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Research Article

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Irradiation of dystrophic host tissue prior to myoblast transfer therapy enhances initial (but not long-term) survival of donor myoblasts

Stuart I. Hodgetts* and Miranda D. Grounds

School of Anatomy and Human Biology, The University of Western Australia, 35 Stirling Highway, Crawley, Perth, WA 6009, Australia

*Author for correspondence (e-mail: [email protected])

Accepted 23 June 2003 Journal of Cell Science 116, 4131-4146 © 2003 The Company of Biologists Ltd doi:10.1242/jcs.00721

Summary There is a massive and rapid death of donor myoblasts (<20% surviving) within hours after intramuscular injection in myoblast transfer therapy (MTT), due to host immune cells, especially natural killer (NK) cells. To investigate the role of host immune cells in the dramatic death of donor myoblasts, MTT experiments were performed in irradiated host mice. Cultured normal C57BL/10ScSn male donor myoblasts were injected into muscles of female C57BL/10ScSn-Dmdmdx host mice after one of three treatments: whole body irradiation (WBI) to eliminate all circulating leukocytes, WBI and bone marrow reconstitution (BMR), or local irradiation (or protection) of one limb. Similar experiments were performed in host mice after antibody depletion of NK cells. Numbers of male donor myoblasts were quantified using a Y-chromosomespecific (male) probe following total DNA extraction of injected muscles. WBI prior to MTT resulted in dramatically enhanced survival (~80%) of donor myoblasts

at 1 hour after MTT, supporting a central role for host inflammatory cells in the initial death of donor myoblasts seen in untreated host mice. BMR restored the massive and rapid loss (~25% surviving) of donor myoblasts at 1 hour after MTT. Local pre-irradiation also resulted in increased donor myoblast numbers (~35-40%) compared with untreated controls (~10%) at 3 weeks after MTT. Preirradiation of host muscle with 10 Gy did not significantly stimulate proliferation of the injected donor myoblasts. Serum protein levels of TNF, IL-1, IL-6 and IL-12 fluctuated following irradiation treatments. These combined results strongly reinforce a major role for host immune cells in the rapid death of injected cultured donor myoblasts.

Key words: Myoblast transfer therapy, Survival, Whole body irradiation, Bone marrow reconstitution, NK cell depletion, Cytokine

Introduction Transplantation of normal muscle precursor cells (myoblasts) into dystrophic host muscle forms the basis of myoblast transfer therapy (MTT), a cell-based transplantation strategy designed to treat myopathies such as Duchenne muscular dystrophy (DMD) (reviewed by Skuk and Tremblay, 2000; Smythe et al., 2000b; Smythe et al., 2001). One major obstacle to the success of MTT is donor myoblast survival because, after injection into the host muscle, cultured donor myoblasts undergo rapid and massive cell death (Beauchamp et al., 1999; Beauchamp et al., 1997; Fan et al., 1996b; Hodgetts et al., 2000; Hodgetts et al., 2003; Qu et al., 1998; Rando et al., 1995) (reviewed by Smythe et al., 2000b; Smythe et al., 2001). Many recent studies attribute this death to the host immune response and the role of some of the major immune cell types is now starting to become apparent (Smythe et al., 2000b; Smythe et al., 2001). The rapid and massive cell death of cultured donor myoblasts after injection contrasts with the excellent long term survival (up to one year) of donor myoblasts using intact or sliced (normal male) grafts of equivalent muscle implanted into female C57BL/10ScSn-Dmdmdx (hereafter known as `mdx') host mice, which are a model for DMD (Fan et al., 1996a; Fan et al., 1997; Smythe et al., 2000a). This striking difference

points to a crucial role for cell isolation and culture in the death of the transplanted muscle cells. Culture conditions themselves (such as media components and serum factors) may alter the antigenicity of the cultured donor myoblasts (Boulanger et al., 1997; Irintchev et al., 1997). Indeed, even brief (~5 minutes) exposure of intact or sliced muscle grafts to tissue culture components results in significantly impaired donor graft survival (Smythe and Grounds, 2000) (reviewed by Smythe et al., 2000b). One way to eliminate all host circulating leukocytes is by whole body irradiation (WBI) (Robertson et al., 1992). Gamma or X-ray irradiation is known to prevent cell proliferation, especially of immune cells, by inducing DNA damage that is lethal if the cell attempts to enter mitosis (Denekamp and Rojas, 1989). Resting lymphocytes die rapidly in interphase after X-irradiation at doses as low as 0.2 Gray (Gy), while T and B lymphocytes activated by mitogens undergo cell death more slowly (Lowenthal and Harris, 1985). Higher doses (such as 1-9 Gy) kill very quickly (within hours) (Lowenthal and Harris, 1985; Lubbe and Zaalberg, 1982; Pecaut et al., 2001) and are associated with damage to the plasma membrane of resting lymphocytes via the generation of highly reactive free radical species (Ashwell et al., 1986). In mdx mice, high doses

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required does vary between mouse strains (Grounds and Davies, 1996; Mitchell et al., 1995). Such irradiation is widely employed to study the contribution of bone marrow-derived stem cells to skeletal muscle (Grounds et al., 2002). A Ychromosome-specific probe (Hodgetts et al., 2000) was used to quantify the numbers of male donor myoblasts after injection into muscles of female mdx host mice, subjected to a variety of irradiation treatments. These included WBI, WBI followed by bone marrow reconstitution (BMR) 1 hour or 24 hours before MTT, local irradiation with one hind limb excluded from WBI, or one hind limb irradiated and the rest of the body excluded. Parallel experiments depleted host NK1.1-positive cells using specific monoclonal antibodies prior to irradiation, in order to optimize the survival of injected donor myoblasts (Hodgetts et al., 2000; Hodgetts et al., 2003). Cytokine assays were performed on sera taken from mice subjected to irradiation (and control mice) to investigate the potential of a `cytokine storm' following irradiation (Budagov and Ul'ianova, 2000; Chang et al., 1997; Hosoi et al., 2001; Neta and Oppenheim, 1991; Van der Meeren and Lebaron-Jacobs, 2001; Van der Meeren et al., 2001; Weill et al., 1996). While irradiation is certainly not a feasible consideration for clinical treatment of DMD boys, it is a powerful experimental tool to investigate factors influencing donor myoblast (i) survival and (ii) proliferation after MTT. Materials and Methods

Animals C57BL/10ScSn-Dmdmdx and C57BL/10ScSn+/+ (the normal parental strain for C57BL/10ScSn-Dmdmdx) mice were obtained from the Animal Resource Centre, Western Australia. All animal procedures were carried out in strict accordance with National Health and Medical Research Council of Australia guidelines and with ethical approval from the Animal Ethics Committee at the University of Western Australia. Tissue culture Skeletal muscles were taken from the hind limbs and lower back of 4- to 6-week old donor male C57BL/10ScSn+/+ (hereafter known as `C57BL/10Sn') mice. Myoblasts were isolated from these muscles by enzymic digestion and filtration through 100 µm nylon gauze as described previously (Fan et al., 1996b), and `pre-plated' for 1 hour on flasks not coated with gelatin in order to preferentially adhere fibroblasts. The myoblast enriched supernatant was then transferred to gelatin-coated flasks and the resulting primary culture was maintained in Hams F10 medium (Trace) supplemented with 20% (v/v) foetal calf serum (FCS) (Trace), 4 mM L-glutamine (Sigma), 100 IU/ml penicillin, 100 µg streptomycin (Sigma) and 25 ng/ml basic fibroblast growth factor (bFGF) (Peprotech Inc., USA). 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 1-2×105 cells per 75 cm2 flask (pre-coated with 1% (w/v) gelatin in phosphate-buffered saline (PBS; pH 7.2) in fresh medium and passaged successively in this way. Irradiation of host mice Female C57BL/10ScSn-Dmdmdx (mdx) host mice (6-8 weeks) anaesthetised by intraperitoneal injection of 100 µl of a mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg) were placed in specially constructed shallow perspex containers to align their bodies correctly and compactly for accurate irradiation. Bolus was packed

