Embed Size (px)
Citation preview
www.elsevier.com/locate/jneuroim
Journal of Neuroimmunolo
Immunomodulation of TGF-beta1 in mdx mouse inhibits connective tissue
proliferation in diaphragm but increases inflammatory response:
Implications for antifibrotic therapy
Francesca Andreetta, Pia Bernasconi, Fulvio Baggi, Paolo Ferro, Laura Oliva, Elisa Arnoldi,
Ferdinando Cornelio, Renato Mantegazza, Paolo Confalonieri *
Department of Neuroimmunology and Neuromuscular Diseases, National Neurological Institute ‘‘Carlo Besta’’, via Celoria 11, 20133 Milan, Italy
Received 4 November 2005; received in revised form 14 February 2006; accepted 6 March 2006
Abstract
Irreversible connective tissue proliferation in muscle is a pathological hallmark of Duchenne muscular dystrophy (DMD), a genetic
degenerative muscle disease due to lack of the sarcolemmal protein dystrophin. Focal release of transforming growth factor-beta1 (TGF-h1)is involved in fibrosis development. Murine muscular dystrophy (mdx) is genetically homologous to DMD and histopathological alterations
comparable to those in DMD muscles occur in diaphragm of older mdx mice. To investigate the early development of fibrosis and TGF-h1involvement, we assessed diaphragms in 6–36-week-old mdx and C57/BL6 (control) mice for fibrosis, and used real-time PCR and ELISA
to determine TGF-h1 expression. Significantly greater fibrosis and TGF-h1 expression were found in mdx from the 6th week. Mice treated
with neutralizing antibody against TGF-h1 had lower levels of TGF-h1 protein, reduced fibrosis, unchanged muscles fiber degeneration/
regeneration, but increased inflammatory cells (CD4+lymphocytes). These data demonstrate early and progressive fibrosis in mdx
diaphragm accompanied by TGF-h1 upregulation. Reduction of TGF-h1 appears promising as a therapeutic approach to muscle fibrosis, but
further studies are required to evaluate long term effects of TGF-h1 immunomodulation on the immune system.
D 2006 Elsevier B.V. All rights reserved.
Keywords: Muscular dystrophy; mdx animal model; Muscle fibrosis; Transforming growth factor-h1; Fibrogenic cytokine; Immunomodulation
1. Introduction
Abnormal connective tissue proliferation following
myofiber degeneration is a major pathologic feature of
Duchenne muscular dystrophy (DMD), a severe genetic
myopathy due to a lack of the sarcolemmal protein
dystrophin, and clinically characterized by progressive and
irreversible degeneration of muscle tissue (Sanes, 1994;
Engel et al., 1994). The proliferation of muscle extracellular
matrix, characterized by deposition of fibronectin and type I
and III collagens in the endomysium and perimysium of
muscle tissue (Foidart et al., 1981; Stephens et al., 1982;
Duance et al., 1980), leads to irreversible derangement of
0165-5728/$ - see front matter D 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.jneuroim.2006.03.005
* Corresponding author. Tel.: +39 02 23942255; fax: +39 02 70633874.
E-mail address: [email protected] (P. Confalonieri).
muscle organization, by impeding the regeneration of
muscle fibers and hindering nutritional support, particularly
in advanced stages when fibers are physically isolated from
their blood supply (Engel et al., 1994; Duance et al., 1980).
Since this fibrotic proliferation is likely to be a major
obstacle to the efficacy of therapies for muscular dystro-
phies, early interventions to prevent it will probably be
necessary as part of an effective treatment protocol.
Abnormal connective tissue proliferation also occurs in
liver cirrhosis, glomerulonephritis, idiopathic lung fibrosis
and systemic sclerosis. In these conditions, focal release of
fibrogenic cytokines, particularly transforming growth fac-
tor-beta1 (TGF-h1) is a key element in promoting fibroblast
proliferation and collagen synthesis (Kovacs, 1991). TGF-h1is a multifunctional cytokine with roles in inflammation,
immunomodulation, and wound healing, as well as fibrosis
(Border and Noble, 1994). A significant correlation between
gy 175 (2006) 77 – 86
F. Andreetta et al. / Journal of Neuroimmunology 175 (2006) 77–8678
fibrosis and TGF-h1 expression in Duchenne and Becker
muscular dystrophies has been reported, supporting a role for
this cytokine in the development of muscle fibrosis, and
suggesting it as target for antifibrotic therapies (Bernasconi et
al., 1995; Yamazaki et al., 1994).
Murine X-linked muscular dystrophy (mdx) and DMD are
genetically homologous conditions characterized by a com-
plete absence of dystrophin due to mutations in the
dystrophin gene. The mdx mouse is a useful animal model
for DMD (Hoffman and Dressman, 2001). Although the limb
muscles of adult mdxmice showmuch less weakness, muscle
degeneration and fibrosis than DMD boys, the diaphragm
exhibits severe degeneration and functional impairment
similar to that seen in DMD (Stedman et al., 1991). In fact,
the extracellular matrix of mdx diaphragm muscle doubles
between 60 and 120 days, and is increased by 8-fold at 240
days (Louboutin et al., 1993). However, the extent of fibrosis
and TGF-h1 expression in early stages of the disease is
poorly documented. Hartel et al. (2001) found increased
TGF-h1 expression by ELISA in the diaphragm at 12-week-
old mdx mice, and suggested its involvement in fibrosis;
while Gosselin et al. (2004) reported early overexpression of
TGF-h1 transcripts in mdx diaphragm, as well as inhibition
of type I collagen mRNA, after decorin administration, but
did not assess muscle fibrosis or TGF-h1 protein expression.Studies on animal models of other diseases characterized
by fibrosis have shown that reduction of TGF-h1 levels in
affected tissues (skin, lung, thyroid and kidney) can limit
fibrotic development (McCormick et al., 1999; Chen et al.,
2002; Ziyadeh et al., 2000). To investigate whether the mdx
mouse diaphragm exhibits a pattern of early fibrosis and
early TGF-h1 overexpression, similar to that in human
dystrophic muscle, and to provide indications as to the
utility of early antifibrotic therapy, we studied mdx and C57/
BL6 mice from 6 to 36 weeks of age, assessing fibrosis by
morphometry (De Luca et al., 2005) and using ELISA and
real-time PCR to determine TGF-h1 expression. Since we
found significantly greater fibrosis and TGF-h1 from 6
weeks, we performed additional experiments to test the
effect of limiting TGF-h1 activity by administering mono-
clonal antibody against the cytokine. In view of indications
that TGF-h1 can have both pro- or antiinflammatory effects
(Wahl, 1992), that inflammation can worsen muscle
degeneration (Chen et al., 2000; Porter et al., 2002) and
may interfere with muscle regeneration (Engel and Arahata,
1986; Spencer et al., 2001), we also investigated effects of
anti-TGF-h1 antibody administration on inflammation,
regeneration and degeneration in mdx diaphragm muscle.
