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Laboratory of
*
Vascular Biology and
Medical Biochemistry, The Picower Institute for Medical Research, Manhasset, NY 11030
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1 (1–10 ng/ml), an important
fibrogenic and growth-regulating cytokine involved in wound healing,
increases the differentiation and functional activity of cultured
fibrocytes. Because fibrocytes home to sites of tissue injury, we
examined the role of chemokine/chemokine receptor interactions in
fibrocyte trafficking. We show that secondary lymphoid chemokine, a
ligand of the CCR7 chemokine receptor, acts as a potent stimulus for
fibrocyte chemotaxis in vitro and for the homing of injected fibrocytes
to sites of cutaneous tissue injury in vivo. Finally, we demonstrate
that differentiated, cultured fibrocytes express 
smooth muscle
actin and contract collagen gels in vitro, two characteristic features
of wound-healing myofibroblasts. These data provide important insight
into the control of fibrocyte differentiation and trafficking during
tissue repair and significantly expand their potential role during
wound healing. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In 1994, a distinct population of blood-borne fibroblast-like cells that rapidly enter sites of tissue injury was described (2). Termed fibrocytes, these cells comprise 0.1–0.5% of nonerythrocytic cells in peripheral blood and display an adherent, spindle-shaped morphology when cultured in vitro. Cultured fibrocytes express the fibroblast products collagen (Col)3 I, Col III, and fibronectin, as well as the leukocyte common Ag (CD45RO), the pan-myeloid Ag (CD13), and the hemopoietic stem cell Ag (CD34). In addition, fibrocytes express MHC class II and costimulatory molecules (CD80 and CD86) and have the capacity to present Ag in vitro and in vivo (3, 4). By their morphology, growth properties, and cell surface markers, fibrocytes appear to be distinct from monocytes/macrophages, dendritic cells, and other known APC types. Cultured fibrocytes do not express typical monocyte/macrophage-specific or B cell markers (such as CD14, CD16, or CD19), nor do they express typical surface proteins of dendritic cells or their precursors (such as CD1a, CD10, CD25, and CD38). In addition, fibrocytes isolated from peripheral blood and cultured ex vivo secrete a unique profile of cytokines, growth factors, and chemokines (5).
Based on their presence in wounds and their secretion of proinflammatory cytokines, chemokines, and extracellular matrix proteins, fibrocytes have been postulated to play a role in wound healing and connective tissue formation. Although initial studies performed in sex-mismatched bone marrow chimeric mice suggested that fibrocytes arose from a relatively radioresistant progenitor population (2), the precise origin of these cells and the wound trafficking signals relevant to their directed migration remain unknown. In this study we identify a differentiation pathway of cultured fibrocytes, characterize the signals for fibrocyte migration to wound sites in vivo, and reveal the potential role of fibrocytes in wound contracture.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c mice (females, 8–12 wk old) were purchased from The Jackson Laboratory (Bar Harbor, ME). All animal procedures were conducted according to guidelines of the institutional animal care and use committee of North Shore University Hospital under an approved protocol.
Antibodies, cytokines, and chemokines
FITC-anti- smooth muscle actin (FITC-anti-SMA) mAb
was purchased from Sigma (St. Louis, MO). Biotinylated rabbit
anti-Col I and biotinylated rabbit IgG were purchased from Rockland
(Gilbertsville, PA). Anti-mouse CCR3, CCR5, CCR7, or CXCR4 polyclonal
Abs and FITC-anti-goat IgG Ab were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA). All other Abs were purchased from BD
PharMingen (San Diego, CA). TGF-1 (active), secondary lymphoid
chemokine (SLC), and stromal-derived cell factor (SDF) were
purchased from R&D Systems (Minneapolis, MN).
Cells
Fibrocytes (human and mouse) were purified from peripheral blood
and cultured as previously described (2, 5). Briefly,
PBMCs were isolated from human Leukopaks (purchased from the Long
Island Blood Center, Long Island, NY) by centrifugation over
Ficoll/Paque (Pharmacia, Piscataway, NJ) following the manufacturer’s
protocol. After 2 days of culture on tissue culture flasks in DMEM
(Life Technologies, Gaithersburg, MD) supplemented with 20% FBS
(HyClone Laboratories, Logan, UT), penicillin, streptomycin, and
L-glutamine, nonadherent cells were removed by gentle
aspiration, and media were replaced. After 10–12 days, adherent cells
were lifted by incubation in ice-cold 0.05% EDTA (in PBS). The crude
fibrocyte preparations (
70–80% pure based on Col I/CD11b staining)
then were depleted by immunomagnetic selection of contaminating T cells
(13%), B cells (3%), and monocytes (11%) using pan-T,
anti-CD2; pan-B, anti-CD19; and anti-CD14 Dynabeads,
respectively (Dynal, Great Neck, NY). The resultant cultured, enriched
fibrocyte populations were 
95% pure based on Col I/CD11b staining,
with T cells and monocytes contributing 3 and 2%, respectively.
