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Monocyte chemoattractant protein 3 as a mediator of fibrosisOverexpression in systemic sclerosis and the type 1 tight-skin mouse.

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Vol. 48, No. 7, July 2003, pp 1979–1991
DOI 10.1002/art.11164
© 2003, American College of Rheumatology
Monocyte Chemoattractant Protein 3 as a Mediator of Fibrosis
Overexpression in Systemic Sclerosis and the Type 1 Tight-Skin Mouse
Voon H. Ong, Lowri A. Evans, Xu Shiwen, Ivan B. Fisher, Vineeth Rajkumar,
David J. Abraham, Carol M. Black, and Christopher P. Denton
Objective. To determine the gene-expression profile in dermal fibroblasts from type 1 tight-skin (Tsk1)
mice, and to examine the expression and potential
fibrotic activity of monocyte chemoattractant protein 3
(MCP-3) in Tsk1 mouse and human systemic sclerosis
(SSc) skin.
Methods. Complementary DNA microarrays
(Atlas 1.2) were used to compare Tsk1 fibroblasts
with non-Tsk1 littermate cells at 10 days, 6 weeks, and
12 weeks of age. Expression of MCP-3 protein was
assessed by Western blotting of fibroblast culture supernatants, and localized in the mouse and human skin
biopsy samples by immunohistochemistry. Activation of
collagen reporter genes by MCP-3 was explored in
transgenic mouse fibroblasts and by transient transfection assays.
Results. MCP-3 was highly overexpressed by
neonatal Tsk1 fibroblasts and by fibroblasts cultured
from the lesional skin of patients with early-stage
diffuse cutaneous SSc. Immunolocalization confirmed
increased expression of MCP-3 in the dermis of 4 of
5 Tsk1 skin samples and 14 of 28 lesional SSc skin
samples, compared with that in matched healthy mice
(n ⴝ 5) and human controls (n ⴝ 11). Pro␣2(I) collagen
promoter–reporter gene constructs were activated by
MCP-3 in transgenic mice and by transient transfection
assays. This response was maximal between 16 and 24
hours of culture and mediated via sequences within the
proximal promoter. The effects of MCP-3 could be
diminished by a neutralizing antibody to transforming
growth factor ␤.
Conclusion. We demonstrate, for the first time,
overexpression of MCP-3 in early-stage SSc and in
Tsk1 skin, and suggest a novel role for this protein as a
fibrotic mediator activating extracellular matrix gene
expression in addition to promoting leukocyte trafficking. This chemokine may be an important early member
of the cytokine cascade driving the pathogenesis of SSc.
Systemic sclerosis (SSc) is a multisystem connective tissue disorder characterized by skin thickening and widespread, but variable, visceral fibrosis.
Its pathogenesis involves immunologic activation and
vascular dysfunction leading to excessive accumulation
of extracellular matrix (ECM) in lesional tissues. A
number of well-characterized animal models have
been used to elucidate the pathogenesis of SSc. The
type 1 tight-skin (Tsk1) mouse develops skin fibrosis
reminiscent of human SSc, with increased synthesis
and accumulation of collagen-rich matrix in the skin.
Activated fibroblasts are implicated in the overproduction of ECM components, but the mechanisms by which
these cells are activated in SSc remain incompletely
Soluble profibrotic mediators, including transforming growth factor ␤1 (TGF␤1) and connective
tissue growth factor (CTGF), have been shown to be
up-regulated in SSc, and a hierarchical cascade of cytokines in which initial induction of proinflammatory
cytokines leads to later expression of profibrotic mediators has been proposed (1). Inflammatory cell recruit-
Supported by The Wellcome Trust, The Arthritis Research
Campaign (UK), and The Raynaud’s and Scleroderma Association
Voon H. Ong, MRCP, Lowri A. Evans, MSc, Xu Shiwen,
PhD, Ivan B. Fisher, BSc, Vineeth Rajkumar, MSc, David J. Abraham,
PhD, Carol M. Black, CBE, MD, PRCP, FACP, Christopher P.
Denton, PhD, FRCP: Royal Free Hospital and University College
Medical School, London, UK.
Address correspondence and reprint requests to Christopher
P. Denton, PhD, FRCP, Centre for Rheumatology, Royal Free and
University College Medical School, Royal Free Hospital, Pond Street,
London NW3 2QG, UK. E-mail:
Submitted for publication December 10, 2002; accepted in
revised form March 12, 2003.
ment is an early feature of SSc and may be involved in
the activation of fibroblasts. Vectorial migration of
inflammatory cells into subendothelial tissues is a multistep process which is mediated by a series of cellular
interactions that establish chemotactic gradients in the
perivascular space. Both resident fibroblasts and extravascular leukocytes may provide the initial migratory
trigger. Members of the chemokine family of proteins
are implicated in the regulation of this process (2).
Chemokines are small, basic peptides having a molecular weight of 8–11 kd. Like other cytokines, they may be
produced constitutively or after induction, and exert
their effect locally or in a paracrine or autocrine manner.
They are classified into 4 groups: CC, CXC, C, and
CX3C, according to the position of 2 highly conserved
cysteine residues (3).
