 5% of donor myoblasts were present.
Apart from time 0, all average survival values were similar to
those in untreated DBA-1 hosts. In CVF-treated DBA-2 hosts
(i.e. C3 depleted and C5 deficient), there was a marked
increase in the average number of donor myoblasts at each
time point (compared to untreated DBA-2 hosts), although
the percentage continued to decline over 1 week. At 0 h
SI Hodgetts and MD Grounds
of the total number of donor cells originally injected) after
injection into various host mice: (a) normal C57BL/10Sn male
donor myoblasts into untreated and cobra venom factor (CVF)-
treated C57BL/10Sn female host mice; (b) normal DBA-1 male
donor myoblasts into untreated and CVF-treated DBA-1 female
host mice; and (c) normal DBA-1 male donor myoblasts into
untreated and CVF-treated DBA-2 (C5-deficient) female host
mice. Standard deviation is shown for each sample set. [n = 6.
*indicates statistical significance (P < 0.005) between the groups
at one time point]. ( ), untreated, (?), CVF-treated.
The time course of myoblast survival (as a percentage
approximately 60% of the injected donor cells had survived
(sixfold over untreated DBA-2 hosts at 0 h). The average
percentage of surviving donor myoblasts declined over time,
and by 24 h was comparable to that observed in untreated
DBA-2 hosts. ANOVA showed statistically significant differ-
ences (P < 0.005) in the average number of donor myoblasts
immediately after, 1 h and at 1 week following MTT between
untreated and CVF-treated DBA-2 host mice.
The data for all three strains presented in Fig. 3a–c are
summarized in Fig. 4 to compare the average survival of
donor male myoblasts between strains following injection
into untreated (a) and CVF-treated (b) hosts.
C3 antibody staining of representative frozen sections of
‘untreated’ and CVF-treated DBA-1 host TA muscles after
MTT as well as control (PBS-injected, no MTT) muscles are
shown in Fig. 5. Extremely similar C3 staining was obtained
in representative muscle sections from C57BL/10Sn and
DBA-2 mice (data not shown). No staining was seen on
sections of controls with no primary antibody incubation
(data not shown). Sections of untreated control TA muscles
(no PBS, no MTT) showed a very slight background staining
of myofibers in the absence of MTT (Fig. 5a) and a pro-
nounced C3 staining around the myofibers of control (PBS-
injected) muscles (Fig. 5b). Following MTT, such C3 staining
was also visible in untreated TA muscles taken at 0 h (Fig. 5c)
and 24 h (Fig. 5e). Blood vessels were strongly stained for C3
in sections from untreated hosts. In marked contrast, in CVF-
treated TA muscles there was no C3 staining around the
myofibers at 0 h (Fig. 5d) or 24 h (Fig. 5f) after MTT. There
was some weak, irregular staining of isolated areas of
myofiber membranes (Fig. 5d,f), possibly indicating that C3
depletion was not complete locally within the TA following
CVF treatment. Some immunosections showed injected
donor (male) myoblasts that were positive for C3 staining
(mainly intracellular), even at 0 h after MTT (data not
The very rapid death of donor myoblasts1–6,9is difficult to
explain. Clearly, an effector mechanism, such as comple-
ment, that is capable of rapid and widespread cell death must
be involved in the process. For this reason we quantitatively
assessed (using the Y-chromosome specific probe) the effect
of complement depletion on the survival of cultured male
donor myoblasts immediately following (and up to 3 weeks
after), their injection into TA muscles of female host mice.
Depletion of host complement C3 using cobra venom
Prior to assessing the impact of host complement C3 deple-
tion on the success of MTT, the amount of C3 remaining in
serum and skeletal muscles was examined using immuno-
diffusion and western blotting. Circulating C3 was effectively
depleted after CVF-treatment in all strains of mice for up to
48 h (Fig. 2). At 1 week after MTT, circulating C3 was
similar to that of control mice, indicating that the effect of a
single CVF depletion (prior to MTT) is only transiently effec-
tive at depleting C3. While differences in the concentrations
of serum38and in allotypic forms of C339between strains
have been reported, the control (untreated) C57BL/10Sn,
DBA-1 and DBA-2 mice showed similar C3 sera ‘levels’ in
the present study, as detected visually by immunodiffusion.