Typically, between 0.4 and 5 x 104
fibrocytes were isolated per milliliter of human blood.
Mouse PBMC were isolated from BALB/c mouse blood (heparinized) obtained
by cardiac puncture following CO2 asphyxiation.
Mouse blood was mixed with PBS (2:1) and layered over Ficoll/Paque
(Pharmacia; 15 ml blood over 30 ml Ficoll) and centrifuged according to
the manufacturer’s protocol. Mouse fibrocytes were cultured in DMEM
supplemented with 10% FBS and 10% mouse serum (Sigma), penicillin,
streptomycin, and L-glutamine, as previously described
(4). After 10–12 days, the adherent crude fibrocyte
preparation (75% pure based on Col I/CD11b staining) were lifted
using 0.05% EDTA in PBS and depleted by immunomagnetic selection of
contaminating T cells, B cells, and monocytes using pan-T
(anti-CD90), pan-B (anti-B220) Dynabeads (Dynal), and
anti-mouse CD14 attached to Dynabeads, respectively. Following
immunodepletion, the cultured, enriched fibrocyte preparations were
verified to be 95% pure by Col
I+/CD11b+ staining as
determined by flow cytometry. Approximately 0.8–4 x
104 fibrocytes/ml mouse blood (1–1.2 ml
blood/mouse) were purified.
Human adult dermal fibroblasts were purchased from Clonetics (San Diego, CA) and cultured according to the manufacturer’s recommendations. The human intestinal smooth muscle cell line was obtained from American Type Culture Collection (Manassas, VA) and cultivated according to recommended procedures.
Analysis of fibrocyte differentiation
Initial studies were aimed toward elucidating the cellular
origin of peripheral blood fibrocytes. Therefore, we fractionated whole
blood supplied as Leukopaks (see Fig. 1
A) and cultured the
various fractions in vitro. Adherent cells were collected from
overnight cultures of human PBMCs (total), and
CD14+ cells were enriched from the PBMC fraction
by depletion of T and B cells (CD14+).
CD14- cells (including all PBMCs except
CD14+ cells) were purified by depletion of the
CD14+ cells from the total PBMC preparation.
Using the Transwell two-chamber system (0.4 µm; Corning Costar,
Cambridge, MA), CD14+,
CD14-, or total cells (3 x
106 cells/ml in DMEM/10% FBS) were cultured in
either the upper or lower chambers, as indicated. After 7 days of
culture, the cells that grew in the lower well were collected and
analyzed for fibrocyte-like differentiation by Col I/CD11b staining and
flow cytometry. Similar results were observed with cells prepared from
three other donors.
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95%, as assessed by flow cytometry using
anti-CD3 Abs (PharMingen). After 7 days of coculture, the resulting
population was analyzed for the percentage of fibrocytes by Col I/CD11b
staining and flow cytometry. Similar results were observed using
fibrocytes isolated from three different donors. Flow cytometric analysis
For single Ab staining, cells (105
aliquots) were resuspended in PBS containing 3% BSA and 0.1% sodium
azide (FACS buffer) and incubated with the indicated Abs (or labeled
isotype control Abs) for 30 min at 4°C. In cases where the primary
Abs were not labeled, cells were washed and incubated with revealing
Abs diluted in FACS buffer. After washing the cells in FACS buffer,
fluorescence data were acquired on a FACSCalibur flow cytometer (BD
Biosciences, San Jose, CA) and analyzed using CellQuest software (BD
Biosciences). At least 5000 cells were analyzed per condition. To
analyze preparations for Col I/CD11b staining, cells were prepared as
described above and first incubated in FACS buffer containing
biotinylated Col I Ab (or biotinylated rabbit control IgG), then washed
and incubated sequentially in FACS buffer containing FITC-strepavidin
(PharMingen) and PE-CD11b (PharMingen). Intracellular staining for
SMA was performed as previously described (6, 7).
