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Immunobiology xxx (2011) xxx–xxx

Contents lists available at ScienceDirect

Immunobiology
journal homepage: www.elsevier.de/imbio

Review

Macrophages in skin injury and repair

Babak Mahdavian Delavary a,b,∗ , Willem M. van der Veer a,b ,
Marjolein van Egmond a,c , Frank B. Niessen b , Robert H.J. Beelen a
a
Department of Molecular Cell Biology and Immunology, VU University Medical Center, Amsterdam, The Netherlands
b
Department of Plastic and Reconstructive Surgery, VU University Medical Center, Amsterdam, The Netherlands
c
Department of Surgery, VU University Medical Center, Amsterdam, The Netherlands

a r t i c l e i n f o a b s t r a c t

Article history: After recruitment to the wound bed, monocytes differentiate into macrophages. Macrophages play a
Received 14 December 2010 central role in all stages of wound healing and orchestrate the wound healing process. Their func-
Accepted 4 January 2011 tional phenotype is dependent on the wound microenvironment, which changes during healing, hereby
altering macrophage phenotype. During the early and short inflammatory phase macrophages exert
Keywords: pro-inflammatory functions like antigen-presenting, phagocytosis and the production of inflammatory
Inflammation
cytokines and growth factors that facilitate the wound healing process. As such, the phenotype of wound
Macrophages
macrophages in this phase is probably the classically activated or the so-called M1 phenotype. During
Phenotype
Remodelling
the proliferative phase, macrophages stimulate proliferation of connective, endothelial and epithelial
Skin tissue directly and indirectly. Especially fibroblasts, keratinocytes and endothelial cells are stimulated by
Wound healing macrophages during this phase to induce and complete ECM formation, reepithelialization and neovascu-
larization. Subsequently, macrophages can change the composition of the ECM both during angiogenesis
and in the remodelling phase by release of degrading enzymes and by synthesizing ECM molecules. This
suggests an important role for alternatively activated macrophages in this phase of wound healing. Patho-
logical functioning of macrophages in the wound healing process can result in derailed wound healing,
like the formation of ulcers, chronic wounds, hypertrophic scars and keloids. However, the exact role
of macrophages in these processes is still incompletely understood. For treating wound repair disorders
more should be elucidated on the role of macrophages in these conditions, especially their functional
phenotype, to find more therapeutic opportunities.
This review summarizes macrophage function in skin injury repair, thereby providing more insight in
macrophage function in wound healing and possible interventions in this process.
© 2011 Elsevier GmbH. All rights reserved.

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Phase I: Hemostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Phase II: Inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Phase III: Proliferation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Phase IIIa: Angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Phase IIIb: Reepithelialization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Phase IV: Remodelling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Conclusion and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

Introduction

∗ Corresponding author at: VU University Medical Center (VUmc), Department

The skin is our largest organ and its primary function is to
of Plastic, Reconstructive and Hand Surgery, Department of Molecular Cell Biology
and Immunology, Postbus 7057, Van der Boechorststraat 7, 1081 BT, Amsterdam,
serve as a protective barrier against infection and excessive water
The Netherlands. Tel.: +31 20 444 8055/20 4448083; fax: +31 20 444 8081. loss. It is also one of the most easily injured human organs. After
E-mail address: b.mahdavian@vumc.nl (B.M. Delavary). injury the skin needs to be repaired to maintain its function. The

0171-2985/$ – see front matter © 2011 Elsevier GmbH. All rights reserved.
doi:10.1016/j.imbio.2011.01.001

Please cite this article in press as: Mahdavian Delavary, B., et al., Macrophages in skin injury and repair. Immunobiology (2011),
doi:10.1016/j.imbio.2011.01.001
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healing process of the injured site, which normally results in the
formation of a scar, is an extremely complex process involving
numerous cell types as well as growth factors, cytokines, and extra-
cellular matrix (ECM) components (Breitkreutz et al. 2009). Wound
healing consists of dynamic processes including inflammation,
granulation tissue formation (proliferation), reepithelialization and
remodelling. None of these phases corresponds to a precisely
defined period of time and all phases overlap (Eming et al. 2007).
When this complex process proceeds normally, a normotrophic
scar is formed. Derailment of wound healing however can cause
pathological conditions like a hypertrophic scar, keloid or chronic
wounds and ulcers (Niessen et al. 1999; van der Veer et al. 2009).
To understand the etiology of these pathological conditions it is
important to understand skin composition and normal wound heal-
ing, and the role of the immune system in the wound healing
process.
The skin can be divided into an epidermal and a dermal layer.
The epidermis mainly contains keratinocytes, whereas the der-
mal part consists of two compartments (Gurtner et al. 2008). The
first is the cellular compartment, which is typically composed of
fibroblasts. The second is the acellular compartment, which mainly
contains the extracellular matrix (ECM). The ECM can further be
functionally divided into a fibrillar fraction, which is characterized
by fibrillar collagen bundles, elastic fibers, and microfibrils, and the
non-fibrillar fraction that consists of glycosaminoglycans and pro-
teoglycans. All these compartments and different components have
their own specific function and work together to protect the body
against invading pathogens and maintenance of other functions like
thermoregulation and protection against water loss.