Monocyte chemoattractant protein (MCP) types
1, 2, 3, 4, and 5 constitute a subfamily within the CC(␤)
chemokines. MCP-1 was first identified as a monocytespecific cytokine, followed by MCP-2, MCP-3, MCP-4,
and MCP-5 (4–7). There is high amino-acid sequence
homology among the 5 MCP chemokines (60–70%)
compared with ⬃40% homology between other, nonMCP CC chemokines. MCP chemokines exert their
putative effects through activation of specific G protein–
coupled 7-transmembrane receptors. They are proinflammatory in nature, and chemotactic for monocytes,
eosinophils, and basophils. Chemokine expression is
increased in a number of different pathologic processes,
including synovial pannus of rheumatoid joints, autoimmune lesions of multiple sclerosis, affected mucosal
surfaces in ulcerative colitis and Crohn’s disease, lung
inflammation in chronic bronchitis, sarcoidosis, and
asthma, and the vascular inflammation that characterizes atherosclerosis (8–12). Overexpression of MCP-1
by SSc fibroblasts was first found to underlie the promotion of leukocyte migration across endothelial cell
monolayers by these cells (13), and other studies have
subsequently confirmed overexpression of MCP-1 in
SSc tissues (14,15) and by lesional fibroblasts in
culture (16). Up-regulation of MCP-1 has also been
demonstrated in several chronic inflammatory disorders (17).
In the present study, we describe, for the first
time, the overexpression of another CC chemokine,
MCP-3, in the Tsk1 mouse model using gene-expression
profiling, and investigate whether this factor is also
overexpressed in human SSc. Since inflammatory cell
infiltration is not a feature of Tsk1 skin, and on the
basis of analogy with MCP-1 (18,19), we have investigated the potential for MCP-3 to promote collagen-gene
expression and thereby operate as a novel mediator of
Patients and controls. All patients and controls
participated in the study after providing their informed consent, in accordance with local institutional guidelines.
Dermal punch biopsy samples were obtained from 22
patients with diffuse cutaneous systemic sclerosis (dcSSc),
6 with limited cutaneous systemic sclerosis (lcSSc), and
11 healthy controls. Skin samples were immediately divided
for tissue culture and snap frozen for histologic study. Patients’
characteristics are summarized in Table 1. Samples were
classified as having early dcSSc (n ⫽ 14) if obtained within
the first 2 years of disease onset, as defined by the appearance
of the first non–Raynaud’s phenomenon symptom. Patients
with established dcSSc (n ⫽ 8) had ⬎2 years of disease.
All SSc patients fulfilled the American College of
Rheumatology (formerly, the American Rheumatism Association) preliminary criteria for the classification of SSc (20).
Patterns of organ involvement at the time of biopsy were
assessed according to standard practice. Briefly, esophageal
involvement was determined by the medical history, the barium swallow test, or scintigraphy. Lung fibrosis was assessed by
high-resolution computed-tomography scanning in the presence of a restrictive pattern of pulmonary function abnormalities. Cardiac involvement was considered present if electrocardiogram alterations, impaired ventricular function, or
pericardial effusion were evident on echocardiography. Pulmonary arterial hypertension (PAH) was determined by an elevated pulmonary artery peak systolic pressure on Doppler
echocardiography, or by other echocardiographic hallmarks of
PAH, and usually confirmed by right heart catheterization. A
creatinine kinase level elevated more than 2-fold defined
skeletal muscle involvement. Renal involvement was identified
by a history of scleroderma renal crisis or significant impairment of creatinine clearance.
Patients were treated with vasodilators and, in most
patients with dcSSc, by immunosuppression using anti–
thymocyte globulin and mycophenolate mofetil (21). Biopsy
samples of skin from patients with early-stage dcSSc were
obtained prior to immunosuppressive treatment. Patients with
lcSSc were receiving vasodilator therapy only at the time of
biopsy. Among patients with established dcSSc, 88% (n ⫽ 7)
were taking immunosuppressant therapy. Three patients
with lcSSc had pulmonary, renal, and muscle involvement
requiring treatment with low-dose corticosteroids and immunosuppression.
Tight-skin and transgenic mice. To examine the profibrotic effects of MCP-3 on extracellular matrix gene expression, we used dermal fibroblasts cultured from transgenic mice
harboring a bacterial ␤-galactosidase reporter gene regulated
by a fibroblast-specific expression cassette (2kb-LacZ). A far
upstream transcriptional enhancer between ⫺17.1 kb and
⫺15.1 kb of the transcription start site and linked to a minimal
endogenous promoter shows a consistently high level of
fibroblast-specific expression in embryonic development and
postnatally. In these mice, basal high-level expression of the
Table 1.
Clinical features of systemic sclerosis patients and healthy controls*
Sex, male/female, no.
Age, mean ⫾ SD years
Duration of disease, mean ⫾ SD months
Duration of Raynaud’s phenomenon
symptoms, mean ⫾ SD months
Organ involvement, %
Other gastrointestinal
Serology, %
Anti–topoisomerase I
Anti–RNA polymerase I/III
Anti–nuclear RNP
(n ⫽ 14)†
(n ⫽ 8)
(n ⫽ 6)
(n ⫽ 11)
49.3 ⫾ 9.9
16.7 ⫾ 4.2
18.9 ⫾ 6.6
57.3 ⫾ 12.4
69 ⫾ 51.2
75 ⫾ 53.2
44.3 ⫾ 16.2
66 ⫾ 58.2
154 ⫾ 49.4
45 ⫾ 14.4
* dcSSc ⫽ diffuse cutaneous systemic sclerosis; lcSSc ⫽ limited cutaneous systemic sclerosis.
† Defined by ⬍2 years’ duration.
reporter gene correlates with expression of the type I collagen
gene. Selective up-regulation of this reporter transgene has
been demonstrated in vivo, using Tsk1 mice (22). Tsk1 heterozygote males were crossed with homozygous pallid females so
that genotyping could be assessed by coat color from 7 days
For gene-expression profiling and assessment of
MCP-3 expression, skin biopsy samples were obtained from
the interscapular region of Tsk1 (n ⫽ 5) and wild-type (pallid
coat color, n ⫽ 5) littermate mice at between 3 days and
12 weeks of age. For most immunostaining experiments,
10-day- and 3-week-old mouse tissues were examined, since
these time points gave consistent and reproducible expression
Fibroblast cultures. Fibroblasts derived from 3
sources were used for these experiments. Tight-skin and
non–tight-skin dermal fibroblasts were cultured from skin
extracted from the upper back of same-sex littermate mice.