Briefly, cells were fixed and permeabilized using the Perm/Fix kit
(PharMingen) according to the manufacturer’s recommendations and
incubated with FITC-anti-SMA mAb (Sigma).
Fibrocyte migration in vivo using a wound model
Cultured, enriched peripheral blood-derived mouse fibrocytes (>96% pure) were stained with a membrane-inserting red dye, PKH-26 (Sigma), following the manufacturer’s protocol. Labeling efficiency, assessed by flow cytometry, and viability, assessed by trypan blue exclusion were >85%. PKH-labeled cells (5 x 105) in 100 µl PBS were administered into the tail vein (i.v.) of BALB/c mice (n = 2/group). Immediately following injection of the enriched fibrocytes, a full-thickness round skin wound (5-mm diameter) was made in the dorsal subscapular area of each recipient mouse by excision with skin punch equipment, as previously described (8). Wound sites were removed 4 days later and examined for the presence of fluorescent fibrocyte cells by microscopic analysis of thin frozen sections and by quantitative flow cytometric analysis following proteolytic digestion. For quantitative flow cytometric analysis, excised skin (250 µg biopsy/animal) was chopped into small fragments, then incubated for 1 h at 37°C in RPMI 1640 containing 10% FBS, 2 mg/ml collagenase, and 20 µg/ml DNase I. The resulting single-cell suspension was examined by flow cytometry to determine the number of fluorescent fibrocytes present using calibration beads as previously described (9).
RT-PCR
Total RNA was isolated from cultured, enriched fibrocytes
(>95% pure) using RNAzol B (Tel-Test, Friendswood, TX). The cDNA was
prepared from 1.0 µg RNA using 0.25 ng
oligo-(dT)12–18 and Moloney murine leukemia
virus reverse transcriptase following the protocol supplied by
the manufacturer protocol (Life Technologies). Two-microliter aliquots
of cDNA were amplified by PCR using Supermix (Life Technologies) in a
Perkin-Elmer model 9600 thermal cycler using specific primers PCR pairs
as previously described: SMA (10); CCR3
(11); CCR4, CCR5, and CXCR3 (12); CCR6
(13); CCR7 (14); CXCR4 (15); and
-actin (sense primer, 5'-GTGGGGCGCCCCAGGCACCA-3'; antisense primer,
5'-CTCCTTAATGTCACGCACGATTTC-3'). Thermal cycling (25–30 cycles, in 25
µl) was performed as follows: denaturation at 94°C for 0.5 min,
annealing at 55°C for 0.5 min, and extension at 72°C for 1 min. PCR
products were separated by electrophoresis through 2% agarose gels and
viewed under UV light after ethidium bromide staining. To control for
potential genomic DNA contamination, PCR were performed without the
reverse transcription step, and no DNA amplification products
were detected.
In vitro fibrocyte chemotaxis assay
Chemotaxis assays were performed using Costar Transwell inserts
(8-µm pore size) according to the manufacturer’s protocol. Cultured,
enriched fibrocytes (95% pure) were resuspended at 1 x
106 cells/ml in DMEM containing 0.1% BSA. Medium
alone (negative control) or medium containing SLC or SDF (600 µl) was
added to individual wells of a 24-well plate. Transwell devices then
were inserted, and the fibrocytes (100 µl) were layered on top of the
membrane (n = 3 wells/condition). After 3 h the
transmigrated cells were collected and counted by flow cytometry using
calibration beads (Coulter, Miami, FL), as previously described
(9). Similar results were observed with two additional
donors. For checkerboard analysis of SLC-directed chemotaxis of
fibrocytes, 100 ng/ml SLC was added to either the top or bottom chamber
alone and to both the bottom and top chambers (see Fig. 5B).
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Immediately following tail vein injection of PKH-labeled enriched fibrocytes (>94% pure; 5 x 105 cells/mouse), BALC/c mice received an i.d. injection of SLC, SDF (0.1 or 1 µg in 50 µl), or PBS alone in the scapular region of the back (shaved). The injected site was excised 4 h later and proteolytically digested to produce a single-cell suspension (as described above). The number of labeled fibrocytes per biopsy sample (250 µg) was estimated by flow cytometry using calibration beads (9). This experiment was repeated twice with similar results.