Besides providing a structural barrier, the skin contains sev-
eral immune cells that can be activated by invading pathogens or
skin damage. One of the most important immune cells involved
in wound healing is the macrophage, which exhibits different
immunological functions in the skin, including phagocytosis and
antigen-presentation. Furthermore, macrophages produce many
cytokines and chemokines that stimulate new capillary growth,
collagen synthesis and fibrosis (Mirza et al. 2009). This immune cell
is thought to orchestrate the wound healing process throughout the
different phases (Mirza et al. 2009; Lucas et al. 2010; Leibovich and
Ross 1975; Devalaraja et al. 2000). Leibovich et al. demonstrated
that mice with non-functioning macrophages display retarded
wound repair (Leibovich and Ross 1975). This retarded wound
repair includes delayed reepithelialization, delayed neovascular-
ization and aberrant granulation tissue formation (Mirza et al.
2009; Lucas et al. 2010).
Macrophages at the site of wound repair consist of two pop-
ulations, which both have their origin in the bone marrow. The
first is the ‘resident’ tissue macrophage that is present in tissues
at all times. Under suitable stimuli it is capable of entering the
mitotic cycle. Normal skin contains resident macrophages at a
low density of approximately 1–2 per mm2 (Dipietro et al. 1995).
MacDonald et al. have demonstrated a minor role for resident tis-
sue macrophages in several processes including wound healing
(Macdonald et al. 2010). They used a macrophage dependant mouse
wound model in which resident tissue macrophages were depleted
using anti-CSF1R. In this model cellular reconstitution occurred
within 7 days and neither the timing of the wound healing process
nor the efficiency of tissue regeneration was affected by anti-CSF1R
treatment (Macdonald et al. 2010).
The other major population is newly recruited from haematoge-
nous precursor cells, called monocytes, which themselves are
derived from a rapidly dividing pool of cells in the bone mar-
row, the promonocytes. The majority of the monocytes circulate
in the bloodstream and the rest is stored in the spleen ready to
be deployed to injured or reactive tissue (Jia and Pamer 2009;
Swirski et al. 2009). Once newly recruited monocytes migrate
Please cite this article in press as: Mahdavian Delavary, B., et al., Macrophages in skin injury and repair. Immunobiology (2011),
doi:10.1016/j.imbio.2011.01.001
through the vessel wall, they release enzymes that fragment ECM
proteins, which creates space for monocytes to migrate into the
wound bed. Subsequently, in reaction to the micro-environment,
monocytes differentiate into macrophages. Macrophage numbers
increase during the phase of inflammation, peak during the phase
of granulation tissue formation and decline during the maturation
phase (Martin and Leibovich 2005).
Macrophages in the wound bed can display different func-
tional phenotypes, which can roughly be divided into two
groups: M1 (classically activated) and M2 (alternatively activated)
macrophages (Mantovani et al. 2002). These phenotypes are two
extremes of a continuum of macrophage function. There are sev-
eral mediators that can stimulate macrophages to differentiate
into M1-macrophages, the most important being bacterial prod-
ucts like lipopolysaccharide (LPS) and inflammatory cytokines like
interferon (IFN)-␥ (Mosser and Edwards 2008; Gordon 2003). M1
macrophages exhibit antimicrobial properties by release of inflam-
matory mediators inducing tumour necrosis factor (TNF)-␣, nitric
oxide (NO) and interleukin (IL)-6. Although this is important in
host defence, the spectrum of expressed cytokines is also capa-
ble of inducing serious collateral tissue damage (Mosser 2003;
Wilson et al. 2005). On the other hand, macrophages activated by
IL-4 and IL-13 develop into so-called alternatively (M2) activated
macrophages (Gordon 2003), which suppress inflammatory reac-
tions and adaptive immune responses. M2 macrophages have been
reported to play an important role in wound healing, angiogenesis
and the defence against parasitic infections (Gordon 2003). How-
ever, despite their beneficial functions, M2 macrophages can also
be involved in different diseases, of which the most prominent are
allergy, asthma and fibrosis (Duffield 2003). These diseases are the
result of a T-helper (Th)-2 response, which is predominated by IL-4
or IL-10 (Mosser and Edwards 2008).
The mentioned macrophage phenotypes will influence the
wound healing process in different ways, depending on the
micro-environment in which they exert their function. As both
macrophage phenotypes are pivotal for the developing wound, it
is likely that the balance between the two phenotypes is impor-
tant in the different phases of wound healing. In the first, more
pro-inflammatory phase of the wound healing process, more M1
macrophages are needed to scavenge debris and to kill possible
invading pathogens. On the other hand, in a later phase in which
new tissue formation is more pronounced, the M2 macrophage
may have a more important role. In derailed healing processes the
balance between the two phenotypes in the phases of repair is prob-
ably disturbed. As such, the role of macrophages in the different
phases of this process will be outlined in detail in this review.