For functional studies of the fibroblast response to MCP-3,
reporter transgenic mouse fibroblasts were cultured from
the 2kb-LacZ mouse line as described above. This allowed
expression of the reporter transgene to be examined in
tissue culture, and allowed the effect of recombinant MCP-3
on the level of transgene expression to be assessed. Human
dermal fibroblasts were cultured from dcSSc patients and from
age- and site-matched control human skin. Neonatal mouse
fibroblasts were used for transient transfection experiments,
since they show consistent responsiveness to recombinant
Cells were grown by explant culture and maintained in
Dulbecco’s modified Eagle’s medium (Gibco BRL, Grand
Island, NY) supplemented with 10% fetal calf serum (Gibco
BRL), 100 units/ml penicillin, and 100 mg/ml streptomycin,
and cultured in a humidified atmosphere of 5% CO2 in
air. For experiments, fibroblasts were generally used at the
first or second passage, because later-passage cells showed
greater interculture variability. All cultures were inspected at
high power using a phase-contrast inverted microscope, to
confirm absence of epithelial cells and a typical fibroblastic
Analysis of gene expression by complementary DNA
(cDNA) microarray. To determine differences in the expression profiles of fibroblasts from Tsk1 and non-Tsk1 mice, total
messenger RNA (mRNA) was prepared from confluent cultures of early-passage neonatal dermal fibroblasts using Trizol
(Gibco BRL), in accordance with the manufacturer’s protocol.
Expression analysis was performed using the mouse Atlas 1.2
array (ClonTech, Palo Alto, CA), which incorporates oligonucleotides specific for 1,176 mouse-gene transcripts. A
cDNA-synthesis gene-specific primer mix, used for probe
synthesis, is enriched for sequences corresponding to the
cDNA for the genes on the array. Hybridization was performed according to the manufacturer’s instructions (ClonTech). Briefly, after DNAse I treatment of the total RNA
to remove any genomic contaminants, 5 ␮g of each paired
sample was incubated with the sequence-specific primer
and reverse transcriptase. The resulting cDNA probes, labeled
by incorporation of ␣32P-dATP, were hybridized with microarrays. The methods are discussed in detail on the manufacturer’s Web site (
Experiments were performed in parallel, and generally, 4 membranes were probed at the same time. Initial
experiments suggested that this gave more reproducible
data when comparing expression patterns between samples.
After hybridization, membranes were washed and radioactivity was determined by phosphorimaging. Differential gene
expression was assessed using AtlasImage software (ClonTech). Normalization of the expression data was achieved by
using the sum of the global intensities of the arrays with the
local background signal for each cDNA spot taken into
account. This analysis used a default difference threshold of
7 for gene detection, and in order for the ratio of the 2
individual samples to be significant, a 2-fold difference in gene
expression was selected, and this difference must have been
observed on at least 3 occasions, including 2 independent
experiments. To minimize the effect of genetic background
and sex, littermate animals were examined and same-sex
(male) mice were used.
Western blot analysis. To extend the cDNA microarray data, fibroblasts from Tsk1 and non-Tsk1 and human
SSc or control samples were used. After overnight incubation
of skin samples in serum-free medium (200 ␮l per well),
culture supernatants were collected and cell layers were
lysed with Laemmli lysis buffer. Sodium dodecyl sulfate–
polyacrylamide gel electrophoresis was performed on 18%
Tris-glycine gels (Invitrogen, Paisley, UK), and the separated
proteins were transferred onto nitrocellulose membranes
at 30V for 90 minutes. Membranes were blocked by incubation for 1 hour with 5% nonfat milk in phosphate buffered
saline (PBS) containing 0.2% Tween 20, and were stained
Figure 1. Constructs used to examine pro␣2(I) collagen gene activation. The minimal promoter sequences were previously defined in
transient transfection, from ⫺354 bp and ⫺378 bp upstream of the
transcription start site for the mouse and human pro␣2(I) collagen
genes, respectively. Larger constructs incorporated additional sequences from the evolutionarily conserved far-upstream transcriptional enhancer that has previously been shown to be a target for
activation in fibrosis. The 6kb-LacZ construct includes the region from
⫺19.5 kb to ⫺13.5 kb upstream of the transcription start site, and the
2kb-LacZ incorporates 2 kb of the upstream region linked to a minimal
murine ⫺354-bp promoter. Construction of the human 5kb-LacZ
contains sequences ⫺22.8 kb to ⫺17.5 kb, which is linked to a minimal
⫺378-bp promoter.
Figure 2. Overexpression of monocyte chemoattractant protein 3
(MCP-3) in type 1 tight-skin (Tsk1) neonatal mouse fibroblasts. A,
Representative neonatal fibroblast gene-expression profiles are shown
using portions of 2 mouse Atlas 1.2 cDNA arrays, corresponding to
cytokine and growth factor or extracellular matrix transcripts. Analysis
using AtlasImage software reveals significant up-regulation (shown in
red) of tissue inhibitor of matrix metalloproteinase 3 (TIMP3), laminin ␤2
subunit (LAMC1), fibronectin 1 (FN1), thrombospondin 1 (TSP1),
MCP-3, cytoplasmic dynein light chain 1 (dynein), Bcl-2 interacting
protein (NIP3), inhibin ␤A subunit (inhibin), insulin-like growth factor
binding protein 6 (IGFBP6), and cholecystokinin A receptor (CCKR).