Collagen lattice contraction assay
Cellular collagen gel contraction assays were performed as
previously described (16). Overnight adherent PBMC
cultures, 10-day-old enriched fibrocytes (95% pure) previously
cultured in the absence or the presence of TGF-1 (10 ng/ml for 7
days before experiment), or normal human dermal fibroblasts were lifted
using cold EDTA/PBS solution. A collagen solution in DMEM was prepared
from rat tail Col I according to the manufacturer’s
instructions, and combined with cells at 2 x
105/ml (n = 3/cell type). The
collagen/cell mixture (400 µl/well) was dispensed into culture plates
and allowed to polymerize at 37°C for 30 min. Immediately after
polymerization, 2 ml DMEM containing 10% FBS was added to each well.
The gels then were detached from the wells by gently shaking the
culture plates at various time points (0, 24, 48, and 72 h), and
the longest and shortest diameters of each gel were measured. The mean
of the linear measurements (n = 3 for each sample)
taken at each time point was used to estimate the contractility of the
cells. The data are presented as percent gel contraction. This
experiment was repeated twice with similar results using cells obtained
from different donors.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine the origin of fibrocytes, we analyzed the growth and
phenotype of adherent human PBMC cultured on plastic (Fig. 1
A). After standard Ficoll
separation, the resulting population was 40–50%
CD14+ cells. Following an overnight adherence
step, the adherent cell population (total) was >70%
CD14+ cells exhibiting no detectable Col I
staining, as assessed by flow cytometry (data not shown)
(5). We have shown in previous studies that after 2 wk
cells in these cultures no longer express CD14, but do express Col I
(5). Importantly, we found that a cell population enriched
for CD14+ cells, (i.e., PBMCs depleted of all T
or B cells by magnetic beads) gives rise to very few Col
I+/CD11b+ spindle-shaped
fibrocytes after 1 wk of culture (data not shown).
Using Transwell culture chambers, we examined the cellular requirements
for fibrocyte differentiation (CD11b/Col I+) in
vitro from circulating blood cell fractions (Fig. 1B). When
a CD14- cell fraction was cultured in the lower
well of a Transwell plate and total PBMCs were cultured in the top
chamber for 1 wk, no fibrocytes appeared in the lower chamber.
Similarly, no fibrocytes appeared in the lower chamber when
CD14+ cells alone were cultured in the bottom
chamber and CD14+ cells or total PBMCs were
cultured in the top chamber for 1 wk. By contrast, when total PBMCs
were cultured in the bottom well of the Transwell chamber and either
CD14- cells or CD14+ cells
(or medium alone, data not shown) were cultured in the top chamber,
numerous spindle-shaped fibrocytes (CD11b+/Col
I+) were observed within 1 wk. These data suggest
that fibrocyte outgrowth from cultured PBMCs requires cellular
interaction between a population of enriched
CD14+ cells and another peripheral blood cell
type or that fibrocyte precursors are only present in the PBMC
fraction.
To examine the requirement of cellular interaction, we then added
either purified, autologous T or B cells to CD14+
cell cultures in various ratios (CD14+:T, 0:1,
1:0, 3:1, 1:1, and 1:3) for 7–10 days and found that cocultures of
CD14+ cells and T cells give rise to fibrocytes
(CD11b+/Col I+; Fig. 1C). We observed that a CD14+cell:T
cell ratio of 3:1 was optimal (Fig. 1C) for culturing
fibrocytes. By contrast, no fibrocytes appeared when T cells were
cultured alone or in cocultures of B and CD14+
cells or when CD14+ cells were cultured with T
cell-conditioned medium (data not shown). Because fibrocytes do not
express T cell markers (CD2, CD3, CD4, and CD8) or typical T cell
cytokines (IL-2, IL-4, and IFN-
), it is unlikely that T cells give
rise to fibrocytes.
TGF-1 accelerates fibrocyte differentiation in vitro
Next, we examined whether TGF-1, a cytokine important for
fibroblast proliferation and extracellular matrix production could
promote the differentiation and accumulation of fibrocytes within PBMC
cultures. The addition of TGF-1 (1–10 ng/ml) to PBMC cultures on
days 3–10 promoted fibrocyte differentiation in vitro, as shown by the
enhanced accumulation of cells with spindle-shaped morphology (Fig. 2, A–C). Treatment of these
cultures with TGF-1 increased the expression of Col I by fibrocytes
within these cultures in a dose-dependent manner (Fig. 2D).