2. Materials and methods
2.1. Animals
All experiments were conducted in accordance with
the Italian Guidelines for the use of laboratory animals,
which conform to European Community Directive 86/
609/EEC. Twenty-five mdx mice (Jackson Laboratories,
Bar Harbor, ME, USA) and 25 C57/BL6 mice (Charles
River, Calco, Italy) were used to investigate the
development of muscle fibrosis and expression of TGF-
h1 between the ages of 6 and 36 weeks. Eighteen
additional mdx mice were used to investigate the effects
of anti-TGF-h1 treatment on muscle fibrosis, TGF-h1expression, degeneration, regeneration and inflammation
at 12 weeks. The animals were sacrificed by cervical
dislocation; the diaphragms were removed rapidly, folded,
rolled up and frozen in isopentane pre-cooled in liquid
nitrogen.
2.2. Anti-TGF-b1 treatment
Eleven mdx mice of age 6 weeks were injected
intraperitoneally with 300 Ag of anti-TGF-h1(1D11.16.8, HB 9849 ATCC, Manassas, VA, USA) on
alternate days to age 12 weeks. Seven mdx mice of the
same age were injected intraperitoneally at the same times
with 300 Ag normal mouse IgG (Pierce, Rockford, IL,
USA). During anti-TGF-h1 treatment, the mdx mice did
not shown abnormal behaviour, or differences in gross
vital functions compared to untreated mdx mice and IgG-
treated mice.
2.3. Morphometric analysis
Morphometric analysis was carried out on C57/BL6
mice as controls and also on untreated mdx mice and on
those treated with anti-TGF-h1 or normal IgG. Serial
cryostat cross-sections (6–8 Am thick) of rolled dia-
phragm were stained with Meyer’s haematoxylin and
eosin or Masson trichrome. The extent of endomysial and
total (endomysial plus perimysial) connective tissue was
determined on haematoxylin and eosin-stained sections
(with Masson trichrome-stained sections used to check
morphology) at �20 magnification using the NIH Image
software. At least three randomly selected fields from each
section were analyzed. The area of connective tissue as a
percentage of total muscle in each field was calculated, and
the mean percentage for each group of animals calculated (De
Luca et al., 2005).
2.4. Immunostaining
Immunohistochemical analysis was carried out on
untreated mdx mice and on those treated with anti-
TGF-h1 or normal IgG. Serial 6–8-Am-thick cryostat
cross-sections of rolled diaphragm were cut, collected
onto polylysine-coated slides, and fixed with ice-cold
acetone for 1 min. Subsequent steps were performed in a
humid chamber at room temperature. The sections were
treated with peroxidase block solution (DAKO, Glostrup,
Denmark) for 5 min followed by protein block solution
0%
5%
10%
15%
20%
25%
30%
6w 12w 18w 24w 36wage
*A
0%
5%
10%
15%
20%
25%
30%
6w 12w 18w 24w 36wage
*
B
% o
f end
omys
ial c
onne
ctiv
e tis
sue
% o
f to
tal c
onne
ctiv
e tis
sue
Fig. 1. Histograms showing development of total (A) and endomysial (B)
fibrosis. Mdx mice are characterized by increasing fibrosis with age, with a
significant increase between 6 and 12 weeks (*P�0.001, Student’s t-test).
Open bars: C57/BL6 controls; black bars: mdx mice.
F. Andreetta et al. / Journal of Neuroimmunology 175 (2006) 77–86 79
(DAKO) for 10 min. The sections were then incubated
for 90 min with primary antibody. The following
antibodies were used: anti-NCAM (Chemicon, Intern,
Germany) a marker of regenerating fibers (Spencer and
Mellgren, 2002); anti-CD11b (BD Pharmingen, San
Fig. 2. Digital images of hematoxylin and eosin-stained diaphragm cross-sections.
weeks. In (A) note the normal organization of muscle tissue, with fibers of similar
the progressive disorganization of fascicle architecture with abnormal variation in
arrowheads indicate expanding endomysial fibrosis. Scale bar: 60 Am.
Diego, CA, USA) marker of degeneration staining
macrophages and neutrophils (Spencer et al., 2000);
anti-CD4 (helper T-lymphocytes) (BD Pharmingen); and
anti-CD8a (suppressor T-lymphocytes) (Cederlane,
Hornby, Ontario, Canada).
Sections were then washed in PBS and incubated with
anti-rat IgG biotinylated secondary antibody (Jackson
ImmunoResearch Laboratories, West Grove, PA, USA)
for 1 hour, followed by avidin biotin horseradish perox-
idase complex (ABC kit, Vector Laboratories Inc, Burlin-
game, CA, USA) for 30 min. The chromogen was DAB
(DAKO) applied for up to 10 min followed by brief (1
min) counterstaining with Meyer’s haematoxylin.