Conversely, there was down-regulation (shown in blue) of cordon bleu
protein (COBL), Cell Division Cycle 46 (CDC46), RAD23 ultraviolet
excision repair protein (RAD23), laminin ␥1 subunit (laminin), CD28
precursor, and interleukin-1 receptor (IL1R). Genes that are similarly
expressed in both samples are indicated in green. Half green/half red and
half green/half blue indicate that although a difference (lower portion)
was observed between the defined samples, the ratio (upper portion) is
nonsignificant. The data are representative of triplicate experiments. B,
Increased MCP-3 protein secretion by Tsk1 fibroblasts. The most highly
overexpressed transcript in neonatal Tsk1 fibroblasts in replicate experiments was MCP-3. Overexpression was confirmed by Western blot
analysis of Tsk1 and control (wild-type [Wt]) fibroblast culture supernatants. Cell-layer ␤-actin was used as a protein-loading control. Protein
levels were quantified in a representative experiment as shown with the
corresponding histogram.
for 1 hour using the following antibodies: goat anti-human
MCP-1 antibody, goat anti-human MCP-3 antibody (100 ␮g/
ml) (all from R&D Systems, Oxford, UK), and goat antimouse MCP-3 antibody (500 gm/ml) (Abcam, Cambridge,
UK) at a dilution of 1:1,000. The nitrocellulose transfers
were washed 3 times before being stained with biotinylated
species-specific secondary antibody for 1 hour, and again
washed 3 times before being stained with biotin substrate
(Vectastain; Vector Laboratories, Peterborough, UK) and
then chemiluminescent substrate (Amersham Pharmacia Biotech, Little Chalfont, UK), and developed against photographic film (Hyperfilm Enhanced Chemiluminescence;
Amersham Pharmacia Biotech). Parallel blots of cell lysates
from each culture were probed with a monoclonal antibody
against ␤-actin (Sigma, St. Louis, MO) to control for variation
in cell number between cultures.
Immunohistochemical staining of serial sections.
For detection of MCP-3 protein, immunohistochemistry
analysis with a biotin/streptavidin-based amplifying system
was performed on mice and human skin sections. Serial
frozen sections (5 ␮m) were cut on a cryostat at ⫺30°C
and air-dried for an hour. Sections were fixed in ice-cold
acetone and covered with 3% hydrogen peroxide for 10
minutes in the dark to block endogenous peroxidase activity.
Slides were then blocked with 20% normal horse serum,
and incubated with goat polyclonal anti-mouse MARC/MCP-3
IgG antibody and goat polyclonal anti-human MCP-3 IgG
antibody (25 ␮g/ml in PBS; R&D Systems) for 1 hour at
room temperature. After washing with PBS, sections were
incubated with biotinylated horse anti-goat IgG diluted in
PBS (7.5 ␮g/ml, BA-9500; Vector Laboratories) for 30 minutes, rinsed, and finally incubated with Vectastain Elite
STR-ABC reagent (Vector Laboratories) for 30 minutes. After
washing, sections were visualized using 3-amino-9ethylcarbazole chromogen and H2O2 as substrate (SK-4200;
Vector Laboratories). Sections were then washed in tap water,
counterstained with Carrazzis hematoxylin, and mounted with
Gelmount (Biomeda, Foster City, CA) for examination using
an Olympus BH-2 photomicroscope. Controls included an
exchange of primary antibodies with goat isotype-matched
Transactivation of pro␣2(I) collagen reporter genes
by recombinant MCP-3. The effect of recombinant murine
MCP-3 was examined using tissue-culture cells. Initially,
transgenic fibroblasts from the 2kb-LacZ line were used,
since these cells provide a highly physiologic assay for
transactivation of a genomic ␤-galactosidase reporter for
which endogenous expression in vivo recapitulates that of
endogenous type I collagen genes. Concentrations between
1 ng/ml and 500 ng/ml of recombinant chemokine were
evaluated in early experiments, and 100–400 ng/ml gave
consistent effects and were used in the majority of later
studies. Transient transfection was used to assess MCP-3
responsiveness of other pro␣2(I) collagen promoter constructs. Analyses were performed in 24-well plates using
Lipofectamine-plus, as described by the manufacturer (Life
Technologies, Gaithersburg, MD). Briefly, 1 ␮g of collagen
reporter plasmid was cotransfected with 0.1 ␮g of pCMVluc control plasmid to allow correction of ␤-galactosidase
expression for transfection efficiency. The reporter constructs used are shown in Figure 1. These included minimal
promoter sequences of mouse or human pro␣2(I) collagen as well as constructs that incorporate sequences
from the far-upstream fibroblast-specific enhancer previously
defined in transgenic mice. This element operates as
a lineage-specific transcriptional enhancer in vivo and is a
target for activation in Tsk1 mice (22). Reporter gene expression was determined using the Galactolight assay kit (Tropix,
Bedford, MA).
TGF␤ neutralization studies. To examine the potential
role of TGF␤ isoforms as downstream effectors of collagen
gene activation by MCP-3, we used a pan-specific anti-TGF␤
monoclonal antibody (1D11; R&D Systems) to block the
effects of recombinant MCP-3 on neonatal mouse fibroblasts
harboring a minimal COL1A2 promoter–reporter construct.
Initial experiments suggested that a concentration of 50 ␮g/ml
blocked the maximal effect of recombinant TGF␤1 (data not
Table 2. Summary of consistently differentially expressed genes in neonatal or adult type 1 tight-skin (Tsk1) mouse fibroblasts*
Prelesional (neonatal)
Established (12 weeks)
Gene name
accession no.
Monocyte chemoattractant protein 3
(MARC-1, CCL7)
Monocyte colony-stimulating factor 1
Cluster differentiation antigen 44
Thrombospondin 1
Hypoxia inducible factor 1␣
Tissue inhibitor of matrix metalloproteinase 3
Fibronectin 1
Transforming growth factor ␤1
Integrin ␤1
Keratinocyte growth factor (FGF7)
* Genes listed are those with consistent differential expression either in early or late-stage Tsk1 in 3 experiments. Values for expression data are
relative to the mean signal for the total signal intensity of expressed genes, after correction for local background signal.