The mean fluorescence intensities for Col I expression were 11, 24, and
63 for fibrocytes in cultures treated with 0, 1, and 10 ng/ml TGF-1,
respectively (Fig. 2D). These Col I+
cells also stained positively for CD11b (data not shown). Furthermore,
there was a dose-dependent increase in the number of fibrocytes within
the cultures that stained positively for Col I in response to TGF-1,
with almost a 40% increase in response to 10 ng/ml TGF-1 compared
with untreated cells. Similar results were observed with fibrocyte
preparations from three other donors, each showing a 30–45% increase
in Col I expression between 0 and 10 ng/ml TGF-1.
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We next sought to quantify the migration into wound sites of
transferred cultured, enriched fibrocytes using a mouse model system.
Cultured, enriched mouse fibrocyte preparations (>96% pure) were
injected (5 x 105/mouse) into the tail vein
of mice. Immediately, full-thickness skin punch biopsy wounds (5 mm in
diameter) were made in the dorsal scapular area in some mice. The wound
sites (and comparable untreated skin tissue) were excised 4 days later,
and biopsy specimens were examined for the presence of labeled
fibrocytes. As shown in Fig. 3A, numerous fluorescent cells
were found by microscopic analysis of the wound tissue at 4 days.
Labeled fibrocytes appeared to be located near newly formed blood
vessels at the edge of the wound. Using another group of mice
(n = 3/group), single-cell suspensions were prepared
from the excised wound or normal tissue (250 µg/biopsy), and labeled
fibrocytes were quantified by flow cytometry. Enumeration of migrated
labeled fibrocytes revealed that wounded tissue contained significantly
more labeled fibrocytes than a similar area of normal skin taken from
the same mouse (Fig. 3B).
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Numerous circulating cells, including, neutrophils, monocytes, and
T cells, are known to migrate into cutaneous wound sites. This process
is organized in part by specific interactions between chemokines and
their receptors. We surveyed cultured enriched fibrocytes for chemokine
receptor mRNA expression by RT-PCR and found CCR3, CCR5, CCR7, and
CXCR4 mRNA (Fig. 4A), but not
CCR4, CCR6, or CXCR3 mRNA expression. We confirmed CCR3, CCR5, CCR7,
and CXCR4 protein expression on the surface of human enriched
fibrocytes by flow cytometry (Fig. 4B). Cultured, enriched
fibrocytes isolated from mouse blood also expressed CCR7 and CXCR4, as
analyzed by cytofluorometric analysis (Fig. 4C).
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A, SLC significantly induced
the migration of fibrocytes, whereas SDF did not. Checkerboard analyses
confirmed the chemotactic (but not chemokinetic) response of cultured,
enriched fibrocytes in response to SLC (Fig. 5B). Based on
these observations, we investigated whether SLC could promote the
migration of transferred, cultured, enriched fibrocytes following an
i.d. injection of the chemokine in vivo. Administered at a dose of 1
µg, SLC dramatically induced the accumulation of prelabeled, ex vivo
cultured fibrocytes in the skin area surrounding the i.d. injection
site compared with the effect of PBS alone (Fig. 5C). By
contrast, SDF injection did not promote fibrocyte chemotaxis in vivo
(Fig. 5C). Immunostaining of a 2-day wound site revealed SLC
chemokine expression by the vascular endothelium (data not shown).