of gamma irradiation inhibit regeneration of the dystrophic muscle from endogenous myoblasts, resulting in mdx muscles more closely resembling the pathology of DMD (Pagel and Partridge, 1999; Quinlan et al., 1997; Quinlan et al., 1995; Wakeford et al., 1991; Weller et al., 1991). Postmitotic (mature) skeletal muscle fibres are not particularly sensitive to radiation and will remain viable unless doses are very high (Gross et al., 1999; Pagel and Partridge, 1999; Quinlan et al., 1997; Quinlan et al., 1995; Wakeford et al., 1991; Weller et al., 1991). Satellite cells, which are the main source of myoblasts in mature muscle, are located outside the myofibre between the basal lamina and plasmalemma of muscle fibres. Satellite cells have the ability to be activated and proliferate (in response to growth or damage) which therefore makes them radiosensitive. When satellite cells are activated they enter S phase after about 24-48 hours, may undergo several cycles of cell division and then differentiate and fuse (by 3 days) to form myotubes and ultimately new myofibres (reviewed by Schultz and McCormick, 1994). The high dose (from 20 to 100 Gy) of irradiation required to inhibit satellite cell activation and muscle regeneration (Gulati, 1987; Scott et al., 2001), means that these myogenic cells may proliferate following lower doses (e.g. below 12 Gy) of irradiation (Ben-Dov et al., 1999; Gross et al., 1999; Quinlan et al., 1995). Gamma irradiation has been successfully used to disable the proliferation of myoblasts in skeletal muscle, with little or no apparent damage to the mature muscle fibre, in studies of muscle regeneration (Mitchell et al., 1995; Morgan et al., 1990; Pagel and Partridge, 1999; Quinlan et al., 1997; Quinlan et al., 1995; Robertson et al., 1992; Wakeford et al., 1991; Weller et al., 1991; Wirtz et al., 1982), hypertrophy (Barton-Davis et al., 1999; Phelan and Gonyea, 1997; Rosenblatt and Parry, 1992) and satellite cell activation (Ben-Dov et al., 1999; Gulati, 1987; Lowe and Alway, 1999). Local irradiation of host skeletal muscle (to prevent endogenous regeneration) has also been used as a pretreatment for MTT (Alameddine et al., 1994; Beauchamp et al., 1999; Gross et al., 1999; Gross and Morgan, 1999; Huard et al., 1994; Kinoshita et al., 1994; Morgan et al., 1996; Morgan et al., 2002; Morgan et al., 1990; Morgan et al., 1993; Wernig et al., 2000). Such pre-irradiation of host muscles increased the efficacy of MTT with enhanced migration (Morgan et al., 1993; Watt et al., 1994) and proliferation (Beauchamp et al., 1999; Morgan et al., 2002) of donor myoblasts. The extent of muscle formed by donor myoblasts depended on the dose rate of irradiation delivered (Gross et al., 1999). The irradiation clearly prevents proliferation of (most) host satellite cells and their participation in muscle regeneration, thus promoting the contribution of donor cells to new muscle formation. It seems that a potent mitogenic environment results from some doses of irradiation (Irintchev et al., 1998; Morgan et al., 2002; Wernig et al., 2000), which (artificially) enhances the proliferation of exogenous donor myogenic cells after transplantation. Such a potent mitogenic effect of the irradiated environment is very interesting although the reasons are not yet clear. In the present study, irradiation with 10 Gy was used to examine the mitogenic effect of locally pre-irradiated host muscle after MTT and WBI was used to eliminate all circulating host immune cells. An irradiation dose of 10 Gy was used because this is a typical lethal dose used to ablate bone marrow for reconstitution experiments, although the dose

Irradiation enhances early donor myoblast survival

around the perspex containers. Mice were irradiated as follows; (a) WBI, (b) WBI with bone marrow reconstitution (BMR), (c) local irradiation with one hind limb excluded from WBI or (d) one hind limb irradiated and the rest of the body excluded. Each mouse received 10 Gy (1000 Rads) of radiation, delivered at 2.5 Gy per minute, to within an accuracy of ±2 mm using a Varian 600C Linear Accelerator with a photon energy of 6 MV. At worst, doses fell to 32% by 1 mm and 10% by 4 mm distance from the edge of the irradiation field. Such accuracy negates the use of lead shielding to `protect' areas of the body from irradiation. Dosimetry considerations were based on measurements in water phantom using ion chamber and diode detectors and were calculated using the superposition algorithm on the Computerised Medical Systems FOCUS planning computer. The V600C accelerator provides a nominal dose rate of 2.5 Gy/minute and this is accurate at any time to within 2%. Potential dose variation for WBI was calculated at 10.4 Gy (at mouse surface facing towards radiation beam) to 9.3 Gy (at mouse surface facing away from radiation beam). For local irradiation, potential dose variation was calculated at 9.1 Gy (at mouse surface facing towards radiation beam) to 8.5 Gy (at mouse surface facing away from the radiation beam) at region of limbs surrounded by the most air space. This was minimised by compactly placing irradiated limbs as close to each other in as small a space as possible. MTT was performed 1 hour, 24 hours or 1 week after irradiation treatments. Another experiment involved depletion of host NK1.1+ cells (Hodgetts et al., 2000) using specific depleting monoclonal antibody followed by local irradiation of one hind limb at 1 hour prior to MTT. A scheme summarising the individual treatments is shown in Fig. 1. Bone marrow reconstitution (BMR) BMR was performed using bone marrow cells taken from the long bones of the legs of 6- to 8-week female mdx mice, killed by cervical dislocation. After removing the ends of each bone, PBS from a 29 G needle was used to flush out the marrow cells. Bone marrow cells were washed twice and resuspended in 200 µl PBS. Female mdx host mice subjected to WBI were slowly reconstituted with approximately 1×107 bone marrow cells from another mouse via tail injection at 1 hour after WBI.

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Blood smears Blood smears were taken from host mice (as described by Robertson et al., 1992), stained with 1% (w/v) Eosin and Haematoxylin and analysed histochemically for the presence of a range of immune cell types such as neutrophils, macrophages, eosinophils and lymphocytes. Cell counts were taken from 10 random fields in triplicate for each treatment. Host NK1.1+ cell depletion In one experiment host mdx mice were injected intraperitoneally with 100 µl (1.5 mg) of anti-NK1.1+ hybridoma supernatant (PK136) on days 5, 3 and 1 before MTT as described by Hodgetts et al. (Hodgetts et al., 2000) to deplete host NK1.1+ cells. To confirm depletion of NK1.1+ cells, a 51chromium-release cytotoxicity assay using YAC-1 target cells was performed on effector host splenocytes from depleted and control mdx mice (Hodgetts et al., 2000). Control depletion: Host mice were injected intraperitoneally with 100 µl (450 µg) of isotypic, non-specific rat IgG (Sigma) resuspended in serum free medium over an identical period of 5 days prior to MTT (Hodgetts et al., 2000; Hodgetts et al., 2003). Fluorescence activated cell sorting (FACS) analysis FACS analysis of CD4+/CD8+ populations of thymii and spleens of mice at 1 and 24 hrs after WBI (10 Gy) and control (non-irradiated) mice was performed as described previously (Hodgetts et al., 2000). A combination of CD4+/CD8+-FITC-conjugated antibodies (rat antimouse CD4+ (L3T4) and rat anti-mouse CD8a+ (53-6.7), Pharmingen) antibodies were used. Samples were analysed with a Becton Dickinson FACSCalibur. Sample acquisition and file analysis was performed using CELLQuest. Files were collected using a gate to count lymphocytes, as defined by forward- and side-scatter measurements, to ensure that each file contained 10,000 lymphocytes for analysis. Cytokine assay Levels of the cytokine tumour necrosis factor alpha (TNF) and

Fig. 1. Schematic representation of irradiation regimes. Female host mdx mice were subjected to one of the following regimes: no treatment (control, white mouse), whole body irradiation (WBI, black mouse) (A), WBI plus bone marrow reconstitution (BMR speckled mouse) (B), WBI with single leg excluded (white leg and black body) (C), single leg irradiation (black leg) (D), or NK cell depletion followed by single leg irradiation (NK­ and black leg) (E). Myoblast transfer therapy (MTT) was performed 1 hour, 24 hours or 1 week after irradiation, and samples taken up to 24 weeks after injection of cultured donor male myoblasts.