† Differential gene expression in Tsk1 fibroblasts compared with non-Tsk1 (pallid) littermate cells.
unpaired t-test, and a P value less than 0.05 was taken as
statistically significant.
Figure 3. Overexpression of monocyte chemoattractant protein 1
(MCP-1) and MCP-3 by lesional systemic sclerosis (SSc) human
skin fibroblasts. A, Fibroblast culture media from representative
normal or SSc dermal fibroblast cultures were analyzed using antibodies to MCP-3 and MCP-1. Cell-layer ␤-actin staining was used as
a loading control, and recombinant human MCP-1 and MCP-3 (rH)
were used to confirm primary antibody specificity. B, Densitometric
analysis summarizing 3 independent experiments shows the mean
and SEM protein levels for MCP-3 and MCP-1 in relative density
units (RDU).
shown), and this concentration was selected for neutralization
experiments using MCP-3.
Statistical analysis. For quantitative variables, the
mean ⫾ SEM results from replicate samples, or from combined independent experiments where between-experiment
variation allowed reliable combination of raw data, were
compared. Means were compared by Student’s paired or
Gene-expression analysis identifies overexpression of MCP-3. Parallel assessment of gene expression
was performed using cDNA microarrays, comparing
fibroblasts from Tsk1 and wild-type (control) littermate skin biopsy samples at 3 time points: neonatal,
6 weeks old, and 12 weeks old. Neonatal fibroblasts
expressed 244 (21%) of the 1,176 genes represented
on the array. At 6 and 12 weeks, 147 genes (13%)
and 94 genes (8%), respectively, were expressed.
Representative portions of the cDNA expression
arrays for neonatal fibroblasts are shown in Figure 2A.
Differential expression was defined by 2-fold differences in hybridization between the control and Tsk1
mRNA samples. Thus, 51 genes were differentially expressed neonatally, whereas the number of differentially
expressed transcripts decreased among fibroblasts from
adult mice. No significant differences were observed between the 6- and 12-week expression profiles.
For 13 genes, there was differential expression at
either the neonatal or 12-week time point or at both of
these time points. These genes, together with the normalized expression relative to the basal wild-type neonatal fibroblast levels, are listed in Table 2. More than 40
of the 51 genes that were differentially expressed neonatally were not expressed consistently in adult samples,
and these were not analyzed further. Conversely, 3 genes
that were not differentially expressed in neonatal fibroblasts were found to be differentially expressed in adult
cells, and these are included. Some genes, including
thrombospondin 1, osteopontin, hypoxia inducible factor 1␣, tissue inhibitor of matrix metalloproteinase 3,
and TGF␤1, showed sustained overexpression at all time
points (prelesional through established). Others, including biglycan, were overexpressed neonatally but suppressed in fibroblasts derived from established fibrotic
In neonatal cultures, MCP-3 was the most overexpressed gene with more than 15-fold greater expression in neonatal Tsk1 fibroblasts, compared with nonTsk1 fibroblasts, although sustained overexpression was
not seen at later time points despite protein upregulation in Tsk1 samples at later time points.
Western blot analysis of MCP-3 protein. Transcript levels may not reflect changes in the gene product,
since many genes are also regulated posttranscriptionally. Therefore, Western blotting of fibroblast culture
greater than that observed for MCP-1 (RDU 3.84 ⫾ 0.73
versus 7.87 ⫾ 0.14, respectively; P ⫽ 0.01), as shown in
Figure 3.
Effect of MCP-3 on collagen reporter gene expression. Analysis of dermal fibroblasts cultured from
2kb-LacZ transgenic mice suggested that transactivation
of type I collagen by MCP-3 occurs in tissue culture.
Using a highly physiologic bioassay, a series of independent experiments (n ⫽ 5) showed that MCP-3 increased
the transactivation of the reporter transgene, with a
maximal mean (⫾SEM) change above baseline levels of
80 ⫾ 31% (P ⫽ 0.01). Similar up-regulation was observed with TGF␤1-treated cells (70 ⫾ 24% above
baseline) (Figure 4). Maximum induction of transgene expression was at 24 hours, with a threshold
concentration that varied between experiments but
Figure 4. Activation of murine pro␣2(I) collagen (Col1a2) gene
expression by monocyte chemoattractant protein 3 (MCP-3). A, Fibroblasts cultured from the skin of transgenic mice with high-level
fibroblast-specific expression of ␤-galactosidase (2kb-LacZ) were used
to examine MCP-3 activation of extracellular matrix gene expression.
Values are the mean and SEM of triplicate samples and are representative of 5 independent experiments. ⴱ ⫽ P ⬍ 0.05 as determined by
Student’s unpaired t-test. B, Transactivation of the murine Col1a2
gene in transient transfection is shown as reporter gene expression in
3 independent experiments for the minimal promoter construct.
Activation is similar to maximum induction by recombinant transforming growth factor ␤1 (TGF␤) (10 ng/ml) at 24 hours. ⴱ ⫽ P ⬍ 0.05;
ⴱⴱ ⫽ P ⬍ 0.01, by Student’s unpaired t-test. RLU ⫽ relative luminescence units.
supernatants was used to assess changes at the protein
level. Up-regulation of MCP-3 was observed in 6-weekold Tsk1 samples in a series of 3 independent experiments (mean ⫾ SEM 235 ⫾ 26% compared with the
non-Tsk1 controls; P ⫽ 0.002) (Figure 2B). Similar
results were observed in parallel analyses of dermal
fibroblast culture media derived from early-stage dcSSc
skin compared with matched human controls. The level
of MCP-3 immunoreactivity in SSc fibroblast supernatants (mean ⫾ SEM relative density units [RDU] 4.92 ⫾
1.12 in controls versus 8.27 ⫾ 0.5 in SSc; P ⫽ 0.02) was
Figure 5. Activation of a human pro␣2(I) collagen (COL1A2)
gene reporter by MCP-3 via a TGF␤-dependent mechanism.