Sections were examined under a Zeiss Axiophot micro-
scope and digital images were obtained with a Leica DC-
500 Firecam digital camera.
2.5. Assessment of regeneration, degeneration and
inflammation
These analyses were carried out on untreated mdx
mice, and on those treated with anti-TGF-h1 or normal
IgG. Consecutive frozen cross-sections of rolled dia-
phragm stained to reveal NCAM and CD11b, were
assessed at �20 magnification using NIH Image soft-
ware. At least three randomly selected fields from each
section were analyzed. Regenerating and degenerating
areas were expressed, respectively, as the percentages of
NCAM- and CD11b-positive areas of total muscle in each
field.
To assess inflammation, the total numbers of CD4+ and
CD8+ lymphocytes were counted on entire cross sections
of rolled diaphragm, and normalised per unit area.
(A) C57/BL6 mice at 12 weeks; (B–F) mdx mice at 6, 12, 18, 24, and 36
size and only thin strands (arrowheads) of connective tissue. In (B–F) note
fiber size (double arrow) and replacement by fibrous connective tissue. The
00,5
11,5
22,5
33,5
4
6w 12w 18w 24w 36wage
*
*A
0
1
2
3
4
5
6
7
8
6w 12w 18w 24w 36wage
B*
TG
F-β1
mR
NA
rela
tive
valu
e(p
g T
GF-
β1/ μ
g to
t. pr
ot.)
TG
F-β1
Fig. 3. Quantitation of TGF-h1 protein (ELISA) and transcripts (RT-PCR)
in diaphragm muscle of mdx and C57/BL6 mice of ages 6–36 weeks. (A)
TGF-h1 protein is significantly upregulated at 12 weeks and 24 weeks
(*P�0.001, Student’s t-test). Open bars C57/BL6 controls; black bars mdx
mice. (B) TGF-h1 transcript is overexpressed in mdx diaphragm only at 24
weeks of age (*P=0.0051, Student’s t-test). Open squares: C57/BL6 mice;
black triangles: mdx mice.
0%
5%
10%
15%
20%
25%
30%
% o
f to
tal c
onne
ctiv
e tis
sue
*
C57/BL6 mdx + IgG + anti-TGF-β1
A
*
0%
5%
10%
15%
20%
25%
% o
f en
dom
ysia
l con
nect
ive
tissu
e
*
C57/BL6 + IgGmdx + anti-TGF-β1
B
*
Fig. 4. Effect of treatment with antibody to TGF-h1 from 6 to 12 weeks
on extent of muscle connective tissue. Histograms showing total (A) and
endomysial (B) connective tissue in 12-week-old animals. Levels of total
and endomysial connective tissue are significantly reduced in mdx mice
treated with anti-TGF-h1 antibody compared to untreated and IgG-treated
mice (*P�0.001, Kruskal–Wallis test). In these mice the amount of
fibrosis was similar to that found in untreated control (C57/BL6) mice.
F. Andreetta et al. / Journal of Neuroimmunology 175 (2006) 77–8680
2.6. Determination of TGF-b1 protein
TGF-h1 protein was quantitated by ELISA, according
to the manufacturer’s instructions (R&D System, Minne-
apolis, MN, USA), on diaphragms from C57/BL6 mice,
untreated mdx mice, anti-TGF-h1-treated mdx mice and
IgG-treated mdx mice. Briefly, 10–20 mg of diaphragm
was homogenized in 500 Al of a solution containing 1%
Triton X-100, 20 mM Tris pH 8.0, 137 mM sodium
chloride, 10% glycerol, 5 mM ethylendiaminetetraacetic
acid and 1 mM phenylmethylsulphonyl fluoride and
treated as described (Hartel et al., 2001). TGF-h1 levels
were expressed as pg of TGF-h1/Ag of total protein.
2.7. Determination of TGF-b1 transcript
Total RNA was extracted from 10 to 20 mg of mdx and
age-matched C57/BL6 diaphragms using RNAwiz reagent
(Ambion, Woodward Austin, TX, USA). The extract was
treated with DNase I (Ambion). Random-primed cDNAwas
prepared using Superscript II reverse transcriptase (Invitro-
gen, Carlsbad, CA, USA) following the manufacturer’s
instructions and stored at �20 -C pending amplification.
For quantitation of TGF-h1, predesigned functionally
tested assay (Mm00441724_m1, Applied Biosystems, Foster
City, CA, USA) was used. cDNA samples (each
corresponding to 100 ng total RNA) were amplified in
triplicate using a GeneAmp 5700 Sequence Detection System
(Applied Biosystems) in a volume of 25 Al containing
TaqMan Universal PCR Master Mix (containing AmpliTaq
Gold DNA polymerase), 1 Al of predesigned TGF-h1 primers
and probes. 18S rRNA (Applied Biosystems) was used as
endogenous control. The appearance of specific fluorescence
from TGF-h1 and 18S mRNAwas analyzed using Sequence
Detector Software (version 1.6, Applied Biosystems). The
expression of TGF-h1 in each diaphragm sample was
normalized to that of 18S and calculated from the formula
2�DDCT as described in the manufacturer’s instructions
(user bulletin #2, Applied Biosystems). As calibrator for
untreated mdx and C57/BL6 mice, the normalized TGF-h1CT value from diaphragm of 6-week-old C57/BL6 mice
was used; for anti-TGF-h1- and IgG-treated mice the mean
normalized CT value for diaphragms from 12-week-old
C57/BL6 mice was used.
2.8. Statistical analysis
Data are expressed as means and standard deviations
(TS.D.). StatView software, release 5.01 (SAS Institute
Fig. 5. Digital images of Masson’s trichrome-stained diaphragm cross-sections from 12-week-old mice. (A) Untreated mdx mice; (B) mdx mice treated
with mouse IgG; (C) mdx mice treated with anti-TGF-h1 antibody. Masson’s trichrome allows good visualization of extracellular matrix and connective
tissue. Note the disorganization of muscle tissue structure in (A) and (B) due to fibrosis (square), and good preservation of syncytial structure in (C)
(square). The arrows indicate endomysial fibrosis which is more evident in untreated and IgG-treated mice than anti-TGF-h1-treated mice. Scale bar:
60 Am.
F. Andreetta et al. / Journal of Neuroimmunology 175 (2006) 77–86 81
Inc., Cary, North Carolina, USA) and Prism software
(GraphPad software, San Diego, California, USA) were
used for the statistical analyses. The two-tailed Student’s
t-test with Bonferroni correction and Kruskal–Wallis with
Dunn’s multiple comparison test were used to assess the
differences between C57 control mice and mdx mice and
between experimental and control groups. P values �0.05were considered significant.