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(designated as 100%). Statistical analysis (two way ANOVA with Student's t-test as post hoc means test) of quantitative DNA data was performed using Minitab software (Minitab Inc.). Y1 probe specificity and sensitivity was confirmed by hybridisation to male and female genomic DNA and dilutions of Y1 plasmid DNA, as well as to different amounts of DNA isolated from donor male primary myoblasts grown in tissue culture.

interleukins (IL) IL-1, IL-6, IL-12 were determined in irradiated (all groups) and control (untreated) sera using an enzyme-linked immunosorbent assay (ELISA) system (Biosource, USA). `Capture' antibody (1 µg/ml) in coating buffer (15 mM Na2CO3, 30 mM NaHCO3, pH 9.6) was incubated in 96-well plates (Sarstedt) for 30 minutes at 37°C. After washing three times in PBS, wells were blocked with 1% (w/v) BSA in PBS for 30 minutes at 37°C and washed three times with 0.5% (v/v) PBS-Tween 20 (PBST). Pooled sera (from four mice at each time point for each treatment) to be assayed and control antigens (purified IL-1, IL-6, IL-12 and TNF) were then incubated for 1 hour at 37°C and washed three times with PBST. Samples of sera were pooled in order to provide sufficient volumes for the ELISA. Biotinylated secondary `detection' antibody (1 µg/ml) was added for 1 hour at 37°C before washing five times with PBST and incubating with streptavidin-HRP (diluted 1/1000 in PBS) for 1 hour at 37°C. After washing 5× with PBST, diaminobenzidene (DAB) substrate was added, and colour reaction allowed to develop for 15 minutes at room temperature (RT). The reaction was stopped with 2% (w/v) oxalic acid and plates read at 420 nm using a BioRad ELISA plate reader. Capture antibodies used were: Rat anti-mouse IL-1 (no. MAB401, R&D Systems, USA), Rat antimouse IL-6 (no. 21201136F, AMS Biotechnology, UK), Rat antimouse IL-12 (no. 554478, BD Pharmingen, USA) and Remicade human/mouse chimeric antibody to TNF (Schering-Plough). Purified antigens used were recombinant murine IL-1 (no. 401ML, R&D Sytems), IL-6 (no. 11240136, AMS Biotechnology), IL-12 (no. 554592, BD Pharmingen) and recombinant mouse TNF (no. 554589 BD Pharmingen). Detection biotinylated secondary antibodies used were: rat anti-mouse IL-1 (no. BAF401, R&D Systems, USA), Rat anti-mouse IL-6 (no. 21201236C, AMS Biotechnology, UK), Rat antimouse IL-12 (no. 554476, BD Pharmingen) and Rat anti-mouse TNF (no. 18122D, BD Pharmingen). Myoblast injection Primary myoblast cultures harvested at passage numbers 3-5 were adjusted to a concentration of 2.5×105/10 µl in PBS and kept on ice. Ten µl of this cell suspension was injected longitudinally into each tibialis anterior (TA) of 6- to 8-week old female host mice using a Hamilton syringe with a 29 G needle. The needle was retracted carefully as the cells were injected in order to minimize the physical trauma of injection. Quantification of male (donor) DNA Female host mice were sacrificed at 1 hour, 24 hours, 72 hours, 3 weeks, 12 weeks, or 24 weeks after injection and TA muscles isolated. The amount of donor male DNA in muscle samples was quantified by hybridisation with the Y-chromosome-specific Y1 probe (Hodgetts et al., 2000) which was random prime-labelled using [-32P]dCTP (DuPoint). In brief, each muscle was homogenised in DNA isolation buffer (50 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, pH 8) using an Ultra-Turrax homogeniser (Janke and Kunkel). Incubation at a final concentration of 0.2% (w/v) SDS at 65°C for 20 minutes was followed by digestion with 200 µg/ml proteinase K (Roche) overnight at 37°C. Total DNA was extracted using phenol (Tris-HCl buffered, pH 7.47.9) (Gibco BRL), 25:24:1 phenol:chloroform:isoamyl alcohol, and 24:1 chloroform:isoamyl alcohol and then ethanol precipitated overnight at ­20°C. DNA pellets were resuspended in double distilled water overnight at 4°C and applied to Hybond-N+ nylon membrane (Amersham) using a slot blot apparatus (Bio Rad) according to the manufacturer's instructions. Quantification was performed by densitometric analysis after exposure to phosphorimaging screens (Fuji) using the MacBas 2500 phosphorimaging system (FujiFilm) and Image Reader Version 1.5E/Image Gauge V3.0 software. The total amount of male donor DNA in samples was measured against DNA isolated from 2.5×105 injected male primary myoblasts

Results Whole body irradiation Circulating leukocytes were effectively removed following WBI, as evidenced by their virtual absence in blood smears from host mice as early as 1 hour after WBI (data not shown). This is supported by FACS analysis which showed that at 1 and 24 hours respectively after WBI, 80.3% and 78.5% of CD4+/CD8+ in the thymii and 88.1% and 94% in the spleens of WBI mice cells were depleted in comparison to (nonirradiated) control mice (data not shown). The average percentage of donor male myoblasts in TA muscles of female mdx host mice subjected to WBI, followed by MTT 24 hours later, is compared with donor myoblast survival in untreated control mdx host mice in Fig. 2A. This control was common to all treatment groups. Very similar results were obtained irrespective of whether MTT was performed at 1 hour or 24 hours after WBI. A dramatic increase in myoblast survival was seen in WBI hosts at 1 hour after MTT, with over 70% of donor male myoblasts being detected compared with only 6% in untreated control hosts. When MTT was performed 24 hours after WBI, approximately 75%, 65% and 30% of donor myoblasts remained at 1 hour, 24 hours and 72 hours respectively (Fig. 2A). Two-way analysis of variance (ANOVA) showed that the decrease between 1 hour and 24 hours was not significant but was between 24 hours and 72 hours (P<0.05). The results were nearly identical in host mice subjected to WBI, followed by MTT only 1 hour later (data not shown). In striking contrast, donor myoblast survival was 6% at 1 hour and 2% at both 24 hours and 72 hours in nonirradiated control mdx host mice. Donor myoblast survival in WBI mice was always significantly (P<0.005) greater than in non-irradiated control host mice. Whole body irradiation and bone marrow reconstitution In order to determine if the re-introduction of leukocytes would `reverse' the benefits of WBI, BMR was performed 1 hour after WBI (Fig. 1B). Myoblasts were injected either 1 hour or 24 hours later, to determine the time required for any potential effect of BMR. On average, threefold higher numbers of leukocytes were visible in blood smears taken from WBI/BMR host mice than in WBI host mice (data not shown). There were markedly fewer cells in WBI/BMR host mice than in blood smears taken from untreated control host mice (which were tenfold higher than WBI hosts, at <1 cell/field). The survival of donor male myoblasts in TA muscles of female mdx host mice subjected to WBI/BMR and then MTT 24 hours after BMR is shown in Fig. 2A. The results were nearly identical to those of host mice subjected to WBI/BMR and MTT only 1 hour later (data not shown). At 1 hour after MTT, approximately 24% of donor myoblasts were detected and values dropped slightly to 11% and 16% by 24 hours and 72

Irradiation enhances early donor myoblast survival

100 ** Untreated WBI ** % donor male myoblasts 80 MTT at 24hrs after WBI ** dd * 20 ** WBI/BMR

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In order to determine if the beneficial effects of WBI persisted over time, a long-term study was carried out and muscles sampled up to 24 weeks following MTT (Fig. 2B). As WBI is lethal after 3-5 days, BMR was required to rescue the host mice. Again, BMR was performed at 1 hour after WBI and MTT performed 1 hour after this (since there appeared to be no difference in the early survival of donor myoblasts following either WBI/BMR regime). After MTT, numbers of donor myoblasts detected were approximately 40%, 25%, 20%, 10% and 5% at 1 hour, 24 hours, 1 week, 3 weeks, 12 weeks and 24 weeks, respectively. ANOVA showed that donor myoblast numbers were significantly higher (P<0.005) following WBI/BMR than in untreated hosts for up to 1 week. There was no difference between numbers of donor myoblasts in treated and untreated hosts at 12 weeks and 24 weeks, indicating that donor myoblasts neither survived above control levels nor proliferated following WBI/BMR. Local irradiation To determine whether donor myoblast survival was affected in irradiated versus non-irradiated tissue within the same animal, two experiments were performed. In the first, host mice were subjected to WBI with a single hind leg excluded or `protected' from irradiation (Fig. 1C), and in the second experiment the whole animal was protected except for one hind leg that was irradiated (Fig. 1D). Within an individual mouse, each hind leg therefore provided a treated (irradiated) or contralateral (nonirradiated or `protected') TA sample. Comparison of the numbers of donor myoblasts detected in each of these tissues would therefore yield information about the local and/or systemic benefit of irradiation. WBI with single leg protected In host mice subjected to WBI with one hind leg excluded from irradiation followed by MTT 24 hours later (see Fig. 1C), donor myoblast survival in irradiated and contralateral (protected) TA muscles was very similar at all times (Fig. 3A). In irradiated muscles, approximately 30%, 32% and 43% of donor myoblasts were detected after 1 hour, 24 hours and 72 hours, respectively. In protected (non-irradiated) muscles of the contralateral leg, numbers of donor myoblasts were 18%, 34% and 44% after 1 hour, 24 hours and 72 hours, respectively. ANOVA showed no difference in donor myoblast survival in irradiated or protected tissues of these mice. In control nonirradiated mdx host mice donor myoblast survival was only 6% at 1 hour, and 2% at 24 hours and 72 hours. All values for irradiated host mice were significantly (P<0.005) greater than for control non-irradiated host mice. From 1-72 hours after WBI with one leg protected, circulating leukocyte numbers were about 35-45% of values seen in control non-irradiated mice (data not shown). Overall, these data suggest that irradiation had a beneficial effect (direct and indirect) on donor myoblast survival and maintenance over time. Single leg irradiated, MTT 24 hours later Where only one leg was irradiated locally followed by MTT 24 hours later, donor myoblast numbers were also very similar in irradiated and contralateral protected TA muscles (Fig. 3B).