Phase I: Hemostasis
Skin injury causes cell damage and injury of blood vessels. To
prevent blood loss, blood vessels constrict within seconds after
wounding, platelets aggregate and clotting and complement cas-
cades are activated. Together, these events are responsible for
the formation of a haemostatic blood clot, mainly composed of
cross-linked fibrin, fibronectin, vitronectin, trombospondin, ery-
throcytes and platelets (Midwood et al. 2004). The clot is a dynamic
matrix of proteins and cells that contribute to hemostasis, serve
as a temporarily protective shield for the wound, act as a net-
work for incoming inflammatory cells and provide a reservoir of
cytokines/growth factors. Platelets are one of the earliest sources
of cytokines which mediate macrophage activation and chemo-
taxis. Once trapped in the fibrin net, platelets release granules
that function as a reservoir for biologically active proteins, such
as ‘Regulated on Activation Normal T cell Expressed and Secreted’
(RANTES or CCL5) (Frank et al. 2000), thrombin, transforming
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Fig. 1. Inflammatory phase: After migration, monocytes differentiate into macrophages. These macrophages probably exhibit a pro-inflammatory phenotype during this
phase and phagocytose debris and dead neutrophils.
growth factor (TGF)-␤, platelet derived growth factor (PDGF) and
vascular endothelial growth factor (VEGF). These early released
proteins act as chemoattractants for several inflammatory cells,
including monocytes (Maraganore 1993). CCL5 is one of the most
important monocyte chemoattractants released by platelets after
injury. Other important cytokines and chemokines that attract
monocytes to the wound bed are MCP-1 (CCL2), MIP-1␣ (CCL3),
TGF-␣, fibronectin, elastin, C5a, C3a, nerve growth factor and ECM-
components as well.
An important and early mediator of clotting released by platelets
is thrombin (He et al. 2010). Thrombin is a serine protease that
plays a central role in hemostasis after tissue injury by convert-
ing soluble plasma fibrinogen into an insoluble fibrin clot and by
promoting platelet aggregation (He et al. 2010). Thrombin not
only mediates clot formation, it also plays a role in inflamma-
tion. The pro-inflammatory effects of thrombin include stimulation
of vasodilation responsible for plasma extravasation, edema, and
an increased expression of endothelial adhesion molecules that
cause monocyte adhesion and infiltration. Thrombin also stimu-
lates release of pro-inflammatory cytokines, like CCL2, IL-6 and IL-8
by endothelial cells, which induce monocyte chemotaxis (Marin
et al. 2001). Thrombin furthermore induces release of inflammatory
cytokines by peripheral blood monocytes, including IL-6, IFN-␥,
IL-1␤ and TNF-␣. These early produced cytokines are typically pro-
inflammatory cytokines, which likely result in differentiation of
monocytes into M1 macrophages.
Please cite this article in press as: Mahdavian Delavary, B., et al., Macrophages in skin injury and repair. Immunobiology (2011),
doi:10.1016/j.imbio.2011.01.001
3
Phase II: Inflammation
Hemostasis and the release of chemoattractants that attract
phagocytic immune cells can be marked as the initiation of the
inflammatory phase (Fig. 1). Recruitment of immune cells is facil-
itated by chemokines, vasodilation and increase in blood vessel
permeability. Initially blood vessels constrict to ensure hemostasis.
In a later stage these vessels dilate, which peaks around 20 minutes
after wounding. The major factor responsible for vasodilation is
histamine. Histamine also causes blood vessel walls to become
porous, which subsequently leads to additional extravasation of
inflammatory cells into the wound area. The initial and most
obvious function of inflammatory cells at the site of injury is to
provide specific and non-specific defence against pathogens. The
first cells that infiltrate the wound are polymorphonuclear cells,
or neutrophils, which remove foreign particles and bacteria from
the wound area. In the skin, neutrophils appear in the wound
bed within hours after injury. After exerting their function, neu-
trophils are extruded with the eschar (the crust containing dead
cells and degradative products of the wound) or phagocytosed by
macrophages. It has been reported that depletion of neutrophils
in mice or guinea pigs does not result in a decreased number of
wound macrophages in normal wound healing (Dovi et al. 2003;
Simpson and Ross 1972; Devalaraja et al. 2000). Thus recruit-
ment of monocytes to the wound in normal wound healing can
also proceed in a normal manner when neutrophils are depleted.
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Additionally, the role of neutrophils in normal wound healing out-
come is inconclusive in our opinion. Some studies suggest a positive
effect of neutrophils in wound healing and some studies suggest
a negative effect. Dovi et al. reported that neutrophil depletion
in the mouse results in accelerated reepithelialization and has
no effect on the wound disruption strength or collagen content
(Dovi et al. 2003). On the other hand, Devalaraja et al. reported
a delayed reepithelialization and decreased neovascularization in
CXCR2 knockout mice (Devalaraja et al. 2000). Neutrophils are nev-
ertheless a major source of several pro-inflammatory cytokines,
such as IL-1␣, IL-1␤, IL-6 and TNF-␣, which can stimulate newly
attracted monocytes to differentiate into M1 macrophages (Hubner
et al. 1996; Werner and Grose 2003).