Table 1
Effect of the anti-TGF-h1 antibody and normal IgG treatment on extent of fibrosis,
numbers of CD4 and CD8 cells per unit area
Mice % of total
fibrosis
% of endomysial
fibrosis
mdx 1 20.53T3.64 15.21T2.282 23.24T1.96 17.78T2.93
3 23.97T1.05 17.70T2.63
4 26.07T0.49 19.29T3.09
5 22.42T1.98 17.14T3.47MeanTS.D. 23.25T2.60 17.42T2.81
mdx+IgG 1 14.86T2.06 11.60T0.89
2 18.81T2.33 13.27T0.71
3 16.28T0.66 12.76T1.10
4 21.65T2.25 18.13T3.165 18.99T1.15 14.65T1.71
6 19.59T2.58 15.51T1.35
7 20.36T0.97 15.76T2.02MeanTS.D. 18.65T2.71 14.53T2.54
mdx+anti-TGF-h1 1 11.68T1.15 9.84T0.70
2 12.50T1.15 10.01T1.38
3 13.34T2.82 9.86T1.18
4 9.24T1.04 7.82T0.665 9.71T1.38 8.51T0.59
6 12.29T1.53 9.84T1.95
7 10.74T0.66 9.62T0.77
8 10.48T1.64 9.92T0.689 12.67T0.58 11.77T1.07
10 10.65T0.81 8.59T1.64
11 12.33T1.36 10.18T1.14MeanTS.D. 11.36T1.66 9.63T1.40
MeansTS.D. for each animal and for each group are shown. The percentages of tot
The data for regeneration and degeneration are more variable, but differences are n
significantly between the three groups and was higher in mdx mice treated with an
CD8 cells did not differ significantly between the three groups (see Fig. 9).
3. Results
3.1. Fibrosis and cytokine expression with age
In diaphragms of mdx mice, the quantity of total and
endomysial connective tissue as a proportion of total
muscle increased significantly from the youngest age
examined (6 weeks) to 12 weeks (P�0.001, Student’s
percentages of regenerating and degenerating cell aspects per unit areas and
% of
regeneration
% of
degeneration
CD4
(mm2)
CD8
(mm2)
5.53T1.75 5.30T0.74 11 3
6.34T3.58 4.60T1.38 9 2
3.65T1.09 3.74T0.62 6 0
7.04T3.69 5.04T1.52 5 1
7.65T5.71 3.91T2.13 4 0
6.04T3.47 4.52T1.35 7T2.9 1T0.6
2.41T0.01 1.22T0.02 6 2
10.07T9.39 2.55T0.49 11 1
6.74T0.02 2.79T0.11 5 1
11.70T0.14 2.63T1.12 1 0
5.84T2.36 2.59T0.80 1 0
4.42T0.36 3.51T2.64 12 0
5.66T4.94 2.98T1.39 1 0
6.69T4.60 2.61T1.43 5T4.7 1T0.7
3.61T0.23 4.09T1.96 16 5
16.46T6.63 4.77T2.49 10 1
6.11T3.42 2.78T0.83 24 2
18.52T21.17 3.93T0.41 10 1
12.33T13.85 2.62T1.30 11 2
4.94T0.10 2.66T0.21 9 1
5.00T1.30 4.45T0.94 22 1
2.81T0.32 5.12T0.12 40 9
9.48T0.37 6.66 T1.88 21 6
12.15T13.43 4.89T1.65 16 2
2.81T1.04 2.68T1.68 33 13
8.57T8.60 4.06T1.73 19T10.1 4T3.9
al and endomysial fibrosis are homogeneous within each group (see Fig. 4).
ot significant (see Fig. 8). The number of CD4 T cells per unit area differed
ti-TGF-h1 antibody than untreated and IgG treated animals. The number of
00.10.20.30.40.50.60.70.80.9
TG
F-β1
(pg
TG
F-β1
/μg
tot.
prot
.)
*
C57/BL6 mdx + anti-TGF-β1+ IgG
A**
00.20.40.60.8
11.21.41.61.8
2
TG
F-β1
mR
NA
rel
ativ
e va
lue
C57/BL6 + IgGmdx + anti-TGF-β1
B **
Fig. 6. Quantitation of TGF-h1 protein (ELISA) and transcripts (RT-PCR)
in homogenized diaphragm from 12-week-old mice. (A) Mdx mice
treated with anti-TGF-h1 antibody had significantly lower protein levels
than untreated (*P�0.01, Kruskal–Wallis test) and IgG-treated mdx
mice (**P�0.05, Kruskal–Wallis test). (B) Mice treated with anti-TGF-
h1 antibody and with normal IgG had significantly lower transcript
levels of TGF-h1 than untreated mdx mice (*P�0.05, Kruskal–Wallis
test).
F. Andreetta et al. / Journal of Neuroimmunology 175 (2006) 77–8682
t-test). Thereafter the extent of total and endomysial
connective tissue remained high and at levels close to
those observed at 12 weeks (Figs. 1 and 2). The extents of
total and endomysial connective tissue were significantly
greater (P�0.001, Student’s t-test) in mdx mice than
C57/BL6 mice at 6 weeks, and in all the older ages
(Figs. 1 and 2).