A, Up-regulation of the COL1A2-regulated reporter gene by MCP-3
is expressed as the mean and SEM percentage of basal expression from 3 independent experiments and corrected for transfection efficiency. B, Anti-TGF␤ antibody significantly reduces
MCP-3–induced activation of human COL1A2 promoter constructs
at 24 hours. Data are representative of 3 independent experiments
and expressed as the mean and SEM of triplicate samples. ⴱ ⫽ P ⬍
0.05 by Student’s paired t-test. See Figure 4 for other definitions.
was generally between 30 ng/ml and 250 ng/ml of MCP-3
and showed very similar dose-response characteristics to
that previously observed for the related chemokine
MCP-1 (19). Results from a series of duplicate experiments revealed that initial induction occurred at 6 hours
with up-regulation of 34 ⫾ 15%, which subsequently
rose and then declined to 45 ⫾ 22% above baseline at 48
Data from transgenic mice were confirmed
using transient transfection of wild-type fibroblasts
using the same 2kb-Col1␣2-LacZ construct (see Figure 1
for details). As shown in Figure 4B, later experiments
evaluating constructs harboring only a minimal Col1␣2
promoter showed a similar dose-dependent upregulation of reporter gene expression with a mean
(⫾SEM) activation above baseline of 72 ⫾ 51% and
85 ⫾ 30% at 200 ng/ml and 400 ng/ml of MCP-3,
respectively, from a series of 3 independent experiments
(P ⫽ 0.02). These data suggest that upstream elements
previously implicated in selective activation of reporter
transgenes in Tsk1 mice are not essential for the effect of
There is substantial sequence conservation of
both proximal and distal regulatory elements of the
mouse and human pro␣2(I) collagen genes (23). We
therefore confirmed our data on mouse constructs
and also tested analogous human promoter–reporter
constructs. Figure 5A summarizes the results from 3
independent experiments using a human minimal
promoter construct, with maximal induction of collagen reporter gene expression of 52 ⫾ 14% (mean ⫾
SEM above basal expression) (P ⫽ 0.02). Further transient transfection experiments showed no greater
induction with larger human 5-kb and murine 6-kb
upstream enhancer constructs, suggesting that lineagespecific upstream enhancer sequences are not important for activation in transient transfection (data not
Figure 6. Up-regulation of monocyte chemoattractant protein 3 expression in type 1 tight-skin (Tsk1) mouse
skin. Frozen skin sections from 3-week-old healthy non–tight-skin (pallid) mice (A and B) and Tsk1 mice at 3
weeks (C) and 10 days (D) old were immunostained. Healthy adult mice show perifollicular (p with arrow)
staining, whereas there is additional dermal expression (d with arrow) in Tsk1 samples. (Original magnification ⫻
240 in A and C; ⫻ 480 in B and D.)
The time course for stimulation of collagen
gene expression (between 16 and 24 hours) suggested
that a second mediator may be elaborated by MCP-3–
activated fibroblasts. Since TGF␤ is a potent stimulator of ECM gene expression in vitro, the effect of
anti-TGF␤ antibody on this stimulation of collagen
reporter gene expression was examined. The results
show that MCP-3 stimulation of fibroblast collagen
synthesis was partially inhibited in the presence of
anti-TGF␤ antibody. In addition, there was a dosedependent response with the antibody, which displayed a
maximal neutralization effect at 50 ␮g/ml, as shown in
Figure 5B.
MCP-3 expression in SSc skin. MCP-3 was detected abundantly in the dermal region in skin biopsy
samples from Tsk1 mice at 10 days and 3 weeks old.
Generalized perifollicular staining was observed using
anti–MCP-3 antibody in both non-Tsk1 control and Tsk1
skin sections. Despite this chemokine overexpression,
there was no demonstrable inflammatory infiltrate in the
skin sections from Tsk1 mice (Figure 6) at all ages
examined, from 3 days to 12 weeks.
In human skin samples, there was a variable
amount of specific immunostaining for MCP-3 in the
lower epidermal layer, in both healthy control and SSc
biopsy samples. However, as with Tsk1 skin, SSc lesional
skin sections showed dermal expression of MCP-3 as
well as additional strong MCP-3 expression in and
around the blood vessels at sites of mononuclear cell
infiltrates (Figures 7A–C). The majority of the inflammatory mononuclear cells in scleroderma skin were
positive for CD68 (Figure 7D). Consistent with the data
on mRNA and protein expression, additional immunostaining studies using anti-AS02 suggested that MCP-3
chemokines localized in dermal cells express this
fibroblast-specific marker well away from vascular or
epithelial structures (data not shown).
There was significantly more MCP-3 expression in the skin of patients with early-onset diffuse
disease compared with that from patients with established diffuse SSc or limited cutaneous SSc,
suggesting that MCP-3 is associated with inflammatory or progressive skin disease. Negative controls
with IgG isotype–matched antibodies showed no
staining. Table 3 summarizes the staining patterns
observed in human and mouse skin biopsy specimens. Immunostaining for MCP-3 was associated
with an inflammatory infiltrate that showed vascular
localization in 10 of the 14 skin sections from
patients with early dcSSc as well as more diffuse dermal
staining. Diffuse dermal staining was absent in the skin
sections from patients with established dcSSc and lcSSc.