Monocytes migrate through the vessel wall in response to
chemotactic stimuli. Transendothelial monocyte migration starts
with the initial contact of monocytes with the vascular endothe-
lium, mediated by adhesion molecules of the selectin family, their
carbohydrate ligands, or by ␣4-integrins (Imhof and Urrand-Lions
2004). This contact results in a weak binding of monocytes to
the endothelium and initiates the adhesion cascade. Subsequently,
selectins, which are cell-surface proteins, interact with glycopro-
tein ligands on monocytes allowing them to bind to endothelial
cells (Imhof and Urrand-Lions 2004). The rolling monocyte is then
stimulated by cytokines and chemokines to engage its surface
integrins that interact with other receptors, like inter-cellular adhe-
sion molecule (ICAM) and vascular cell adhesion molecule (VCAM),
expressed by endothelial cells. This results in a firm adhesion to
the endothelial wall, followed by transmigration into the wound
site.
Monocytes migrate along defined chemotactic gradients and
accumulate within avascular hypoxic areas of the wound bed.
With their integrin receptors and selectines, they bind to spe-
cific proteins, like CAMs of the extracellular matrix (Brown 1995).
After migration to the ECM, monocytes are stimulated by several
cytokines like IL-4, IL-10, IFN-y, IL-13, bacterial product like LPS and
ECM-components to differentiate into macrophages, which peaks
approximately 42 hours after wounding. Monocytes immigrating
into the wound usually develop an inflammatory or debriding phe-
notype, depending on the signals generated in the surrounding
tissue (Stout et al. 2009). It has recently been described by Daley
and co-workers that early (day 1) infiltrating macrophages in the
wound mainly (85%) display a M1 phenotype in a sponge mouse
wound model (Daley et al. 2010).
Within three to five days after injury, macrophages are the most
prominent cells in the healing tissue. Lucas et al. demonstrated that
depletion of macrophages during the inflammatory phase resulted
in significant delay of wound repair in a mouse model (Lucas et al.
2010). Macrophages are firstly important in clearance of senescent
cells and debris within the wound. In case of pathogen spreading
in the wound bed, macrophages phagocytose these pathogens and
present antigens to T-cells. Macrophage involved in clearance of
cells or dead tissue undergo apoptosis. Macrophages that survive
and do not undergo apoptosis, remain in the wound bed area and
exert other functions that influence the wound healing process, like
stimulation of collagen production, angiogenesis and reepithelial-
ization (Baum and Arpey 2005). This change in functional activity
appears to be initiated predominantly by phagocytosis of apop-
totic cells (Stout 2010). Moreover, it has recently been reported by
Khallou-Laschet et al. that macrophages can phenotypically con-
vert from a M1 to a M2 phenotype (Khallou-Laschet et al. 2010).
They clearly demonstrated a phenotype switch of bone-marrow
derived macrophages in an atherosclerosis model in ApoE KO mice
due to environmental changes in cytokine expression. As such,
macrophages play a pivotal role in the transition of the inflamma-
tory phase to the proliferative phase in which they coordinate and
sustain the wound healing events (Singer and Clark 1999).
Please cite this article in press as: Mahdavian Delavary, B., et al., Macrophages in skin injury and repair. Immunobiology (2011),
doi:10.1016/j.imbio.2011.01.001
Phase III: Proliferation
The proliferative phase of cutaneous wound healing, also called
granulation phase, is characterized by active fibroplasia, epider-
mal regeneration, wound contraction and angiogenic sprouting
(Fig. 2). It has been reported that upon phagocytosis of apoptotic
cells M1 macrophages revert to that of M2 macrophages (Duffield
2003; Gordon 2003). It has furthermore been suggested that M2
macrophages derived from M1 macrophages contribute to the res-
olution of inflammation and the wound healing process (Goerdt
and Orfanos 1999; Porcheray et al. 2005). Additionally, Lucas et al.
recently demonstrated that depletion of macrophages during the
proliferative phase significantly disturbed the transition of the mid
stage to the late stage of repair response (Lucas et al. 2010). It can
be concluded from these findings that macrophages have a great
role in the resolution of the inflammatory phase and consequently
the transition of the inflammatory phase to the proliferative
phase.
Within a few days of wound debridement by inflammatory
cells, the injured dermis starts to gain volume by the formation
of granulation tissue. As recently reported by Lucas et al. the dom-
inating macrophage population in the wound at day 5 is the M2
macrophage population (Lucas et al. 2010). In the normal wound
the M2 macrophage probably dominates the wound very early
in the healing process and the inflammatory phase is very short
due to absence of bacterial product or excess dead tissue material.
Furthermore, it has been reported that M2 macrophages suppress
inflammatory responses and adaptive Th1-immunity, but scavenge
debris, and promote angiogenesis, tissue remodelling and repair.