3.2. TGF-b1 protein in mouse diaphragm with increasing
age
The quantity of TGF-h1 in the diaphragm of C57/BL6
mice did not change significantly with age (Fig. 3A). The
quantity of TGF-h1 protein was significantly greater in mdx
diaphragms than C57/BL6 mice at 12 weeks (0.610T0.102vs. 0.236T0.071; P�0.001, Student’s t-test), and 24 weeks
(2.516T0.982 vs. 0.317T0.029; P�0.001, Student’s t-test)
(Fig. 3A). Differences were not significant at 6, 18 and 36
weeks.
3.3. TGF-b1 transcript in mouse diaphragm with increasing
age
TGF-h1 transcript levels were quantified by real-time
PCR in mdx and C57/BL6 mouse diaphragms taken at
different ages (from 6 to 36 weeks of age; with an average
of three animals per group investigated); as calibrator the
normalized CT mean TGF-h1 value (17.26) in 6-week-old
C57/BL6 mice was used. In C57/BL6 mice TGF-h1 was
transcribed at constant rate at all ages (Fig. 3B). In mdx
mice TGF-h1 transcript levels at 6, 12 and 18 weeks
were similar (TGF-h1 relative value: 0.873T0.196; 1.042T0.597; 0.958T0.237) compared to C57/BL6 mice; at 24
weeks, levels were 6-fold greater than C57/BL6 of the same
age (P=0.0051, Student’s t-test) and declined thereafter
(Fig. 3B).
3.4. Effects of treatments with anti-TGF-b1 antibody and
normal mouse IgG
3.4.1. Morphometric evaluation of connective tissue
Twelve-week-old mdx mice treated for 6 weeks with
anti-TGF-h1 antibody had significantly less total and
endomysial connective tissue than untreated mdx mice
(P�0.001, Kruskal–Wallis test). Mdx mice treated with
normal mouse IgG did not differ in terms of extent of
connective tissue from untreated mdx mice, but differed
significantly from mice treated with anti-TGF-h1 antibody
(P�0.001, Kruskal–Wallis test) (Figs. 4 and 5, Table 1).
Note that treatment with anti-TGF-h1 antibody brought
levels of connective tissue close to those of untreated
C57/BL6 control mice (Fig. 4).
3.4.2. TGF-b1 protein and transcript
Twelve-week-old mdx mice treated for 6 weeks with
anti-TGF-h1 antibody had significantly less (P�0.01,
Kruskal–Wallis test) TGF-h1 protein in diaphragm than
untreated mdx mice (0.247T0.118 vs. 0.610T0.102) or
mice treated with normal IgG (0.522T0.295: P <0.05,
Kruskal–Wallis test). Protein levels in anti-TGF-h1-treatedmice were similar to those in diaphragm from C57/BL6
control mice (0.200T0.073) (Fig. 6A).Levels of TGF-h1 transcript were significantly lowered
by both anti-TGF-h1 antibody and normal IgG treatments
(0.605T0.155, P�0.05, and 0.666T0.403, P�0.05, Krus-
kal–Wallis test) (Fig. 6B).
3.4.3. Muscle regeneration, degeneration and inflammatory
cell content
Twelve-week-old mdx mice diaphragms contained
NCAM-positive regenerating and CD11b-positive degener-
ating fibers. Each fiber type was usually present in clumps.
Sometimes regenerating and degenerating fibers were
localised close to each other (Fig. 7B, E and C, F).
Quantitation of regenerating and degenerating areas did not
reveal significant differences between mdx mice treated
and not treated with anti-TGF-h1 antibody (Fig. 8 and
Table 1).
0%
5%
10%
15%
20%
% o
f re
gene
ratio
n an
dde
gene
ratio
n ar
eas
mdx + anti-TGF-β1+ IgG
Fig. 8. Evaluation of muscle fiber regeneration/degeneration in mdx
untreated and treated mice. Treatment with normal mouse IgG or with
antibody against TGF-h1 did not cause significant changes in the extent of
regeneration (NCAM staining=open bars) or degeneration (CD11b stai-
ning=black bars).
F. Andreetta et al. / Journal of Neuroimmunology 175 (2006) 77–86 83
Twelve-week-old untreated and IgG-treated mdx mice
had low numbers of CD4+ and CD8+ T lymphocytes
scattered in perimysial and endomysial connective tissue.
These lymphocytes were never found in groups of more
than three cells and never appeared to be invading muscle
fibers (Fig. 7G, J and H, K). Mdx mice treated with anti-
TGF-h1 antibody have significantly more CD4+ T cells per
unit area of muscle than untreated (P <0.05, Kruskal–
Wallis test) or normal IgG-treated ( p <0.01, Kruskal–Wallis
test) mdx mice. These cells were occasionally present in
groups in the endomysium, but again there appeared to be
no invasion of the muscle fibers. The number of CD8+ T
cells was also numerically greater in the diaphragm of
antibody-treated mdx mice without reaching statistical
Fig. 7. Digital images of consecutive diaphragm sections of untreated and IgG- and anti-TGF-h1-treated mdx mice showing staining with NCAM for
regenerating fibers (A–C) (arrows), CD11b for degenerating areas (D–F) (arrows), CD4 (G–I) and CD8 (J–L) (arrows). Note that degeneration, regeneration
and inflammatory cells are often localized in the same areas. In anti-TGF-h1-treated mdx mice an increase of CD4 T cells is evident (I). Scale bar: 60 Am.
0
5
10
15
20
25
30
35
CD
4 an
d C
D8
T-c
ells
/ m
m2
*
mdx + anti-TGF-β1+ IgG
**
Fig. 9. Quantitative evaluation of numbers per unit area of CD4 and CD8
cells in untreated mdx mice and those treated with normal IgG and antibody
against TGF-h1. Treatment with anti-TGF-h1 antibody was associated withsignificantly more CD4 cells than untreated (*P�0.05, Kruskal–Wallis
test) and IgG-treated mice (**P�0.01, Kruskal–Wallis test). Open bars:
CD4 T-cells; black bars: CD8 T-cells.