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Fig. 2. (A) Number of donor male myoblasts after MTT of untreated (control), whole body irradiated (WBI) and WBI followed 1 hour later by bone marrow reconstitution (BMR) hosts. All mdx host mice are female. MTT was performed 24 hours after WBI and muscles sampled at 1, 24 and 72 hours. Schematics of the irradiation treatment of host mice as described in Fig. 1 are indicated in the top right-hand corner. Bars indicate standard deviation. *P<0.05 and **P<0.005 (untreated to treated samples) using ANOVA; d and dd indicates significance of P<0.05 or P<0.005, respectively, between treatment groups at each time point using ANOVA; n=6 unless otherwise stated. (B) Number of donor male myoblasts after MTT of untreated (control), WBI and WBI/BMR, female mdx host mice. MTT was performed 1 hour after BMR and muscles sampled at 1 hour, 24 hours, 1, 3, 12 and 24 weeks after MTT. Bars indicate s.d. *P<0.05, **P<0.005 (untreated to treated samples) using ANOVA; d and dd indicate significances of P<0.05 or P<0.005, respectively, between treatment groups at each time point using ANOVA (n=6 unless otherwise stated).

hours respectively. ANOVA showed no significant differences between all 3 times. These data indicate that the cellular component removed by WBI can be successfully re-introduced by BMR and results in substantial donor myoblast death even at 2 hours after BMR. Despite the very high donor myoblast death seen after WBI/BMR, the numbers of donor myoblasts did remain significantly higher than in untreated (nonirradiated) host mice, indicating that there was still some protection of donor myoblast survival following total irradiation.

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% donor male myoblasts

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These data are similar to those for WBI with one leg protected and support the beneficial effect of local irradiation on donor myoblast survival and maintenance. They suggest that local irradiation has a greater initial benefit to myoblast survival than no irradiation of muscle in the same (irradiated) host.

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Fig. 3. (A) Numbers of donor myoblasts in irradiated and contralateral (protected) TA muscles in mdx host mice subjected to WBI with one leg protected. MTT was performed 24 hours after WBI and muscles sampled at 1 hour, 24 hours and 72 hours after MTT. Schematics of the irradiation treatment of host mice (see Fig. 1) are indicated in the top right-hand corner. Bars indicate s.d. * P<0.05, **P<0.005 (untreated to treated samples) using ANOVA; d and dd indicate significances of P<0.05 or P<0.005, respectively, between treatment groups at each time point using ANOVA (n=6 unless otherwise stated). (B) Numbers of donor myoblasts in irradiated and contralateral (protected) TA muscles in mdx host mice subjected to local irradiation of one leg only. MTT was performed 24 hours after irradiation and muscles sampled at 1 hour, 24 hours and 72 hours after MTT. Bars indicate s.d. *P<0.05, **P<0.005 (untreated to treated samples) using ANOVA; d and dd indicate significances of P<0.05 or P<0.005, respectively, between treatment groups at each time point using ANOVA (n=6 unless otherwise stated).

Single leg irradiated, MTT after 1 hour or 1 week The interval between local irradiation and MTT was altered in order to see if the mitogenic effect on donor myoblasts reported for irradiated muscle (Morgan et al., 2002) was rapidly induced (1 hour) or sustained over time (1 week). Muscles were also sampled up to 24 weeks following MTT to determine if irradiation (with 10 Gy) promoted long-term survival and proliferation of donor myoblasts. Donor myoblast survival in host mice with a single leg irradiated and subjected to MTT at 1 hour or 1 week after irradiation is shown in Fig. 4A and 4B respectively. It should be noted that at 24 hours after MTT (Fig. 4A,B), donor myoblast numbers were similar (25-35%) to that seen in locally irradiated mice subjected to MTT at 24 hours after irradiation (Fig. 3A,B). The 24 hour time point is common to all experiments in Figs 3 and 4. Donor myoblast numbers at all times up to 24 weeks were similar, in both locally irradiated and contralateral protected muscles, irrespective of whether MTT was performed at 1 hour or 1 week after irradiation (Fig. 4). Almost a sevenfold increase in donor myoblast numbers (35%) was seen at 24 hours compared to that seen in untreated (non-irradiated) control host mice. The numbers of donor myoblasts declined steadily after 3 weeks, where values were almost as low as those in untreated (non-irradiated) host mice. By 12 and 24 weeks, numbers of donor myoblasts were extremely similar (5-10%) regardless of treatment or host. The only statistically significant differences between locally irradiated and contralateral (protected) muscles was a slightly lower donor myoblast survival at 24 hours and 12 weeks after MTT (P<0.05, ANOVA) in the contralateral muscles of host mice subjected to local irradiation and MTT 1 week later. The expected trend of increasing donor myoblast numbers from 1 hour to 72 hours as seen in Fig. 3A and B was not seen after 1 week. Beyond this time (up to 24 weeks) there was a loss of donor myoblasts and no indication of any superior survival or mitogenic effect in irradiated hosts. Single leg irradiation in NK-depleted host mice To enhance the initial survival of donor myoblasts, host mice were depleted of NK cells (Hodgetts et al., 2000; Hodgetts et al., 2003) before being subjected to local irradiation of one leg followed by MTT 24 hours later (Fig. 1E). Comparison is made with donor myoblast survival in untreated controls, as well as with previous data (Hodgetts et al., 2000; Hodgetts et al., 2003) from NK-depleted mdx host mice (Fig. 5). Data for NKdepleted host mice were similar regardless of irradiation. NK depletion significantly enhanced donor myoblast survival at 1 hour after MTT in irradiated and contralateral tissues compared with irradiated host mice without NK depletion (Fig. 3A,B), supporting a beneficial effect of NK depletion over irradiation. At 24 hours after MTT, numbers of donor myoblasts were effectively the same (20-30%) in all tissues of NK-depleted hosts and significantly higher than for untreated (nonirradiated) control host mice.

In locally irradiated muscles, numbers of donor myoblasts were 24%, 31% and 45% at 1 hour, 24 hours and 72 hours, respectively, although ANOVA showed that this increase was not significant. In contralateral protected muscles, donor myoblast numbers were 26%, 18% and 45% at 1 hour, 24 hours and 72 hours respectively. ANOVA showed no difference in donor myoblast survival in irradiated or protected tissues of these mice. Numbers of donor myoblasts in either leg of these locally irradiated host mice were significantly (P<0.005) greater than in non-irradiated (untreated) control host mice.

Irradiation enhances early donor myoblast survival

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Fig. 5. Numbers of donor myoblasts in irradiated and contralateral (protected) TA muscles in NK-depleted mdx host mice where MTT was performed at 1 hour after local irradiation and muscles sampled at 1 hour, 24 hours, 1 week, 3 weeks, 12 weeks and 24 weeks after MTT. Schematics of the irradiation treatment of host mice (see Fig. 1) are indicated in the top right hand corner. Bars indicate s.d. *P<0.05, **P<0.005 (untreated to treated samples) using ANOVA; d and dd indicate significances of P<0.05 or P<0.005, respectively, between treatment groups at each time point using ANOVA (n=6 unless otherwise stated).