Additionally, they promote a Th2 response, which has been linked
to fibrinogenesis (Wynn 2008). M2 macrophages are furthermore
a prominent source of TGF-␤, which promotes many aspects of
wound repair including inflammation, chemotaxis, wound contrac-
tion, angiogenesis and ECM deposition. TGF-␤ not only stimulates
chemotaxis of and cytokine production by macrophages, it is one
of the most important cytokines that influence fibroblast func-
tion, chemotaxis and ECM deposition. This multipotent protein
promotes fibrosis by directly stimulating mesenchymal cells and
fibroblasts (Martin and Leibovich 2005). TGF-␤ not only stimulates
collagen production, but also reduces degradation of the wound
matrix by collagenase and through increased inhibition of MMPs
via increased expression of TIMPs. TGF-␤ has three isotypes (TGF-
␤1, -␤2 and -␤3), which all stimulate infiltration of inflammatory
cells and fibroblasts. However, at gestational ages associated with
scarless repair, low levels of TGF-␤1 and high levels of TGF-␤3 are
expressed. Furthermore, in fetal wound experiments, a high TGF-
␤3/TGF-␤1 ratio was associated with scarless healing (Bullard et al.
2003), suggesting that the relative proportion of each subtype may
be crucial for scarring.
TGF-␤ signals through transmembrane receptors that trigger
intracellular regulatory proteins known as Smads. A key cyto-
plasmic mediator of TGF-␤ signalling is Smad3. Recent studies
suggest that attenuation of the Smad3 signalling improves the
healing of wounds with an inhibition of fibrosis. Additionally,
wounds in Smad3-null mice exposed to irradiation show decreased
wound width, enhanced epithelialization and reduced numbers of
myofibroblasts compared to wounds of irradiated wild-type mice
(Ashcroft et al. 1999). These findings suggest an important role for
TGF-␤ and Smad3 in wound healing and scar formation.
The involved transcription factors regulate transcription of tar-
get genes, including procollagen I and III (Roberts et al. 2003).
Hypertrophic scar formation is mainly associated with overexpres-
sion of TGF-␤1 and -␤2 (Ishida et al. 2008; Shah et al. 1995; Wang
et al. 2000). Counteracting the effect of TGF-␤1 and -␤2 significantly
reduced scarring in several animal wound models (Choi et al. 1996;
Huang et al. 2002; Shah et al. 1995).
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Fig. 2. The proliferative phase (including reepithelialization): During this phase active fibroplasia, neovascularization and reepithelization occurs. Dependent on the macrophage
phenotype more or less ECM is produced via fibroblasts and myofibroblasts.
The role of fibroblasts in wound healing has been extensively
studied over the past years. Fibroblasts and myofibroblasts mainly
produce the new ECM necessary for supporting cells and blood
vessels, which provides nutrients and oxygen needed for cell
growth and proliferation. Some fibroblasts are already present
in the wound area, scattered within the tissue without forming
cell–cell contacts, while others are derived from nearby undiffer-
entiated mesenchymal cells (Ross et al. 1970). Both TGF-␤ and
PDGF can mediate the transition of these mesenchymal cells into
myofibroblasts (Kalluri and Neilson 2003; Werner and Grose 2003).
Furthermore, both cytokines stimulate these (myo)fibroblasts to
produce collagen and other ECM components.
Myofibroblasts are characterized by ␣-smooth muscle actin
(␣-SMA) expression and are involved in wound contraction
(Martinez-Ferrer et al. 2010). Compared to normal dermal fibrob-
lasts, myofibroblasts produce higher amounts of ECM components
(Huet et al. 2008). Collagen production by fibroblasts is initiated
between 3 and 5 days after injury and is stimulated by TGF-␤, PDGF,
Fibroblast Growth Factor 2 (FGF2) and Insulin Like Growth Factor
1 (IGF-1) (Ishida et al. 2008; Vogler et al. 2003; Werner and Grose
2003), which are mainly produced by macrophages.
Simultaneously, fibroblasts and endothelial cells start to migrate
over the provisional wound matrix into the wound space with the
help of matrix metalloproteinases (MMPs). The migrated endothe-
lial cells provide the formation of new vessels in the wound bed.
An important cytokine that promotes this migration is IL-1, which
stimulates the release of MMPs and synergistically induces collage-
nase activity in conjunction with IFN-␥ and TNF-␣. Increased levels
of IL-1 may consequently result in ECM degradation and slower
wound healing (Niessen et al. 2001).
Macrophages provide an ongoing source of pro-inflammatory
cytokines, including IL-1␣, IL-1␤, IL-6 and TNF-␣, which are not
only responsible for the control of inflammatory cell adhesion and
migration, but are also important for the stimulation of fibroblast
Please cite this article in press as: Mahdavian Delavary, B., et al., Macrophages in skin injury and repair. Immunobiology (2011),
doi:10.1016/j.imbio.2011.01.001
5
and keratinocyte proliferation (Werner and Grose 2003). Ablation
of macrophages in the wound consequently results in decreased
expression of TGF-␤, reduced proliferation of fibroblasts and less
ECM deposition (Mirza et al. 2009).
Eventually, during the proliferative phase, the fibrin clot is trans-
formed into connective tissue rich in blood vessels, underlying the
pink granular appearance. This transformation requires an accurate
balance between matrix degradation and production.
Phase IIIa: Angiogenesis
The formation of new capillaries from pre-existing blood ves-
sels during wound healing mainly presents as angiogenic sprouting
and is an essential component of wound healing because of the
increased nutrient demand (Fig. 3). This process proceeds through
a series of steps. Macrophages play an important role in this pro-
cess, which has been illustrated in for instance the cornea, the rabbit
ear (Thakral et al. 1979), and in vitro (Greenburg and Hunt 1978).