These results suggest that fibrocytes migrate into early wound sites in
part due to an interaction between vascular endothelium-derived SLC and
fibrocyte CCR7. Fibrocytes contract collagen gels
Based on their presence within the wound and their expression of
Col I and III, we have postulated that fibrocytes mediated wound
healing and fibrosis. Gabbiani and coworkers have previously described
a population of wound fibroblasts that differentiate into
myofibroblasts in the presence of TGF- (Ref. 17 ,
reviewed in Ref. 18). These cells are characterized by
expression of SMA, the activity of contracting collagen gels in
vitro, and their proposed role in wound closure, inflammation, and
fibrosis (reviewed in Ref. 19). Recognizing that TGF-1
enhances Col I expression by cultured fibrocytes (Fig. 2D)
and that fibrocytes are present in wound tissue for days
(20), we next examined whether cultured, enriched
fibrocytes express SMA and exhibit a contractile force. As shown in
Fig. 6A, unstimulated,
cultured, enriched fibrocytes were found to express SMA mRNA, but
freshly isolated PBMCs did not. Unstimulated cultured, enriched
fibrocytes also express SMA protein, and the addition of TGF-1
(10 ng/ml) increased SMA levels by about 4-fold (Fig. 6B). Next, we examined the contractile activity of cultured,
enriched fibrocytes. We found that untreated cultured, enriched
fibrocytes significantly contracted the collagen gels in vitro by
20%, whereas PBMCs did not (Fig. 6C). Pretreatment of
fibrocytes with TGF-1 (10 ng/ml) for 7 days before the assay further
increased their contractile activity (Fig. 6C). This
increase in gel contraction by TGF-1-treated fibrocyte cultures
correlated to the enhanced expression of SMA by fibrocytes in
response to TGF-1.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fibrocytes differentiated from an adherent population of
CD14+-enriched peripheral blood cells when
cultured in DMEM and FBS (with no additional growth factors). This
differentiation process appears to require T cell interaction. Further
studies will be necessary to identify the molecules involved in
functionally significant interactions between T cells and fibrocytes
that are required for fibrocyte maturation. Similarly, several studies
have demonstrated the differentiation of CD1a+
dendritic cells from GM-CSF- and IL-4-treated
CD14+ peripheral blood monocytes
(22, 23, 24, 25). The T cells requirement observed for fibrocyte
differentiation is reminiscent of the maturation of dendritic cells,
also known for their ability to process and present Ags
(26). Recent studies have shown that the final step in the
maturation of dendritic cells occurs during their association with
CD4+ T cells; this contact diminishes their
responsiveness to IFN- by down-regulation of their IFN- receptors
(27).
Interestingly, the addition of TGF-1, a multifunctional cytokine
that plays a central role in tissue repair and fibrosis, to crude
fibrocyte-evolving cultures facilitated fibrocyte differentiation. The
role of exogenous TGF- in fibroblast proliferation and collagen
production is well documented (reviewed in Ref. 28).
TGF- significantly up-regulates collagen expression by dermal
fibroblasts in vitro (29), by myofibroblasts
(30), as well as by proliferative scar xenografts in vivo
(31). Many laboratories have confirmed that TGF- plays
a role in the natural wound healing process and that TGF- is
expressed in rodent wound chambers during the early to mid phases (days
4–7) of wound healing (32). Furthermore, in vivo gene
transfer with TGF-1 cDNA into the skin of rats significantly
enhanced the rate of wound repair (33). Consistent with
these prior observations, we postulate that circulating fibrocyte
precursor cells interact with activated T cells, which permits their
early differentiation (toward the fibrocyte phenotype), and they then
migrate to the wound site (Fig. 7).
Within the wound site, these early differentiated fibrocytes might
further interact with recruited T cells and fully differentiate and
mature following exposure to TGF-. These fully differentiated,
mature fibrocytes express increased levels of SMA and produce
collagen and other extracellular matrix proteins that promote wound
healing and contracture.
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overexpression in fibrosis of the skin
(35) and lungs (35, 36). In addition, TGF-
overexpression has been associated with enhanced myofibroblast
activity in animal models of pulmonary fibrosis (37). Our
findings that TGF-1 enhanced proliferation, collagen production, and
SMA expression by cultured fibrocytes potentially implicates this
circulating cell type in TGF--dependent fibrotic responses
in vivo. A role for fibrocytes in wound healing and connective scar tissue formation has been postulated based on their accumulation in wound sites (2). However, the molecular signals that mediate the trafficking of fibrocytes to the wound has not yet been investigated. We examined chemokine receptor expression (mRNA and protein) by cultured enriched fibrocytes and revealed the presence of CCR3, CCR5, CCR7, and CXCR4 and the absence of CCR4, CCR6, and CXCR3. Further studies showed directed chemotaxis of cultured, enriched fibrocytes in response to the ligand of CCR7, SLC (also known as 6Ckine, Exodus-2, and TCA-4), in vitro and in vivo. SLC, a C-C chemokine family member, has been shown to be involved in the organization of lymphoid tissue during development by attracting T cells and mature dendritic cells (38). SLC expression has been observed in sites of inflammation (39). We observed SLC expression by the vascular endothelium within the wound sites. Based on these observations it would be interesting to examine the role of fibrocytes in wound responses using mutant mice lacking SLC expression (40, 41, 42).
The functional role of fibrocytes in wound healing has not been
investigated previously. TGF- has been shown to be the most
important cytokine for the trans-differentiation of
fibroblasts to contractile wound myofibroblasts that exhibit increased
SMA staining, elevated collagen secretion (reviewed in Ref.