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Fig. 4. Numbers of donor myoblasts in locally irradiated and contralateral (protected) TA muscles in mdx host mice subjected to local irradiation of one leg. MTT was performed at 1 hour (A) or 1 week (B) after irradiation and muscles sampled at 24 hours, 1 week, 3 weeks, 12 weeks and 24 weeks after MTT. Schematics of the irradiation treatment of host mice (see Fig. 1) are indicated in the top right hand corner. Bars indicate standard deviation. * and ** indicates significance of P<0.05 or P<0.005, respectively, between untreated and treated samples using ANOVA; d and dd indicates significance of P<0.05 or P<0.005, respectively, between treatment groups at each time point using ANOVA, n=6 unless otherwise stated.

legs of locally irradiated, NK-depleted host mice. This was also apparent in the contralateral (protected) leg at 1 week. This difference is difficult to explain, but might be due to changes in cytokine profile following irradiation (see below). However, the combined effect of NK depletion prior to irradiation still results in higher donor myoblast survival (almost twofold) from 3 weeks to 24 weeks than following irradiation alone. Again, there was no evidence of any increase in donor myoblast numbers over time (up to 24 weeks). Cytokine profiles following irradiation Protein levels of the circulating cytokines TNF, IL-1, IL-6, and IL-12 in serum from irradiated host mice are shown in Fig. 6. In host mice subjected to WBI (Fig. 6A), TNF levels were greatly enhanced within 2 hours, being elevated to 550%, 700% and 15% of control mdx (no MTT) levels at 2, 25 and 73 hours after WBI (which represents 1, 24 and 72 hours after MTT) respectively. In WBI/BMR mice, TNF levels were initially elevated to 600%, before dropping to 10% and 1% of control values at 3, 26 and 74 hours after WBI (representing 1, 24 and 72 hours after MTT) respectively (Fig. 6A). In marked contrast, WBI greatly reduced levels of IL-1 to 10% of control levels from 1-72 hours in WBI host mice (Fig. 6B). Similar IL-1 levels were seen in WBI/BMR mice at 1 and 24 hours after MTT, but there was a dramatic return at 72 hours to 120% of control levels (Fig. 6B). Levels of IL-6 in WBI host mice (Fig. 6C) increased slowly and transiently, being 100%, 270% and 140% of control values at 2, 25 and 73 hours after WBI (representing 1, 24 and 72 hours after MTT) respectively. In WBI/BMR host mice, IL-6 levels decreased over time, being 160%, 25% and 2% of control values at 3, 26 and 74 hours after WBI (representing 1, 24 and 72 hours after MTT)

At 1 week after MTT, numbers of donor myoblasts were essentially unchanged for all groups except for contralateral (protected) legs of locally irradiated, NK-depleted hosts where myoblast numbers dropped significantly (P<0.05) to about 20%, although this value was still significantly higher (P<0.005) than in untreated (non-irradiated) hosts (under 5%). Between 3 and 24 weeks after MTT, donor myoblast numbers also decreased in locally irradiated, NK-depleted hosts to about 15% and remained at this level in both legs of irradiated hosts, apart from a slight drop in irradiated legs at 12 weeks (P<0.05). Donor myoblast numbers in untreated (non-irradiated) control hosts remained low (around 5%) from 24 hours to 24 weeks. Up until 1 week, the results were very similar for muscles from all NK-depleted hosts. Unexpectedly, at 3 weeks a striking drop in myoblast numbers was seen in both irradiated and contralateral (protected)

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TNF-alpha Levels in Sera from Host Mice Con mdx WBI WBI/BMR

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respectively (Fig. 6C). IL-12 levels dropped to (and stayed below) 10% of control levels from 1-72 hours after MTT in WBI host mice, but BMR reversed this decrease and IL-12 levels were 70%, 85% and 10% of control values at 1, 24 and 72 hours after MTT respectively (Fig. 6D). In mice where only a single leg was locally irradiated (followed by MTT 1 hour later), TNF levels were similar (120%) to control values at 24 hours but increased to 200% and 500% of control values at 1 week and 3 weeks, respectively. If MTT was conducted 1 week after local

irradiation, TNF levels increased dramatically from just above control values at 24 hours (i.e. 8 days after local irradiation) to 400%, 700%, 900% and 500% above controls at 1, 3, 12, and 24 weeks post MTT, respectively. The comparison of samples injected at different times after irradiation shows that the relative timing of MTT per se affects cytokine levels. It is noted that the pattern of TNF increase is markedly different to that seen after WBI (Fig. 6A) where TNF was strikingly elevated by 1 hour and returned to control levels by 3 days.

Irradiation enhances early donor myoblast survival

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Fig. 6. Profiles of circulating cytokines TNF, IL-1, IL-6 and IL-12 in sera from mdx host mice subjected to WBI and WBI/BMR (A-D), local irradiation (single leg) followed by MTT 1 hour or 1 week later (E-H), or NK depletion with or without local irradiation of a single leg (IL). Levels of cytokines are expressed as a percentage of that detected in untreated control mdx (no MTT) and shown at various times after MTT. The scale for the amounts of cytokines changes between the data sets. Samples represent the value of pooled sera from four mice at each time point.

IL-1 levels in locally irradiated mice subjected to MTT at 1 hour after irradiation (Fig. 6F) dropped below control levels (to 40%) at 24 hours, increased to 250% at 1 week and decreased to about 30% by 3 weeks after MTT. In contrast, IL1 levels stayed below control levels (30-50%) up to 1 week

after MTT in mice subjected to MTT at 1 week after local irradiation: levels then increased to 500% at 3, 12 and 24 weeks after MTT (Fig. 6F). The striking elevation of IL-1 above control levels contrasts with the reduced or control levels seen after WBI (Fig. 6B).

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after 1 hour (~80%) compared to control untreated host mice (~10%). Numbers of donor myoblasts remained high (70%) for up to 24 hours after MTT, irrespective of whether MTT was performed at 1 hour or 24 hours after WBI. These results of very early myoblast survival in WBI mdx hosts are in agreement with a previous study (Beauchamp et al., 1999) using nude (athymic immunologically compromised mice lacking T cells) mdx hosts, which reported survival values of around 85% at time 0 following MTT (72 hours after irradiation) although few myoblasts (20%) remained by 24 hours. A central role for host blood borne cells in rapid donor myoblast death was strongly supported by studies in host mice perfused with saline immediately prior to MTT, where excellent donor myoblast survival was seen at time 0 (Hodgetts et al., 2003). The striking survival of myoblasts in WBI hosts is most likely also due to the absence of circulating leukocytes at the time of MTT, as leukocytes numbers were greatly reduced (<10%) in blood smears even at 1 hour after WBI. It is known that after WBI, peripheral lymphocytes, polymorphonuclear leucocytes and monocytes all disappear rapidly from circulation, and are usually completely absent within 24 hours of irradiation, by way of leakage through the gut without replenishment by bone marrow cells (Robertson et al., 1992). Resident immune cells (e.g. macrophages, dendritic cells) may still be present within irradiated tissue for much longer (Robertson et al., 1992), but after irradiation are unable to replicate. The decrease in numbers of donor myoblasts at 72 hours after WBI (to approximately 30%) is most likely due to the declining health of host mice, which usually die within a week of lethal WBI unless rescued by BMR. The irradiation dose (10 Gy) used in these experiments typically results in extremely rapid lymphocyte death (Lowenthal and Harris, 1985; Lubbe and Zaalberg, 1982; Pecaut et al., 2001) with death of the host animals around 4-7 days (our unpublished observations). There is also a possibility that the cytokine storm resulting from irradiation (and also MTT) (see below) may have contributed to the death of donor myoblasts by 3 days. Bone marrow reconstitution BMR was performed to restore circulating cells after WBI and rescue host mice from inevitable death, thereby allowing long term studies to examine the reported mitogenic effect of irradiation (Beauchamp et al., 1999; Morgan et al., 2002) on the injected donor myoblasts. Donor myoblast survival in WBI host mice with BMR was greatly reduced compared with WBI host mice. This indicates that rapid reconstitution of the bone marrow-derived immune cells of the host (confirmed by increased numbers of leukocytes in blood smears taken at all times from mice subjected to WBI/BMR) was responsible for the increased donor myoblast death. This striking effect was observed irrespective of whether MTT was performed at 1 hour or 24 hours (allowing more time for host cell replacement) after BMR. Within 3 weeks, numbers of donor myoblasts were the same in treated and untreated control mdx host mice. This suggests that BMR is sufficient to restore the factor (within 1 hour) that causes the death of donor myoblasts. It is well documented that there are strain-specific differences in the reconstitution of leucocytes (Mitchell et al., 1995) and the proportion of neutrophils and lymphocytes may not reflect that