Data from immunohistochemical staining of frozen TA
sections in these host mice suggest that, although circulating
C3 was effectively depleted, extremely low amounts of local-
ized C3 component were still present in the skeletal muscle
of CVF-treated mice (Fig. 5d,f). Immunosections showed that
myoblasts were positive for C3 staining (mainly intra-
cellular), even at 0 h after injection, suggesting that comple-
ment fixation rapidly occurred at the injection site following
MTT. This result is in accord with the observations of Skuk
and Tremblay30who also showed transplanted immortalized
donor myoblasts that had fixed complement. The strong C3
staining in control (PBS injected, Fig. 5b) host muscles sug-
gests that the physical process of injection itself may be
responsible for C3 fixation in host muscle, especially when
one considers the weak staining observed in untreated con-
trols (no PBS, no MTT, Fig. 5a). It is unlikely that sterile PBS
Complement and myoblast transfer therapy
(a) untreated and (b) cobra venom factor (CVF)-treated
C57BL/10Sn (), DBA-1 (?) and DBA-2 (C5-deficient) (?)
mice. Standard deviation is shown for each sample set. [n = 6.
*indicates statistical significance (P < 0.005) between the groups
at one time point].
Donor male myoblast survival after injection into
would activate the complement cascade, especially as endo-
toxin-tested PBS was used in this study, although no animals
were subjected to needle insertion without injection. The
limit of detection of C3 could be more sensitive on immuno-
sections than in double immunodiffusion plates and it is pos-
sible that any recovery of C3 component has not yet reached
the level of detectability using double immunodiffusion.
Indeed, Skuk and Tremblay reported that CVF-treated host
mice subjected to MTT showed ‘very slight or absent’ C3
staining on sections of muscle immediately following MTT.30
Sewry et al.25also indicated that there may be a discrepancy
between the levels of complement detectable in circulating
serum, as compared to complement localized in specific
DNA quantification of surviving donor myoblasts
The percentage of donor male myoblasts surviving after in-
jection into untreated C57BL/10Sn, DBA-1 and C5-deficient
DBA-2 host mice (Fig. 4a) shows a classical ‘death curve’
compared to that obtained in a previous report (Hodgetts
et al. 2000).9There was a characteristically rapid and massive
death of cultured male donor myoblasts within minutes of
injection into all three strains, and survival declined further
over 3 weeks to within 5–10%, as has been described pre-
viously for C57BL/10Sn and mdx host mice.1,2,4,5,9While the
overall trend was similar for C57BL/10Sn, DBA-1 and DBA-
2 mice there were significant strain-specific differences in
the initial numbers of surviving donor myoblasts. These data
suggest that there may be some variation in the survival of
donor myoblasts between strains of host mice used.
Further differences in early myoblast survival between
strains were noted in host mice treated with CVF. Deoxyri-
bonucleic acid quantification data using the Y1 probe reveals
that host depletion of C3 (or decomplementation) increased
the average percentage of surviving donor male myoblasts
twofold (to approximately 20%) in CVF-treated C57BL/10Sn
host mice (Fig. 3), and that this survival was maintained
SI Hodgetts and MD Grounds
staining for complement C3 on
frozen sections of muscle. Sections
of immunostained tibialis anterior
(TA) muscle fibres from (a) control
(untreated, no injection) muscle;
(b) control PBS injected [no myo-
blast transfer therapy (MTT)]
muscles; (c) untreated [no cobra
venom factor (CVF), MTT] DBA-1
hosts at 0 h and (e) 24 h after
MTT; and CVF-treated DBA-1
hosts at (d) 0 h and (f) 24 h after
MTT are shown. C3 staining is
evident around the myofibres of
untreated (no CVF) host muscle
(b, c, e). Minimal C3 staining is
seen in muscles of CVF-treated
mice (d, f) and blood vessels are
denoted by an asterix (b). Scale
bar indicates 50 µm.
over 1 week. Donor myoblast survival in CVF-treated DBA-
1 and DBA-2 mice was significantly higher immediately after
MTT. The number of donor myoblasts remained high (at
approximately 85–90%) in DBA-1 mice at 1 h. After 1 h all
strains showed a similar progressive ‘death curve’ and
myoblast numbers at 3 weeks were similar (less than 10%) in
all three strains. The reasons for these differences in donor
myoblast survival in different strains of CVF-treated mice
within the first 24 h after MTT are not known, but they
emphasize that genetic factors can influence the success of
MTT. Despite these differences, the effect of C3 decomple-
mentation by CVF does suggest a role for C3 in the death of
donor myoblasts following MTT. The loss of this beneficial
effect after 3 weeks is expected given that a single C3 deple-
tion using CVF is only temporary.