F. Andreetta et al. / Journal of Neuroimmunology 175 (2006) 77–8684
significance; these cells never appeared to invade muscle
fibers (Figs. 7I, L and 9 and Table 1).
4. Discussion
Our first finding is that connective tissue formed a
greater proportion of the diaphragm muscle of mdx mice
(15.5%) than of control C57/BL6 mice (11%) at the
earliest age studied (6 weeks). The difference was
significant at 12 weeks (23% vs. 9%) and reached 25%
at 36 weeks (Fig. 1, Table 1). Thus the fibrotic process
(Fig. 2) begins at a very early stage in mdx diaphragm,
just as it does in the limb and other muscles of DMD boys
(Bernasconi et al., 1995). We found that the fibrosis was
present in perimysial septa surrounding muscle fascicula,
and also in the endomysium surrounding individual muscle
fibers, thereby hindering nutritional support by isolating
the myofibers from the blood supply, as also reported in
DMD (Engel et al., 1994).
Our second main finding is that the fibrogenic cytokine
TGF-h1 was upregulated in young mdx mice diaphragm,
with levels numerically higher than control from 6 weeks, a
significant difference at 12 weeks, and a peak difference at
24 weeks (Fig. 3A). These findings enlarge the data of a
previous study that investigated TGF-h1 in diaphragm from
mdx mice at 12 weeks only, finding upregulation of the
protein at that time (Hartel et al., 2001).
Note, however, that we found significantly increased
transcript levels at 24 weeks only (Fig. 3B). A previous
study reported that TGF-h1 transcript levels were signif-
icantly upregulated in mdx diaphragm at 6 and 9 weeks,
but not 12 weeks, and were apparently related to increases
in collagen type I mRNA (Gosselin et al., 2004). The
difference in the timing of TGF-h1 transcript peaks
between the two studies might be related to the fact that
we used real time PCR, whereas the other study used
competitive PCR. Furthermore, we normalized TGF-h1values to those of 18S transcript to obviate differences due
to quantity of starting material. Despite this discrepancy,
several studies indicate that TGF-h1 expression in mdx
mouse diaphragm changes with time, as it does in DMD
limb muscle (Bernasconi et al., 1995).
In view of the similarity of the pathological changes in
mdx diaphragm and DMD limb muscle, particularly in
terms of early development of fibrosis and concomitant
upregulation of TGF-h1, we administered monoclonal
antibody against TGF-h1 to determine whether it was able
to inhibit the development of fibrosis. Treatment from 6
to 12 weeks of age resulted in much lower levels of the
cytokine protein in the diaphragm of treated than
untreated and IgG-treated mdx mice (Fig. 6A). In
addition, TGF-h1 mRNA was inhibited in both treatments,
probably because of the known immunomodulatory effect
of IgG in muscle, as reported in patients treated with high
dose intravenous immunoglobulin for inflammatory myo-
pathies (Amemiya et al., 2000). More importantly, a
significant reduction of muscle fibrosis compared to
untreated mdx mice and also mdx mice treated with
normal IgG (Fig. 4, Table 1) was found. A similarly
successful immuno-modulatory approach to the in vivo
control of this overexpressed cytokine has been reported
in animal models of diseases involving excessive fibrosis
of skin, lung and thyroid (McCormick et al., 1999; Chen
et al., 2002).
Our finding that TGF-h1 immunomodulation inhibits
fibrosis, together with the data of Gosselin et al. (2004)
indicating that the use of decorin to inhibit the biological
activity of TGF-h1 results in the downregulation of type I
collagen mRNA, constitute additional evidence for the
involvement of TGF-h1 in the development of fibrosis,
but more importantly suggest that TGF-h1 inhibition may
be useful as an early clinical approach to the inhibition of
fibrosis in dystrophic disease.
It is noteworthy that treatment with anti-TGF-h1antibody did not eliminate the cytokine from diaphragm
muscle, but brought it to levels similar to those of healthy
control mice (Fig. 6). This finding could be important in
view of the fact that TGF-h1 is involved in multiple
biological processes (Wahl, 1994), has various, sometimes
opposing effects on immune system cells (Wahl, 1992),
may be produced by various cell types including
lymphocytes and macrophages and also platelets (Barnard
et al., 1990) and could be involved in muscle degeneration
and regeneration of muscle fibers, as shown by the close
relationship between TGF-h1 expression and inflammatory
infiltrates in dystrophic muscle (Gosselin et al., 2004), and
by the known inhibitory effects of this cytokine on myoblast
differentiation (Heino and Massague, 1990).
The continuous fiber regeneration that occurs in mdx
muscle is considered the main reason for the good clinical
outcomes in this animal model, while the transient and
limited muscle regeneration that occurs in DMD muscle is
F. Andreetta et al. / Journal of Neuroimmunology 175 (2006) 77–86 85
unable to counteract the continuous fiber loss (Granchelli et
al., 2000). In our mice, the 6 weeks of treatment with anti-
TGF-h1 antibody did not affect muscle regeneration or
degeneration (Fig. 8, Table 1) although numbers of CD4 T
cells in the muscle endomysium were increased. The CD8 T
cells did not show signs of direct cytotoxicity (invasion of
non-necrotic muscle fibers) against muscle fibers. This
inflammatory cell pattern is consistent with data reported by
others in limb muscles of untreated mdx mice (Spencer et
al., 1997).