IL-6 levels remained very similar to control mdx (no MTT) values for up to 3 weeks when myoblasts were injected at 1 hour after irradiation (Fig. 6G). When MTT was performed at 1 week after irradiation, IL-6 levels increased 500% above control values at 1 week and remained 15-fold higher than controls from 3 to 24 weeks (Fig. 6G). Again the timing and pattern of cytokine change differs from that seen after WBI (Fig. 6C) and is also strongly influenced by the time the donor myoblasts are injected after local irradiation. Levels of IL-12 remained lower than control values (20%) for up to 3 weeks after MTT, regardless of when myoblasts were injected (Fig. 6H). In NK-depleted host mice, TNF levels decreased gradually from control values to about 10% after 3 weeks, regardless of whether host mice were locally irradiated or not and it is noted that this was seen even when no myoblasts were injected (Fig. 6I). This cytokine pattern after NK-depletion has little resemblance to that seen with the other irradiation regimes (Fig. 6A,E). IL-1 levels in NK-depleted hosts stayed below control levels (30-50%) for up to 3 weeks after local irradiation and MTT, and were further reduced (<20%) by 12 weeks in hosts that were only NK depleted (Fig. 6J). A tendency for decreased IL-1 serum levels was also seen for some of the other irradiation treatments (Fig. 6B,F). Levels of IL-6 (Fig. 6K) increased dramatically from over 10-fold above control mdx (no MTT) values at 24 hours to 17-fold by 3 weeks, when NK-depleted host mice were locally irradiated, but were always below control values (5-20%) if not locally irradiated. (Fig. 6K), implying a very potent systemic effect of local irradiation in NK-depleted host mice. Transient IL-6 increases were also seen with some, but not all, of the other irradiation treatments (Fig. 6C,G). Finally, IL-12 levels remained lower than control values (<10%) for up to 3 weeks after MTT, if locally irradiated and, in NK-depleted (non-irradiated) host mice, only around 45-60% at any time with or without MTT (Fig. 6L). It should be noted that these cytokine assays illustrate the flux of potentially influential cytokines in the serum following irradiation but appeared to have no relationship to the numbers of surviving donor myoblasts detected over time for each treatment. Discussion Quantification of male donor myoblast DNA using the Ychromosome specific (Y1) probe provides accurate information on the number of donor myoblasts surviving immediately after MTT as a baseline to detect any subsequent increase in myoblast numbers. The amount of male donor DNA present over time will reflect the potential for donor myoblast survival, in combination with myoblast death and proliferation. Measurement of long term myoblast proliferation (overall increase in numbers of male donor myoblasts) after MTT is a major objective of this study. Whole body irradiation In contrast to the typically rapid and massive death of donor myoblasts (Beauchamp et al., 1999; Beauchamp et al., 1997; Fan et al., 1996b; Hodgetts et al., 2000; Qu et al., 1998; Rando et al., 1995; Smythe et al., 2000b; Smythe et al., 2001), a single 10 Gy dose of whole body irradiation (WBI) prior to MTT dramatically increased the survival of donor male myoblasts

Irradiation enhances early donor myoblast survival

seen in untreated (non-irradiated) hosts: the total number of leukocytes in peripheral blood of untreated (non-irradiated) SJL/J and BALB/c mice was about twofold higher than in irradiated/BMR mice after 24 days, and there was a significant (P<0.05) decrease in lymphocytes and increased neutrophils in BALB/c (but not SJL/J) mice compared to untreated (nonirradiated) controls (Mitchell et al., 1995). In other studies, repopulation of 88% of leukocytes was seen by 4 weeks after BMR in female C57BL/6 mice (Han et al., 2001), whereas only 68% replacement was seen at 8 weeks in BALB/c hosts (Stewart et al., 2001). In mdx mice (background strain C57BL/10SnSc) the results suggest that BMR is able to reconstitute sufficient numbers of immune cells, and/or circulating factors that are responsible for the rapid death of donor myoblasts within hours. In addition to reconstituting a host cellular immune component, BMR appears to disturb the cytokine levels following WBI (see below). The effect of local irradiation Local irradiation (10 Gy) of the host muscle resulted in increased (nearly 10-fold higher) survival of injected donor male myoblasts at 1 hour after MTT in either the irradiated or contralateral (protected) muscles of host mice compared to

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untreated (non-irradiated) controls. This was followed by a tendency for donor myoblast numbers to increase slightly (from ~25 to 45%) between 1 and 3 days (Fig. 3) but numbers decreased by 1 week after MTT (Fig. 4) and continued to gradually decrease to values in untreated (non-irradiated) controls by 3 weeks (Fig. 4). These results indicate that irradiation of even one leg of the host had a systemic effect and increased the initial survival of transplanted donor myoblasts (in both legs). While it would be interesting to determine the minimum amount of tissue required for irradiation to produce this protective effect, such experiments were not undertaken in the present study. Unfortunately, the initial survival of donor male myoblasts was not maintained and there was no evidence of donor myoblast proliferation for up to 24 weeks after MTT. This contrasts with other studies that show enhanced donor myoblast proliferation in irradiated muscles (Beauchamp et al., 1999; Morgan et al., 2002; Morgan et al., 1993) although these studies used nude mdx mice (see Table 1). The studies in nude mdx mice show that the vast majority of transplanted myoblasts die, but suggest that pre-irradiation of the host muscle enhances the proliferation of a small proportion of injected myoblasts (mooted to be stem cells), which undergo rapid and extensive proliferation after MTT. This was demonstrated by an initial massive drop (to ~10% of controls

Table 1. Summary of selected irradiation experiments in skeletal muscle

Total dose (Gy) 10 Dose rate (Gy/minute) 2.5 Host strain mdx (6-8 weeks old) Treatment WBI, WBI+BMR, local irradiation, WBI with 1 leg protected (+/­NK host cell depletion). MTT at 1 hour, 24 hours or 1 week later (C57BL/ 10 primary donor myoblasts) Local irradiation, MTT 3, 28 or 100 days later (C2C12 donor myoblasts) Local irradiation with/without notexin (1-20 weeks after irradiation), single muscle fibre analysis Local irradiation, MTT at 2 days later (C57BL/10primary donor myoblasts) Local irradiation, MTT 1-10 days later (H2Kb-tsA58 primary donor myoblasts) Local irradiation, muscle grafts Local irradiation, MTT 3 days later (H2Kb clone 18 immortal cell line donor myoblasts) Local irradiation, MTT 3 days later (C57BL/10 primary donor myoblasts) Local irradiation, WBI with one leg protected, muscle grafts Local irradiation, muscle grafts WBI, WBI+BMR, local irradiation, crush injury at 21 days, muscle grafts Local irradiation, MTT 3-4 days later (C57BL/10 or mdx primary donor myoblasts) WBI, WBI+BMR, local irradiation, crush injury at 24 hours or 48 hours before or after, muscle grafts Local irradiation, muscle grafts Local irradiation, MTT 3-5 days later (C57BL/10, C57BL/6J or mdx primary donor myoblasts) Isolated EDL muscle irradiation and grafting WBI+BMR, muscle grafts at 3 weeks later Sample* Reference

1 hour-24 weeks This study (Hodgetts et al., 2003) 21 days (Morgan et al., 2002)

4.5, 9, 18 18, 25 16 16, 18 18 18 16-20 16 12, 18, 24, 30 8, 10 18 16 16 18 6.5, 20, 100 8

0.7 1.29 2.3 1.29 0.5 unavailable 1.29 or 0.73 8.32 unavailable 8.32 unavailable 8.32 unavailable unavailable 0.75 8.32

mdx nu/nu, beige/nu/Xid, C5­ (3 weeks old) mdx, C57BL/10 mdx (3 weeks old) mdx nu/nu (3-6 weeks old) mdx, C57BL/10 (16, 105 days old) mdx nu/nu (3-4 weeks old) mdx nu/nu (25-30 days old) SJL/J, BALB/c (6-8 weeks old) mdx, C57BL/10 (12, 21, 42 days old) SJL/J (10 Gy), BALB/c (8 Gy) (6-8 weeks old) mdx nu/nu (16 days old) SJL/J (10 Gy) (6-8 weeks old) mdx, C57BL/10 (15-23 days old) mdx nu/nu (16 days old) Fischer rat C57BL/6J, 129/ReJ, 129/ ReJ-dy (6-8 weeks old)

1 or 3 weeks (Heslop et al., 2000) (after notexin) 67-69 days (Wernig et al., 2000) 21-93 days 4-42 days 100 days 21 days 1-3 days 8 weeks 3-10 days (Gross and Morgan, 1999) (Pagel and Partridge, 1999) (Beauchamp et al., 1999) (Gross et al., 1999) (Grounds and Davies, 1996) (Quinlan et al., 1995) (Mitchell et al., 1995)