Macrophage depletion leads to delayed and abnormal neovascular-
ization.
Firstly, the ECM and basement membrane are degraded to facil-
itate migrating endothelial cells. Degradation is mediated by MMPs
produced by several cells including macrophages, which are a
rich source for MMPs and serine protease. Besides macrophages,
endothelial cells themselves also produce a number of proteases
by which they locally degrade the basement membrane. Subse-
quently, endothelial cells migrate into the wound by adherence to
integrin cell surface receptors on the ECM. Migration is stimulated
by among others VEGF, FGF, Angiopoietins and TGF-␤. The expres-
sion of integrin receptors on endothelial cells is also stimulated
by VEGF, FGF and TGF-␤. TGF-␤ not only stimulates endothelial
migration, differentiation and tubule formation, but also amplifies
the latter mentioned processes.
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6 B.M. Delavary et al. / Immunobiology xxx (2011) xxx–xxx
Fig. 3. Angiogenesis: This is part of the proliferative phase. angiogenesis is predominantly regulated by macrophages, endothelial cells and keratinocytes. The most important
mediator during this phase is VEGF.
Simultaneously, these secretory proteins regulate capillary
branching and neovascularization during ischemia reperfusion.
Among them is plasminogen activator (PA), which can degrade ECM
molecules resulting in pro-angiogenic fragments like hyaluronic
acid and fibrin (van Hinsbergh et al. 1997). Inhibition of PA conse-
quently leads to suppression of angiogenesis.
Once recruited to the wound, macrophages induce angiogene-
sis. This is predominantly exerted by releasing TNF-␣ and VEGF.
TNF-␣ may in turn induce VEGF expression by keratinocytes
and fibroblasts (Frank et al. 1995). Depletion of macrophages in
the wound results in reduced vascularization (Mirza et al. 2009)
and also leads to severe hemorrhage, fibrin and serum exudates
in macrophage depleted granulation tissue (Lucas et al. 2010).
This has been reported to be mainly caused by withdrawal of
macrophage-derived TGF-␤1 and VEGF (Mirza et al. 2009; Lucas
et al. 2010), supporting an important role for macrophages in pro-
moting angiogenesis via their secretory products. Furthermore,
there is an important relation between macrophages, monocytes
and endothelial cells which originates from embryogenesis. Both
monocytes and endothelial cells have the same precursor cell, the
hemangioblast. Monocytes and macrophages have the ability to
acquire endothelial properties when placed in angiogenic condi-
tions. Moreover, cells with a hybrid phenotype, positive for both
monocytic marker CD45 and endothelial V-cadherin, were isolated
from tumours (Conejo-Garcia et al. 2005).
For induction of angiogenesis, macrophages first have to be acti-
vated in a specific way. Several studies have reported that M2
macrophages promote angiogenesis. Under several stimuli, like
low oxygen tension or high concentrations of lactate pyruvate or
hydrogen ions that have also been observed in the wound area,
macrophages can become angiogenic (Knighton et al. 1983).
Another very important function has recently been suggested to
be conducted by (resident) tissue macrophages in angiogenesis, as
these cells are located near vessel branches. Specialized endothe-
lial cells at the forefront of the sprout, the so called tip cells, are
thought to navigate to their target. The formation of tip cells is pre-
Please cite this article in press as: Mahdavian Delavary, B., et al., Macrophages in skin injury and repair. Immunobiology (2011),
doi:10.1016/j.imbio.2011.01.001
dominantly stimulated by M2 macrophages, especially through the
formation of VEGF (Tammela et al. 2008). Previous studies mainly
focussed on understanding how tip cells initiate vessel sprouting
while very little is known about how these cells fuse with neigh-
bouring sprouts to form a perfused vessel (Schmidt and Carmeliet
2010). Recently it has been reported by Fantin et al. that con-
trary to expectation, tip cells lack the ability to recognize, find and
fuse with other tip cells. Instead, macrophages serve as guidance
and bridge cells in this fusion process (Fantin et al. 2010). These
findings further support an important role for macrophages in
angiogenesis.
VEGF is one of the most important pro-angiogenic mediators
and stimulates multiple components of the angiogenic cascade. It
is present in wound fluid in high concentrations (Howdieshell et al.
1998), and it is secreted by different cell types in response to ker-
atinocyte growth factor (KGF), TGF-␤ (Ferrara et al. 1995; Ferrara
1995), hypoxia (Shweiki et al. 1992), bFGF (Stavri et al. 1995) and
PDGF (Frank et al. 1995). Platelets are one of the first cells to
appear in the wound site followed by neutrophils and macrophages.
Platelets release VEGF after stimulation, particularly after thrombin
stimulation (Mohle et al. 1997), leading to stimulation of angiogen-
esis in the early phase of wound healing.
The endothelium of the microvasculature exhibits several
important responses to extracellular VEGF. The first is increased
endothelial proliferation, which leads to a greater population size.
The second is increased permeability, exerted through an unknown
change in endothelial cell structure and function (Dvorak et al.