Identification of MCP-3 as a highly overexpressed gene in fibroblasts from neonatal Tsk1 mice
raised the possibility that this chemokine might have a
role in the pathogenesis of fibrosis in these mice. Because the related CC chemokine MCP-1 has been shown
to be overexpressed in SSc fibroblasts, and since the
Tsk1 strain is regarded as a model for human SSc, we
extended our study to examine skin biopsy specimens
and fibroblasts cultured from a series of SSc patients.
We found overexpression of MCP-3 in SSc, and confirmed that it may operate as a profibrotic mediator with
the potential to activate ECM gene expression, at least
in part via induction of TGF␤1, in addition to promoting
an inflammatory cellular response. Although multiple
mediators are likely to be involved in the complex,
multifaceted pathology of SSc, the identification of
MCP-3 as a soluble factor capable of regulating 2 key
aspects of pathology, leukocyte migration and ECM
overproduction, is intriguing. It is plausible that early
mediators such as MCP-3 might induce other factors
such as TGF␤1, and perhaps other downstream candidates such as CTGF or platelet-derived growth factor
are induced later (1). The ability of fibroblasts to secrete
these factors supports a model of autocrine or paracrine
local regulatory pathways in pathogenesis. Potential
consecutive induction of fibroblast-derived mediators
also emphasizes the importance of disease stage–specific
approaches to targeted molecular therapies in SSc.
There are a number of approaches for determining broad differences in gene expression in different
tissue or cell samples. Other studies have used a variety
of technologies including differential-display reverse
transcription–polymerase chain reaction, ribonuclease
protection assay, and Northern blotting to examine
gene-expression profiles in connective tissue diseases,
including SSc. Candidate genes suggested by these approaches have included fibronectin, protease nexin 1,
interleukin-1␣, and CTGF (24,25). However, MCP-3
was not identified in these earlier studies. This may
reflect intrinsic differences in the SSc skin biopsy samples examined or in the fundamental properties of each
of the methods used for examining differential gene
expression, and emphasizes the value of using multiple
approaches to address the same question.
Overexpression of chemokines and related receptor genes, including MCP-3, has been described in
Figure 7. Increased monocyte chemoattractant protein 3 (MCP-3) expression in early-stage
diffuse cutaneous systemic sclerosis (dcSSc) skin biopsy samples. In skin sections from patients with
early dcSSc (A–D), vascular MCP-3 localization with dermal immunostaining is observed in A and
B, respectively, and MCP-3 protein is detected in the inflammatory perivascular infiltrate (C); most
of these samples are positive for the monocyte/macrophage marker CD68 (D). There is a variable
amount of epidermal staining (e with arrow) in skin sections from patients with established dcSSc
(E) and in clinically uninvolved early dcSSc skin (F) or limited cutaneous SSc (lcSSc) skin (G) and
in skin from healthy controls (H). Minimal vascular MCP-3 immunostaining is detected in
uninvolved skin from patients with established dcSSc (E) and lcSSc (G). (Original magnification ⫻
Table 3. Immunostaining patterns for human or mouse skin using specific anti–monocyte chemoattractant protein (MCP-3) antibody*
Human skin
Mouse skin
(n ⫽ 11)
Early dcSSc†
(n ⫽ 14)
Established dcSSc
(n ⫽ 8)
(n ⫽ 6)
(n ⫽ 5)
(n ⫽ 5)
Inflammatory infiltrate
Diffuse dermal
* Values are the no. of samples displaying the respective staining patterns for MCP-3. dcSSc ⫽ diffuse cutaneous systemic sclerosis; lcSSc ⫽ limited
cutaneous systemic sclerosis; Tsk1 ⫽ heterozygous type 1 tight-skin mouse; pallid ⫽ homozygous pallid (non–tight-skin) mouse.
† Defined by ⬍2 years’ duration.
gene-cluster analysis of data from high-density genechip experiments studying whole-lung samples in the
bleomycin-induced model of murine pulmonary fibrosis
(26). Also, in a recent analysis by Luzina et al (27) of
gene-expression profiles in bronchoalveolar lavage cells
from SSc patients with lung inflammation, there was
increased expression of chemokines and chemokine
receptor genes associated with a greater risk for lung
fibrosis. These studies suggest that cDNA microarray
approaches are valuable as a screening approach to
identify potential targets for further study. However,
results must be interpreted with caution, and repetition
of studies is important, including validation with independently prepared samples.
Homogenous populations of dermal fibroblasts
from Tsk1 and wild-type mice were used to reduce the
biologic variability, which may otherwise limit data
analysis. However, even with this starting material, there
was substantial variation when independently prepared
samples were compared, presumably due to differences
in cell cycle and other biologic parameters. Thus, many
of the differentially expressed genes initially seen in
neonatal fibroblasts were observed in only some littermate pairs. Replication of data is essential to reduce
background differences in gene expression that are
independent of fundamental differences in the cell lines,
even in genetically homogeneous samples such as sexmatched littermates of inbred mouse lines. Only a few
genes were consistently differentially expressed, and
low-density microarrays such as those used for screening
in this study have relatively few transcripts. More complete assessment using high-density gene chips may be
more amenable to formal statistical and bioinformatic
In addition to prompting the present study of
MCP-3, our gene-expression analysis of Tsk1 skin provides some additional information regarding the pathogenesis of the tight-skin mouse phenotype. Representing
one of the best-characterized genetically determined
animal models for SSc (28), the Tsk1 mouse model
demonstrates cutaneous hyperplasia and abnormal connective tissue architecture in the dermis and visceral
organs (29). Although heterozygous Tsk1 mice are normal at birth, in the second week of life, skin tightness
develops in the interscapular region with increased
numbers of high collagen-expressing fibroblasts. The
histologic appearances of marked thickening of the
dermis and excessive deposition of thick collagen fibers
extending into the subdermal adipose tissue are clearly
apparent from 3 weeks of age. Generalized dermal
fibrosis with adherence to subcutaneous tissues develops
by 12 weeks.