35, 49, 250 days (Morgan et al., 1993) 4-5 days 7, 14, 70 days 7-67 days 4-30 days 30-120 days (Robertson et al., 1992) (Wakeford et al., 1991) (Morgan et al., 1990) (Gulati, 1987) (Grounds, 1983)

Gy, Gray (1 Gy, 100 Rads); MTT, myoblast transfer therapy; WBI, whole body irradiation; BMR, bone marrow reconstitution; local irradiation, single leg irradiated only. Abbreviated strains of mice used are: C57BL/10, C57BL/10ScSn(+/+); mdx, C57BL/10ScSn-Dmdmdx; mdx nu/nu, C57BL/10ScSn mdx/mdx(nu/nu); beige/nu/Xid, beige/nu/Xid; C5­, C5­/ chain-deficient/Rag2­; *, after last treatment (MTT, grafting or irradiation)

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this dose appears insufficient to induce the mitogenic effect that results in proliferation of donor myoblasts after MTT. Dosage difference may also account for the lack of enhanced survival seen in irradiated but not contralateral (protected) muscles of locally irradiated host mice (Morgan et al., 2002). What are the effects of local and whole body irradiation? Local irradiation is known to affect levels of many growth factors, which may enhance donor cell proliferation. However, no changes in protein levels within muscles were seen at 3 days (after 18 Gy) for bFGF, FGF-4, FGF-6, HGF, MMP-2 or MMP-9 (Morgan et al., 2002). Irradiation may affect many important molecules within the muscle environment, including adhesion molecules and extracelluar matrix although little information is available. Ionizing radiation (2-6 Gy) increases beta1-integrin in human lung tumour cell lines in vitro (Cordes et al., 2002) and irradiation of human umbilical endothelial cells induces the expression of adhesion proteins such as intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin (Son et al., 2001). An earlier study using the same umbilical cells revealed both a time- (from 2 to 10 days) and dose- (from 2 to 10 Gy) dependent up-regulation of cell-surface expression of ICAM1, but no induction of VCAM-1 and E-selectin (Gaugler et al., 1997). The possibility of systemic clastogenic activity in serum as a consequence of `non-targeted' potential radiation-induced bystander effects (Lorimore et al., 2001; Lorimore and Wright, 2003; Mothersill and Seymour, 2001) must also be considered. Such effects may include irradiation-induced genomic instability expressed in non-irradiated neighbouring cells close to the area of irradiation and subsequent secretion of serum factors (such as cytokines or reactive oxygen species) that could possibly contribute to the death (or survival) of donor myoblasts. Ionizing radiation can mimic cytokine signals and can lead to immune cell activation (McKinney et al., 1998; Uchimura et al., 2000). It is possible that irradiation may have activated macrophage (and neutrophil?) activity which in turn may have contributed (directly or indirectly through cytokinemediated pathways) further to donor myoblast death. It is well documented that exposure to ionizing radiation selectively induces expression of some cytokines. The changing profiles of cytokine expression following the irradiation regimes used in the present study have no obvious correlation with the numbers of surviving donor myoblasts detected over time for each treatment (Fig. 6). However, given the highly complex nature of interactions relating to circulating cytokines, and the influence on the host's immune response, such changes become important when considering the potential role of cytokines in donor myoblast death. Our results showed different cytokine profiles depending on the amount of host tissue irradiated. The data demonstrate the complex response of 4 key cytokines to irradiation, myoblast injection and depletion of host NK cells. No clear pattern emerges. While there was an overall trend for increased TNF and IL-6 and decreased IL-1 and IL-12 after WBI, this was not universally true; it was influenced by the time after irradiation when myoblasts were injected and was generally reversed by reconstitution with bone marrow cells. TNF, IL-1 and IL-6 are known as the `inflammatory triad' being powerful systemic

at 24 hours after MTT) of male DNA detected with the Y1 probe followed by an increase in the amount of male DNA (to between 20-25%) after 4 days in irradiated, but not contralateral muscles (Beauchamp et al., 1999). In these experiments, 18 Gy was used to locally irradiate the muscle of nude mdx host mice (Beauchamp et al., 1999). Subsequent work using the myogenic C2 cell line as donor myoblasts showed that the mitogenic effect of irradiation was dose dependent, with 18 Gy being reliably mitogenic, 4.5 Gy ineffective, and 9 Gy mitogenic in four out of five mice (Beauchamp et al., 1999; Morgan et al., 2002; Morgan et al., 1993). Thus it would be expected that 10 Gy would be an effective mitogenic dose. However, the dose rate used in the present study (2.5 Gy/minute) was nearly twice that used in studies where effective mitogenic effects were reported (Beauchamp et al., 1999; Morgan et al., 2002; Morgan et al., 1993). Local irradiation (5-15 Gy, at 8.4 Gy/minute) was also reported to increase proliferation of proximal tubule cells in both irradiated and contralateral kidneys (Otsuka and Meistrich, 1993). It was postulated that this was due to induction of a humoral growth factor, although this occurred about 3 months after irradiation. That the factor produced by 18 Gy irradiation is long lived was also supported by the work of Morgan et al., who demonstrated a potent mitogenic effect at 100 days after MTT (Morgan et al., 2002). There is probably some interplay of negative factors and positive mitogenic stimuli as a result of irradiation. The timing of myoblast proliferation induced by the mitogenic effect of irradiation (Beauchamp et al., 1999; Gross et al., 1999; Morgan et al., 2002) could be different in WBI and locally irradiated mice. The latency effect of irradiation may also be different in these different treatments. Evidence for latency comes from a variety of tumour-related studies involving irradiation. For example, local pre-irradiation of the inoculation site of tumour bed cells decreased both the latent period and rate of tumour growth (van den Brenk et al., 1977). There is considerable evidence that the amount of radiation, as well as the dose rate may be important for the mitogenic effects of irradiation on skeletal muscle. Clearly, experimental differences in the total dose and dose rate between different research studies may account for differences in results, apart from differences between mdx and nude mdx host mice. Dose rates range from 1.29 Gy/minute or 0.73 Gy/minute (Beauchamp et al., 1999; Gross et al., 1999; Morgan et al., 2002) to 2.3 Gy/minute (Wernig et al., 2000), 2.5 Gy/minute in the present study and 8.32 Gy/minute (Robertson et al., 1992). Total dose ranges from 10 Gy (this study), 16 Gy (Robertson et al., 1992; Wernig et al., 2000) to 18 Gy (Beauchamp et al., 1999; Gross et al., 1999; Morgan et al., 2002). Low doses (<5 Gy) are sufficient to eliminate circulating immune cells (Lowenthal and Harris, 1985; Lubbe and Zaalberg, 1982; Pecaut et al., 2001), and 7 Gy (at 1.81 Gy/minute) can effectively inhibit proliferation of vascular smooth muscle and adventitial cells (Scott et al., 2001). Higher doses (16-20 Gy) are considered necessary to completely prevent muscle regeneration (Gulati, 1987; Robertson et al., 1992) and also to improve the muscle formation from transplanted donor cells (Beauchamp et al., 1999; Gross et al., 1999; Morgan et al., 2002). While 10 Gy, used in this study, is sufficient to eliminate the host cellular immune response and clearly results in increased initial survival of donor myoblasts,