1995). This increased permeability results in a provisional extra-
cellular matrix necessary for angiogenesis. Macrophages can also
increase vascular permeability by releasing vaso-active substances
such as vascular permeability factor (Berse et al. 1992), sub-
stance P (Pascual and Bost 1990), platelet activating factor and
prostaglandins (Middleton and Thatcher 1998). These findings sup-
port the ability of the monocyte/macrophage lineage, especially
the M2 macrophage (the major source of TGF-␤), to contribute to
angiogenesis during wound healing.
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Phase IIIb: Reepithelialization
Reepithelialization includes the re-establishment of an intact
epidermis over the newly formed (scar) tissue (Fig. 2). In intact
skin, the basal keratinocyte layer is attached to the basal lamina.
After injury, keratinocyte organization is disturbed and has to be
restored. This restoration process initiates within hours after injury
and is performed by keratinocytes that move across the granula-
tion tissue. This process, called contact guidance, continues until
keratinocytes from the opposing sides of the wound re-establish
contact. Before migration, keratinocytes change shape, become flat-
ter and elongated, and extend cellular processes like lamellipodia
and fillopodia (Santoro and Gaudino 2005).
TGF-␤1 is one of the most important ligands for epithelial
cell migration during reepithelialization (O’Kane and Ferguson
1997), as it stimulates expression of integrin subunits that pro-
mote keratinocyte migration on the provisional ECM. Mirza et al.
have recently shown that depletion of macrophages in the wound
results in delayed reepithelialization (Mirza et al. 2009), sup-
porting that macrophage-derived TGF-␤ plays a crucial role in
reepithelialization. Several other growth factors are important for
keratinocyte proliferation at the wound edge, including epidermal
growth factor (EGF), TGF-␣, keratinocyte growth factor (KGF) and
heparin-binding epidermal growth factor (HB-EGF), all acting as
ligands for the EGF receptor. In order to create a path through the
fibrin clot, the keratinocytes in the wound edge have to dissolve
the fibrin barrier. The major fibrinolytic enzyme in this process
is plasmin, which is derived from activated plasminogen out of
the bloodstream. Furthermore, various MMPs play a crucial role
in this process. These MMPs are upregulated by keratinocytes and
macrophages in the wound edge. Degradation of ECM components
by MMPs is required to remove and reorganize provisional matrices
and to allow migration (Ravanti and Kahari 2000).
The reepithelialization process is made easier by contraction of
the underlying connective tissue, which brings the wound mar-
gins towards each other. This contraction process is performed by
myofibroblasts, activated by TGF-␤ and PDGF (Frank et al. 1995).
Once the wound surface is covered by a monolayer of keratinocytes,
epidermal migration ceases and a new stratified epidermis with
underlying basal lamina is re-established from the margins of the
wound inwards. At this moment, the defect is filled with gran-
ulation tissue and covered by a newly formed epidermal layer.
Nevertheless, the wound healing process, particularly the remod-
elling phase, can still go on for months. The remodelling process is
crucial for the scar strength and appearance.
Phase IV: Remodelling
The remodelling phase is the last and longest phase of the
wound healing process and continues for weeks to months (Fig. 4).
During this phase cell proliferation slows down, protein synthesis
decreases and remodelling of collagen into larger, more organised
fibrils occurs. As the nutrient demand within the tissue decreases,
a regression of recently formed capillaries occurs, and the red-
ness of the scar will fade. Most endothelial cells, macrophages and
myofibroblasts undergo apoptosis, or exit the wound (Gurtner et al.
2008).
The wound remodelling process is a balance between ECM
production, breakdown and remodelling. This balance is probably
determined by the microenvironment of the scar by means of a Th2
or Th1 dominated environment (Tredget et al. 2006) and is proba-
bly maintained by the T-cells that infiltrate the wound and signal
macrophages and other immune cells.
It has been reported that T-cells do not play a prominent role
in wound healing in case of minor damage. In case of major dam-
Please cite this article in press as: Mahdavian Delavary, B., et al., Macrophages in skin injury and repair. Immunobiology (2011),
doi:10.1016/j.imbio.2011.01.001
7
age or when wound healing is complicated by infection or other
activity which activates the adaptive leukocyte system, T-cells play
an important role (Tredget et al. 2006). Macrophages and T-cells
cross-talk via cytokines and co-stimulators, including cell surface
molecules, which can result in a wide range of outcomes in function
of these cells (Doherty 1995). Moreover, macrophages stimulate T-
cell expansion and differentiation to Th1 and Th2 cells. In this case,
T-cells make wound healing more complex and may prolong or
derail the wound healing process in several aspects. This may result
in necrosis, fibrosis, ulcera formation and granuloma formation.
T-lymphocytes infiltrate the wound bed in the late inflamma-
tory phase of wound repair, suggesting that these cells are involved
in either the late proliferative or remodelling phase. In skin wounds,
T-cell numbers peak at day 7, but remain in the scar for up to
months after wounding (unpublished data). Studies in mice lack-
ing T-cells suggest that the primary function of T-cells in incision
wounds is downregulation of fibrous tissue growth. This suggests
that more Th1 cells populate the normal wound during the remod-
elling phase, because a Th2 response results in the production of
ECM. Th1 cells mainly produce IFN-y which results in the differen-
tiation of macrophages into M1-macrophages.