19), and increased stress fibers (17) in
response to TGF-. Myofibroblasts are transiently found in early to
mid phase wound tissue and have been proposed to exert a critical
contractile force that is required to close wounds. Neither the origin
of myofibroblasts nor any trafficking signals necessary for
myofibroblast migration to injured tissue are well understood.
Myofibroblasts have been postulated to derive from progenitor stem
cells, resident tissue fibroblasts, or tissue smooth muscle cells.
However, a plausible alternative is that myofibroblasts differentiate
from a circulating, rather than a resident, precursor cell
type.
In this paper we show that blood-borne, ex vivo cultured, precursor
fibrocyte cells have the capacity to differentiate into
SMA+, TGF-1-responsive fibrocyte cells that
exhibit characteristics similar to those of wound-healing
myofibroblasts. Differentiated fibrocytes and myofibroblasts share many
common features: transient presence within the wound, production of
numerous proinflammatory cytokines and growth factors, secretion of
collagen and other extracellular matrix proteins, and enhanced collagen
production in response to TGF-1. Furthermore, we observed that
cultured fibrocytes, like myofibroblasts, express SMA protein that
is enhanced by TGF-1 treatment and exert a contractile force that
would aid in reducing the amount of denuded surface area of wounded
tissue. The question remains of whether fibrocytes and myofibroblasts
are distinct populations. However, it is reasonable to suggest that
fibrocytes derived from a circulating precursor population play an
important role during the resolution and repair phase of wound
healing.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Christine N. Metz, The Picower Institute, 350 Community Drive, Manhasset, NY 11030. E-mail address: cmetz{at}picower.edu
3 Abbreviations used in this paper: Col, collagen; SLC, secondary lymphoid chemokine; SMA, smooth muscle actin; SDF, stromal-derived cell factor.
Received for publication November 14, 2000. Accepted for publication April 5, 2001.
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N. C. Direkze, S. J. Forbes, M. Brittan, T. Hunt, R. Jeffery, S. L. Preston, R. Poulsom, K. Hodivala-Dilke, M. R. Alison, and N. A. Wright Multiple Organ Engraftment by Bone-Marrow-Derived Myofibroblasts and Fibroblasts in Bone-Marrow-Transplanted Mice Stem Cells, September 1, 2003; 21(5): 514 - 520. [Abstract] [Full Text] [PDF]
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M. Schmidt, G. Sun, M. A. Stacey, L. Mori, and S. Mattoli Identification of Circulating Fibrocytes as Precursors of Bronchial Myofibroblasts in Asthma J. Immunol., July 1, 2003; 171(1): 380 - 389. [Abstract] [Full Text] [PDF]
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H Nakayama, H Enzan, E Miyazaki, and M Toi {alpha} Smooth muscle actin positive stromal cells in gastric carcinoma J. Clin. Pathol., October 1, 2002; 55(10): 741 - 744. [Abstract] [Full Text] [PDF]
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Y. Li and J. Huard Differentiation of Muscle-Derived Cells into Myofibroblasts in Injured Skeletal Muscle Am. J. Pathol., September 1, 2002; 161(3): 895 - 907. [Abstract] [Full Text] [PDF]
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M Brittan, T Hunt, R Jeffery, R Poulsom, S J Forbes, K Hodivala-Dilke, J Goldman, M R Alison, and N A Wright Bone marrow derivation of pericryptal myofibroblasts in the mouse and human small intestine and colon Gut, June 1, 2002; 50(6): 752 - 757. [Abstract] [Full Text] [PDF]
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N. Scholler, M. Hayden-Ledbetter, A. Dahlin, I. Hellstrom, K. E. Hellstrom, and J. A. Ledbetter Cutting Edge: CD83 Regulates the Development of Cellular Immunity J. Immunol., March 15, 2002; 168(6): 2599 - 2602. [Abstract] [Full Text] [PDF]
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), cultured, enriched fibrocytes
(untreated (
) and TGF-
)), or dermal fibroblasts
(
) were resuspended in a Col I solution at 105 cells/ml.
The contraction assay (n = 3) was performed as
described in Materials and Methods. The data are percent
gel contraction (from beginning of experiment) ± SE (some error
bars are smaller than the symbol). *, p < 0.05,
as determined by Student’s t test, comparing
experimental to PBMCs (