Irradiation enhances early donor myoblast survival

and local mediators of inflammation (Kulmatycki and Jamali, 2001; Mosser and Karp, 1999; Taga, 1997). In addition, IL-12 plays a central role in regulating the balance between Th1 and Th2 responses, as well as exerting immunoregulatory effects on NK cells and T cells (Brunda, 1994; Gately et al., 1998). Very low doses of gamma irradiation can also induce immunological responses through the release of interleukin-4 (IL-4) and interferon-gamma (IFN) from lymphocytes, in addition to IL-1 and TNF from monocytes (Galdiero et al., 1994). IL-4 is also known to down-regulate the acute inflammatory response after irradiation in mice (Van der Meeren and Lebaron-Jacobs, 2001; Van der Meeren et al., 2001). Induction of IL-1 and IL-6 mRNA by low doses (2 Gy) of ionizing radiation in murine peritoneal macrophages in vitro was seen by 1 to 2 hours after irradiation and then mRNA levels decreased below the basal level after 4 hours (Hosoi et al., 2001). Sublethal (7.75 Gy) gamma-irradiation of murine bone marrow and spleen (Chang et al., 1997) showed that IL1 mRNA levels were significantly increased in bone marrow at days 2 and 4 post-irradiation and at day 7 in spleen, whereas, post-irradiation, IL-6 mRNA levels were significantly increased at day 2 in bone marrow and at days 7 and 10 in spleen. TNF mRNA levels exhibited a significant increase at day 2 post-irradiation in bone marrow, but in spleen no difference between control and irradiated samples was observed on any day post-irradiation (Chang et al., 1997). The transient increase in serum TNF and IL-6 seen at day 1 in the present study after WBI (Fig. 6C) endorses some elevation of these circulating cytokines by such WBI. Macrophages, harvested from mouse bone marrow and spleen did not increase cytokine production within the first 3 days following gammairradiation (7 Gy), whereas peritoneal macrophages revealed enhanced IL-6 and IL-1 protein production (Budagov and Ul'ianova, 2000). When exposed to low doses (4 Gy) of gamma-irradiation, enriched human peripheral blood T lymphocytes, rapidly express TNF mRNA (increasing almost 3-fold in 30 minutes). This response decreases at doses of 10 Gy, strongly suggesting that T lymphocytes, the most radiosensitive cells of the body, are responsible in blood for the irradiation-induced expression of TNF (Weill et al., 1996). It is noted that many other cell types such as skeletal muscle produce cytokines including TNF (Collins and Grounds, 2000), IL-6 (Steensberg et al., 2002; Steensberg et al., 2000) and leukaemia inhibitory factor (LIF) (Kurek et al., 1996; Kurek et al., 1998; White et al., 2001) and this local production of cytokines by muscle cells must be taken into account. It has been shown that irradiation (16 Gy) of SJL/J muscle 2 days prior to injury ablated the production of soluble chemotactic factors by damaged muscle (although the chemotactic signals were not diminished in the contralateral protected leg in WBI mice) (Grounds and Davies, 1996). Furthermore, PCR amplification studies of mRNA for IL-6 and LIF in muscle showed that the marked increase in these local cytokines normally seen at 3 days after crush injury was unaffected by preirradiation (WBI or local) (Grounds and Davies, 1996). Cytokines are also radioprotective; IL-1 and TNF, given before irradiation, can protect mice from doses of radiation (~7 Gy) that would normally be fatal (Neta and Oppenheim, 1991). Furthermore, IL-1, IL-4, IL-6, TNF, interferon and LIF can promote recovery when administered after low dose irradiation

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(Neta and Oppenheim, 1991). Clearly the complex increase of some cytokines and decrease in others may have many important consequences. They may also influence the ability of donor myoblasts to remain viable following MTT in irradiated hosts, either in the short or long term, and this may in turn depend on the dose (and perhaps rate) of radiation administered. The effect of local irradiation and host nk cell depletion on donor myoblast survival The use of specific antibodies to deplete host NK1.1 cells results in enhanced survival and maintained numbers of donor myoblasts after MTT, as has been reported previously (Hodgetts et al., 2000; Hodgetts et al., 2003). This initial treatment regime forms the basis of a potential strategy to improve the efficacy of MTT in the clinical situation. Local irradiation (10 Gy) had no additional benefit on donor myoblast numbers compared to NK1.1+ host cell depletion alone (no irradiation) prior to MTT (Hodgetts et al., 2000; Hodgetts et al., 2003). However, the addition of NK-depletion prior to local irradiation (Fig. 5) resulted in higher initial and long-term donor myoblast survival (almost twofold at 24 weeks after MTT) than following irradiation alone (Fig. 4). In contrast, high doses (18 Gy) of local irradiation in Beige/nu/Xid host mice did not result in significantly more donor muscle cells than in non-irradiated muscles at 100 days after MTT (Morgan et al., 2002) [Beige mice have defective NK activity (Hodgetts et al., 2003; Ramey et al., 1996) and Beige/nu/Xid are a nude athymic mouse strain)]. These data suggest that possibly there are multiple effects at work in NK-depleted, irradiated mice and that NK depletion and irradiation work through different, but as yet unclear, mechanisms. Little information is available relating to the effects of irradiation on NK cell activity and the role of such a combined effect on survival of donor muscle cells. However, it is interesting to note that decreasing leukocyte and lymphocyte numbers in C57BL/6 mice occur with varying doses (0.5-3 Gy) and dose rates (0.01 and 0.8 Gy/min) of whole-body gammaradiation on lymphoid cells, so that CD4+ T helper and CD8+ T cytotoxic cell counts decrease, whilst NK1.1+ natural killer cell numbers and proportions remain relatively stable in both blood and spleen (Pecaut et al., 2001). NK cells are believed to be a primary obstacle to successful allogeneic bone marrow engraftment (Engh et al., 2001; Liu et al., 2000; Yu et al., 1992), and there is strong evidence that acute bone marrow transplant rejection in lethally irradiated mice is caused by NK cells (Warner and Dennert, 1985). Our results suggest that a circulating `death factor' is present in sufficient quantity to effect a massive reduction in remaining myoblast numbers after local irradiation irrespective of whether the site of transplantation is protected or has been directly irradiated. Only WBI (10 Gy) is effective, although removal of circulating host cells by WBI or perfusion (Hodgetts et al., 2003) is the most effective way to protect donor myoblasts, host NK depletion is also highly beneficial (Hodgetts et al., 2000; Hodgetts et al., 2003). The apparent recovery of donor myoblast numbers after 72 hours in our initial experiments (Fig. 3A,B) suggested that the positive mitogenic effect of irradiation might be responsible for this increase. However, this was not sustained. The subsequent

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expertise and assistance of Ms Marilyn Davies (School of Anatomy and Human Biology, UWA) and Drs Rajin Nathan and Peter Lanzon (Department of Radiation Oncology, QEII Medical Centre, UWA) are gratefully acknowledged.

decrease in donor myoblast numbers might be due to the onset of immune responses (involving T cells, neutrophils, macrophages and dendritic cells), and may also be linked with changes in cytokine profiles following irradiation. The absence of any pronounced proliferation of donor myoblasts over and above the initial survival following MTT might also relate in part to the isolation of donor myoblasts. It is possible that the early passage primary myoblasts used in our experiments did not possess the rare population of putative stem cells that have the potential to proliferate after MTT in irradiated hosts (Beauchamp et al., 1999). Beauchamp et al., used the conditionally immortal myoblast cell line (H-2Kb clone 18) and such myoblasts may behave differently (as is the case for the C2 myoblast cell line) to primary cultures in vivo. Whether only certain sources of donor myoblasts are suitable for potential clinical applications needs to be considered when critically evaluating success in experimental MTT (Skuk and Tremblay, 2000; Smythe et al., 2000b; Smythe et al., 2001; Tremblay and Guerette, 1997). The normal primary muscle cultures used in the present study were not subjected to exhaustive pre-plating techniques that have recently been reported to select for muscle precursor cells with enhanced proliferative capacity and the possession of `stem cell-like' properties (Lee et al., 2000; Qu-Petersen et al., 2002). Indeed, only one pre-plating step was routinely employed in the present study in order to remove contaminating fibroblasts. However, it would be expected that the enriched (>65%) myoblast population would have an excellent capacity for proliferation in vivo regardless of the presence of a minority putative `stem cell' population. Other experiments in our laboratories are now employing the interesting pre-plating technique (Lee et al., 2000; Qu-Petersen et al., 2002) to quantify the survival and proliferation of such `late preplate' myogenic cells and determine their efficacy for MTT. Conclusion Irradiation of dystrophic host muscles is certainly not a therapeutic option in the clinical situation since it would destroy the remaining inherent regenerative capacity of dystrophic muscle. However, irradiation is an important tool to investigate the factors determining the survival and subsequent proliferation of donor myoblasts. Such studies highlight the roles of immune cells that are normally involved in rapid death of donor myoblasts, and demonstrate striking changes in circulating cytokines that may contribute to the short and long term survival of donor myoblasts after MTT. With the dose (10 Gy) and design of the present experiments no pronounced mitogenic effect on donor myoblast proliferation was observed. However, even local irradiation increased initial donor myoblast survival in both the irradiated and contralateral (nonirradiated) leg. A central role for host NK cells in the initial survival of donor myoblasts was further endorsed by these studies. Such information is crucial for the development of interventions designed to increase the success of clinical MTT.

This work was made possible by funding support from the International Parent Project for Duchenne Muscular Dystrophy (http://www.parentmd.org/), Aktion Benni & Co e.V. (http://www.abc-online.org/) and the Association Française Contre les Myopathies (http://www.afm-telethon.asso.fr/). The technical

References

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