Macrophages also play an important role in modulating the
capacity of other cell types to secrete neutral proteinases that
degrade matrix macromolecules. ECM breakdown is fundamental
for wound healing and tissue remodelling. One of the most impor-
tant components of ECM is collagen which increases the strength
of the wound. The important processes in the remodelling phase
are the rearrangement and cross-linking of initially deposited col-
lagen fibers and substitution of type III collagen by type I collagen,
which is a stronger collagen fibril. In vivo, collagen is primarily pro-
duced by (myo)fibroblasts. Increasing quantities of ECM signals the
fibroblasts to decrease subsequent collagen production. Further-
more, IFN-␥ and TNF-␣ stimulate fibroblasts to decrease collagen
synthesis.
MMPs and their natural inhibitors (TIMPs) are generally
accepted as being important mediators of proteolytic activity dur-
ing many physiological and pathological remodelling processes,
including tissue repair. The MMP family has the combined capacity
to degrade virtually all ECM components and basement mem-
brane macromolecules. Macrophages are a rich source of MMPs
and serine proteases. They are especially involved in MMP-2, MMP-
12 and MMP-19 expression, and they synthesize tissue inhibitors
of metallo- and serine proteases. MMP-12, for example, is a
macrophage-specific metallo-elastase (Shapiro et al. 1993), which
has been suggested to play a role in capillary regression in skin
wounds, resulting in a decrease of erythema (Madlener et al. 1998).
Furthermore, macrophages have been reported to be able to com-
plete the hydrolysis of the matrix proteins into small molecules
(Jones and Scott-Burden 1979). Proteinases, including PA, can
also mediate extracellular protein degradation by macrophages
(Gordon et al. 1974; Jones and Scott-Burden 1979). Besides pro-
moting the formation of ECM, macrophages also promote tissue
integrity by producing type VIII collagen (Weitkamp et al. 1999)
and some matrix proteins (Gratchev et al. 2001).
The remodelling phase is primarily dependent on tissue break-
down and ECM production and macrophages play a crucial role in
both processes. The breakdown is directly regulated and the ECM
production is mainly regulated via fibroblasts. As recently pub-
lished, in the last phase of normal wound healing, the number of
wound macrophages is decreased, which will result in less ECM
production. Beside the changed macrophage numbers, different or
reduced activity of macrophages in the last phase of wound healing
could also result in less ECM production. These changes, together
with contraction of the wound, will result in a decrease in wound
size and volume. In pathological conditions macrophage numbers
and function could be changed resulting in aberrant scarring.
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8 B.M. Delavary et al. / Immunobiology xxx (2011) xxx–xxx
Fig. 4. Remodelling phase: During this phase the balance between ECM-breakdown and -formation is important and determines the eventual scar result. The balance is
determined by among others the micro-environment, macrophage phenotype and T-cell response.
Conclusion and perspectives
The phases of wound healing normally proceed in a predictable
timely manner. In humans, problems with the wound healing can
manifest as either delayed or excessive. Delayed wound healing
can result in a chronic wound and excessive wound healing results
in a hypertrophic scar or keloid scar formation. Excessive scar for-
mation is characterized by an elevated ECM deposition and scar
vascularization. Excessive scars can cause disfigurement and func-
tional impairment. In burns, for example, joint function can be
severely impaired due to the excessive scar. Much research has
been conducted, primarily focussing on fibroblasts and TGF-␤, to
elucidate the wound healing process, and to find novel treatment
possibilities. Unfortunately, studies on wound healing have not yet
led to substantial advances in patient care.
Until now, macrophage phenotype has not been regarded to play
a substantial role in the outcome of the wound healing process.
Since the role of macrophage phenotype has been highlighted in
fibrosis in several studies and some studies underline the impor-
tance of macrophages in wound healing, the interest in macrophage
function in wound repair is rising.
Based on the mentioned studies in this paper we hypothesize
that the inflammatory phase in excessive scarring is prolonged and
therefore more macrophages are attracted to the wound bed. The
phenotype of newly attracted macrophages will first be more pro-
inflammatory (M1) and will switch to a more pro-fibrotic (M2)
phenotype. The M2 macrophages could furthermore be more active
in excessive scarring because of more intense stimuli.
As macrophages play an important role in wound healing,
intervention in macrophage function in several pathological con-
ditions may result in less ulceration and chronic wounding
and may prevent hypertrophic scar and keloid formation. For
intervention, more knowledge on the role of macrophages in
would healing, especially their functional phenotype (M1/M2) in
the different phases and in different pathological conditions is
needed.
Please cite this article in press as: Mahdavian Delavary, B., et al., Macrophages in skin injury and repair. Immunobiology (2011),
doi:10.1016/j.imbio.2011.01.001
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Please cite this article in press as: Mahdavian Delavary, B., et al., Macrophages in skin injury and repair. Immunobiology (2011),
doi:10.1016/j.imbio.2011.01.001