Our quantitative data clearly demonstrate a role for C3 in
donor myoblast death and contrast markedly with the con-
clusions of the study by Skuk and Tremblay.30The rapid death
of donor myoblasts in untreated DBA-2 mice shows that a
lack of complement C5 makes no difference to donor
myoblast survival. C5-deficient mice suffer a deletion in the
gene encoding functional C5 so that the mRNA codes for an
abnormal protein31,32,40and the rest of the complement
cascade are blocked. One report suggests that C5 fragments
are important in polymorphonuclear leucocyte accumulation
at inflammatory sites in both C5-sufficient and C5-deficient
strains of mice.41Our results indicate that the full comple-
ment cascade per se, and especially C5b-C9, does not play a
central role in the rapid death of myoblasts. Skuk and Trem-
blay also reported little evidence of MAC formation follow-
ing MTT in CVF-treated host mice.30The existence of
functional C3a/C5a fragments in C5-deficient mice, as well
as opsonic C3b/C3bi molecules, may be involved in the gen-
eration of peptides that are chemotactic for neutrophils and
NK cells, which are thought to be involved in the death of
donor myoblasts following MTT.9,30,42,43Recent studies in our
laboratory emphasize a key role for NK cells in donor
myoblast survival (SI Hodgetts and MD Grounds, unpubl.
data, 2001). However, the removal of C3 complement with
CVF did result in significantly enhanced donor myoblast
survival following MTT. These data could be explained if C3
were normally involved in donor myoblast death without the
need for C5. In addition, a more general immunosuppressive
effect of CVF resulting from the generation of complement
cleavage products, may also impair the secretory functions
and activation states of immune cells, such as macrophages,
and their subsequent contribution to humoral (T-cell depen-
dent) responses.44–46While the mechanism underlying the
protective effect of CVF is unclear, the influence was only
transitory as evidenced by generally similar numbers of
myoblasts at 3 weeks in CVF treated and control muscles.
Exposure to tissue culture prior to transplantation in vivo
can adversely affect the donor myoblasts.14It is possible that
culture conditions may reduce expression of regulatory
proteins [such as Crry, CD59, decay accelerating factor
(CD55) or membrane cofactor protein (CD46)] on myoblasts
that inhibit complement activation (reviewed in Nangaku47).
Expression of such regulatory proteins has been shown to
protect cells from complement mediated injury.48–52In addi-
tion, activated C3a/C3b fragments are generated from native
C3 following treatment with trypsin,53which is used in the
regular passaging of cells in culture. Further work is required
to address the role of such complement regulatory proteins in
the death of donor myoblasts in MTT after exposure to tissue
culture conditions (including trypsin). The use of primary
myoblasts (as in the present study) to test regimes such as
complement depletion, as opposed to the use of immortalized
clones of donor myoblasts (as used in Skuk and Tremblay30),
are important to confirm the potential relevance to clinical
MTT. The ‘only transient’ benefit of regimes such as CVF
treatment on MTT highlight the multifactorial processes that
are probably involved in the death of cultured donor
myoblasts in the in vivo environment. Identification of the
cellular events involved in these early events is of critical
importance, in order to develop strategies to enhance long-
term survival of donor myoblasts in MTT.
This work was made possible by generous support from
the Parent Project for Duchenne Muscular Dystrophy
(http://www.parentdmd.org/), the Association Française
Contre les Myopathies and the National Health and Medical
Research Council of Australia. The authors gratefully
acknowledge the technical expertise and assistance of Ms
Marilyn Davies (Department of Anatomy and Human
Biology, UWA). The constructive and helpful criticism of the
referees was greatly appreciated for this manuscript.
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