TGF-h1 is a potent inhibitor of lymphocyte activation
and proliferation (Miyazono, 2000; Wahl et al., 1988). Its
inhibition following skeletal muscle injury results in
macrophage infiltration (Lefaucheur et al., 1996). Further-
more TGF-h1-null mice are characterized by dramatic
inflammation (Kulkarni and Karlsson, 1993). The in-
creased inflammatory cells in the muscle of mice treated
with antibody against TGF-h1 is therefore probably due
to reduction in level of the cytokine (TGF-h1), and could
plausibly result in increased muscle degeneration and
fibrosis, particularly in the prolonged treatment that would
be required in DMD therapy. For these reasons, future
antifibrotic protocols with antibodies against TGF-beta1
might also include immunosuppressive agents. In fact it
has been shown that antibody depletion of CD8 and CD4
cells significantly reduces histologically discernable pa-
thology in mdx muscle (Spencer et al., 2001), and that
mdx mice with nu/nu genotype (T cell-deficient) have
reduced muscle fibrosis (Morrison et al., 2000), although
these data were not confirmed by a recent paper
(Morrison et al., 2005). It seems clear that secondary
chronic inflammation could contribute to disease progres-
sion in DMD patients: indeed this is the rationale for
immunomodulatory therapy with glucocorticoids that is
currently the mainstay of treatment in the disease (Skuk et
al., 2002). It is important therefore that proposed anti-
fibrotic treatments for DMD do not exacerbate this
chronic inflammatory state. Our results support the utility
of systemic immunomodulation to inhibit TGF-h1 in
muscle, and hence fibrosis, and provide further evidence
that TGF-h1 plays an important role in the development
of muscle fibrosis, at least in the murine form of x-linked
muscular dystrophy. TGF-h1 inhibition therefore appears
as a promising therapeutic strategy for fibrosis inhibition
in DMD, particularly since no side effects were observed
in the animals during the 6 weeks of treatment. However
effects of TGF-h1 inhibition must be evaluated over the
long-term, paying attention to effects on muscle degener-
ation, muscle fibrosis, and the immune system. Finally,
since muscle fibrosis is likely to be the result of multiple
mechanisms involving molecules other than TGF-h1, datafrom expression profile studies on laser-microdissected
areas of muscle extracellular matrix may identify other
cytokines or growth factors whose modulation may
prevent or reduce fibrosis development without adversely
affecting the immune system.
Acknowledgements
The authors thank Dr. Marina Mora and Prof. Annamaria
De Luca for the helpful suggestions and Donald Ward for
help with the English.
References
Amemiya, K., Semino-Mora, C., Granger, R.P., Dalakas, M.C., 2000.
Downregulation of TGF-beta1 mRNA and protein in muscles of
patients with inflammatory myopathies after treatment with high-dose
intravenous immunoglobulin. Clin. Immunol. 94, 99–104.
Barnard, J.A., Lyons, R.M., Moses, H.L., 1990. The cell biology of TGF-
beta. Biochem. Biophys. Res. Commun. 163, 56–63.
Bernasconi, P., Torchiana, E., Confalonieri, P., Morandi, L., Brugnoni, R.,
Barresi, R., Mora, M., Cornelio, F., Mantegazza, R., 1995. Expression
of transforming growth factor-beta1 in dystrophic patient muscles
correlates with fibrosis: pathogenetic role of fibrogenic cytokines. J.
Clin. Invest. 96, 1137–1144.
Border, W.A., Noble, N.A., 1994. Transforming growth factor-beta in tissue
fibrosis. N. Engl. J. Med. 331, 1286–1292.
Chen, Y.W., Zhao, P., Borup, R., Hoffman, E.P., 2000. Expression profiling
in the muscular dystrophies: identification of novel aspects of molecular
pathophysiology. J. Cell Biol. 151, 1321–1336.
Chen, K., Wei, Y., Shrap, G.C., Braley-Mullen, H., 2002. Inhibition of
TGFbeta1 by anti-TGFbeta1 antibody or lisinopril reduces thyroid
fibrosis in granulomatous experimental autoimmune thyroiditis. J.
Immunol. 169, 6530–6538.
De Luca, A., Nico, B., Liantonio, A., Di donna, M.P., Fraysse, B., Pierno,
S., Burdi, R., Mangeri, D., Rolland, J.F., Camerino, C., Zallone, A.,
Confalonieri, P., Andreetta, F., Arnoldi, E., Courdier-Fruh, I., Mayar,
J.P., Frigeri, A., Pisoni, M., Svelto, M., Conte-Camerino, D., 2005. A
multidisciplinary evaluation of the effectiveness of Cyclosporine A in
dystrophic mdx mice. Am. J. Pathol. 166, 477–489.
Duance, V.C., Stephens, H.R., Dunn, M., Bailey, A.J., Dubowitz, V., 1980.
A role for collagen in the pathogenesis of muscular dystrophy? Nature
284, 470–472.
Engel, A.G., Arahata, K., 1986. Mononuclear cells in myopathies:
quantitation of functionally distinct subsets, recognition of antigen-
specific cell-mediated cytotoxicity in some diseases. Human Pathol. 17,
704–721.
Engel, A.G., Yamamoto, M., Fischbeck, K.H., 1994. Dystrophinopathies.
In: Engel, A.G., Franzini-Armstrong, C. (Eds.), Myology, vol. 2.
McGraw-Hill, New York, pp. 1133–1187.
Foidart, M., Foidart, J.M., Engel, W.K., 1981. Collagen localization in
normal and fibrotic skeletal muscle. Arch. Neurol. 38, 152–157.
Gosselin, L.E., Williams, J.E., Deering, M., Brazeau, D., Koury, S.,
Martinez, D.A., 2004. Localization and early time course of TGF-beta1
mRNA expression in dystrophic muscle. Muscle Nerve 30, 645–653.
Granchelli, J.A., Pollina, C., Hudecki, M.S., 2000. Pre-clinical screening of
drugs using the mdx mouse. Neuromuscul. Disord. 10, 235–239.
Hartel, J.V., Granchelli, J.A., Hudecki, M.S., Pollina, C.M., Gosselin, L.E.,
2001. Impact of prednisone on TGF-beta1 and collagen in diaphragm
muscle from mdx mice. Muscle Nerve 24, 428–432.
Heino, J., Massague, J., 1990. Cell adhesion to collagen and decreased
myogenic gene expression implicated in the control of myogenesis by
transforming growth factor beta. J. Biol. Chem. 265, 10181–10184.
Hoffman, E.P., Dressman, D., 2001. Molecular pathophysiology and
targeted therapeutics for muscular dystrophy. Trends Pharmacol. Sci.