The genetic basis for the phenotype has recently
been identified as an in-frame genomic duplication
within the fibrillin 1 gene (30), resulting in a mutant
transcript that is 3 kb larger than the wild-type transcript. The link between expression of a mutant fibrillin
1 protein and generalized matrix overproduction is
uncertain. Our data indicate major differences in expression profiles neonatally, with up-regulation of MCP-3
and monocyte colony-stimulating factor 1, although
some of these differences were not sustained at later
time points. A smaller number of genes, mostly related
to ECM turnover, were consistently up-regulated, including TIMP3 and TGF␤, at all time points. There was
significant similarity with the gene-expression profile of
TGF␤1-stimulated wild-type fibroblasts (data not
shown). Array data suggest that MCP-3 is overexpressed
neonatally in Tsk1 skin and not up-regulated at 12
weeks. Immunostaining confirmed this in mouse tissue.
These findings are consistent with the more frequently
observed MCP-3 overexpression in early dcSSc skin as
compared with biopsy samples of skin with established
disease. This would suggest that MCP-3 may be an
important initiator in the cascade of mediators leading
to dermal fibrosis. This is similar to the characteristics of
other profibrotic mediators, including TGF␤1, in skin
samples, which often show little expression in established lesional skin of SSc.
The significance of the differences in staining
patterns for MCP-3 in human and mouse skin is unclear.
Diffuse dermal expression was observed in both mouse
and human skin samples. Similar perifollicular staining
and a variable amount of epidermal staining was seen in
both control and lesional samples from mice and humans, and thus these patterns are unlikely to be of
pathogenic significance, although it is apparently specific
and therefore probably reflects a physiologic function
for MCP-3. The vascular and perivascular staining observed in early-stage dcSSc may reflect a role for
fibroblast-derived MCP-3 in mononuclear cell extravasation, and the colocalization with CD68-positive cells
supports this. It is possible that MCP-3 overexpression
recruits macrophages into the SSc skin, leading to the
initiation of skin fibrosis and production of other inflammatory or fibrotic cytokines, as outlined above. The
absence of mononuclear cell infiltrates in Tsk1 skin
despite overexpression of a potent chemoattractant is
surprising. A possible explanation may be that increased
metalloproteinase activity that has previously been demonstrated in Tsk1 mouse skin samples might proteolytically cleave MCP-3 and render it inactive. Analogous
modification has been identified for human MCP-3 and
has been proposed to be a mechanism by which acute
inflammation is down-regulated during wound healing
or scar formation (31).
There is growing evidence that chemokines may
affect the homeostasis of ECM, and excessive synthesis
of type I collagen by fibroblasts in the dermis is a
pathologic hallmark of SSc. Given the lack of inflammatory infiltrate in Tsk1 skin, we studied the effect of
MCP-3 on ECM biosynthesis. Our data showed increased pro␣2(I) collagen promoter activity in response
to MCP-3 stimulation with similar dose-response characteristics to those previously observed for MCP-1 (19).
Although relatively high concentrations of recombinant
cytokine were needed for maximal effect, the threshold
for activation was often an order of magnitude lower,
and likely to reflect expression levels in the pericellular
space in vivo (8). For the promoter assay, we initially
used transgenic mouse fibroblasts containing a 2-kb
upstream enhancer fragment driving high-level
fibroblast-specific expression to reporter genes. Such use
of transgenic cells represents a highly physiologic approach to studies of collagen-gene activation in vitro,
since the transgene is stably integrated in chromatin and
is known to reflect endogenous collagen-gene expression
in vivo.
Reporter genes regulated by different murine
and human promoter sequences were also examined in
transient transfection studies with early-passage fibroblast cultures. Transactivation of Col1a2 in Tsk1 skin has
previously been shown to depend on far-upstream
fibroblast-specific elements as well as sequences within
the proximal promoter (22), with both proximal and
distal regulatory sequences appearing to be responsive
to TGF␤. In contrast, the present study suggests that
MCP-3–induced Col1a2 activation may be independent
of the upstream enhancer. This raises the possibility that
the profibrotic effects of MCP-3 are only partly mediated via TGF␤1. Collagen-gene up-regulation in Tsk1
skin is likely to occur via a number of pathways that may
depend both on TGF␤1 and on other factors. Antibody
neutralization experiments showed that the stimulatory
effect of MCP-3 on collagen-gene expression is partly
dependent on TGF␤. Consistent with this observation is
the time course of maximal induction, which was between 16 and 24 hours, with little effect before 6 hours.
It nevertheless remains possible that TGF␤ may be a
co-factor as well as a potential downstream mediator of
collagen-gene activation, especially since collagen-gene
activation was not completely abrogated by a high
concentration of anti-TGF␤ antibody. Conversely, there
are recent reports suggesting that overexpression of the
CC chemokine pulmonary and activation-regulated chemokine (known as PARC) may directly activate ECM
gene expression (32).
In summary, our findings implicate MCP-3 as a
potential mediator of dermal fibrosis in SSc, although
several issues remain unresolved, especially concerning
the interplay in vivo between MCP-3 and other profibrotic factors. It is possible that different mediators are
important at various stages or in different clinical subsets of SSc. A microsatellite polymorphism in the promoter region of MCP-3 has been described in association with certain patterns of multiple sclerosis, another
heterogeneous chronic inflammatory and sclerotic disease (33), and it is possible that polymorphic variants
may associate with the clinical phenotype of SSc or other
conditions such as inflammatory bowel disease (34).
Further studies to better define the regulation and
function of MCP-3 overexpression in SSc are currently
in progress.
The 2kb-LacZ transgenic mice were generated in
collaboration with Dr. Benoit de Crombrugghe’s laboratory at
the University of Texas M. D. Anderson Cancer Center in
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