22, 465–470.
Kovacs, E.J., 1991. Fibrogenic cytokines: role of immune mediators in the
development of scar tissue. Immunol. Today 12, 17–23.
Kulkarni, A.B., Karlsson, S., 1993. Transforming growth factor-beta 1
knockout mice: a mutation in one cytokine causes a dramatic
inflammatory disease. Am. J. Pathol. 143, 3–9.
F. Andreetta et al. / Journal of Neuroimmunology 175 (2006) 77–8686
Lefaucheur, J.P., Gjata, B., Lafont, H., Sebille, A., 1996. Angiogenic and
inflammatory responses following skeletal muscle injury are altered by
immune neutralization of endogenous bFGF, IGF1 and TGF-beta1. J.
Neuroimmunol. 70, 37–44.
Louboutin, J.P., Fichter-Gagnepain, V., Thaon, E., Fardeau, M., 1993.
Morphometric analysis of mdx diaphragm muscle fibers. Comparison
with hindlimb muscles. Neuromuscul. Disord. 3, 463–469.
McCormick, L.L., Zhang, Y., Tootell, E., Gilliam, A.C., 1999. Anti-TGF-
beta treatment prevents skin and lung fibrosis in murine scleroderma-
tous graft-versus-host disease: a model for human scleroderma. J.
Immunol. 163, 5693–5699.
Miyazono, K., 2000. Positive and negative regulation of TGF-beta
signaling. J. Cell. Sci. 113, 1101–1109.
Morrison, J., Lu, Q.L., Pastoret, C., Partridge, T., Bou-Gharios, G., 2000. T-
cell-dependent fibrosis in the mdx dystrophic mouse. Lab. Invest. 80,
881–891.
Morrison, J., Palmer, D.B., Cobbold, S., Partridge, T., Bou-Gharios, G.,
2005. Effects of T-lymphocyte depletion on muscle fibrosis in the mdx
mouse. Am. J. Pathol. 166, 1701–1710.
Porter, J.D., Khanna, S., Kaminski, H.J., Rao, J.S., Merriam, A.P.,
Richmonds, C.R., Leahy, P., Li, J., Guo, W., Andrade, F.H., 2002. A
chronic inflammatory responses dominates in skeletal muscle molecular
signature in dystrophin-deficient mdx mice. Hum. Mol. Genet. 11,
263–272.
Sanes, J.R., 1994. The extracellular matrix. In: Engel, A.G., Franzini-
Armstrong, C. (Eds.), Myology, vol. 1. McGraw-Hill, New York, pp.
242–260.
Skuk, D., Vilquin, J.T., Tremblay, J.P., 2002. Experimental and therapeutic
approach to muscular dystrophies. Curr. Opin. Neurol. 15, 563–569.
Spencer, M.J., Mellgren, R.L., 2002. Overexpression of a calpastatin
transgene in mdx muscle reduces dystrophic pathology. Hum. Mol.
Genet. 11, 2645–2655.
Spencer, M.J., Walsh, C.M., Dorshkind, K.A., Rodriguez, E.M., Tidball,
J.G., 1997. Myonuclear apoptosis in dystrophic mdx muscle occurs by
perforine-mediated cytotoxicity. J. Clin. Invest. 99, 2745–2751.
Spencer, M.J., Marino, M.W., Winckler, W.M., 2000. Altered pathological
progression of diaphragm and quadriceps muscle in TNF-deficient,
dystrophin-deficient mice. Neuromuscul. Disord. 10, 612–619.
Spencer, M.J., Montecino-Rodriguez, E., Dorshkind, K., Tidball, J.G., 2001.
Helper (CD4(+)) and cytotoxic (CD8(+)) T-cells promote the pathology
of dystrophin-deficient muscle. Clin. Immunol. 98, 235–243.
Stedman, H.H., Sweeney, H.L., Shrager, J.B., Maguire, H.C., Panettieri,
R.A., Petrof, B., Narusawa, M., Leferovich, J.M., Sladky, J.T., Kelly,
A.M., 1991. The mdx mouse diaphragm reproduces the degenerative
changes of Duchenne muscular dystrophy. Nature 352, 536–539.
Stephens, H.R., Duance, V.C., Dunn, M.J., Bailey, A.J., Dubowitz, V., 1982.
Collagen types in neuromuscular diseases. J. Neurol. Sci. 53, 45–62.
Wahl, S.M., Hunt, D.A., Wong, H.L., Dougherty, S., McFarney-Francis, N.,
Wahl, L.M., Ellingsworth, L., Schmidt, J.A., Hall, G., Roberts, A.B.,
Sporn, M.B., 1988. Transforming growth factor-beta is a potent
immunosuppressive agent that inhibits IL-1 dependent lymphocyte
proliferation. J. Immunol. 140, 3026–3031.
Wahl, S.M., 1992. Transforming growth factor beta (TGF-h) in inflamma-
tion: a cause and a cure. J. Clin. Immunol. 12, 61–74.
Wahl, S.M., 1994. Transforming growth factor beta: the good, the bad, and
the ugly. J. Exp. Med. 180, 1587–1590.
Yamazaki, M., Minota, S., Sakurai, H., Miyazono, K., Yamada, A.,
Kanazawa, I., Kawai, M., 1994. Expression of transforming growth
factor-beta1 and its relation to endomysial fibrosis in progressive
muscular dystrophy. Am. J. Pathol. 144, 221–226.
Ziyadeh, F.N., Hoffman, B.B., Han, D.C., Iglesias-De la Cruz, M.M., Hong,
S.W., Isono, M., Chen, S., McGowan, T.A., Sharma, K., 2000. Long-
term prevention of renal insufficiency, excess matrix gene expression,
and glomerular mesangial matrix expansion by treatment with mono-
clonal antitransforming growth factor-beta antibody in db/db diabetic
mice. Proc. Natl. Acad. Sci. U. S. A. 97, 8015–8020.
