Download Human embryonic epidermis contains a diverse Langerhans cell

© 2014. Published by The Company of Biologists Ltd | Development (2014) 141, 807-815 doi:10.1242/dev.102699
RESEARCH ARTICLE
STEM CELLS AND REGENERATION
Human embryonic epidermis contains a diverse Langerhans cell
precursor pool
Christopher Schuster1, Michael Mildner2, Mario Mairhofer3, Wolfgang Bauer1, Christian Fiala4, Marion Prior1,
Wolfgang Eppel3, Andrea Kolbus3, Erwin Tschachler2, Georg Stingl1 and Adelheid Elbe-Bürger1,*
KEY WORDS: Langerhans cell, Precursor, Development, Skin,
Skin equivalent
INTRODUCTION
Langerhans cells (LCs) are antigen-presenting cells (APCs) that play
important roles as immune sentinels in skin (Romani et al., 2012a).
During human skin development, their precursors are first identified
in the epidermis at 7-9 weeks estimated gestational age (EGA),
based on the expression of CD45 (PTPRC – HGNC), HLA-DR,
CD1c and ATPase (Foster et al., 1986; Fujita et al., 1991; Schuster
et al., 2009). The distinguishing features of LCs in adult human
epidermis, namely Birbeck granules, CD207/langerin, and CD1a are
detectable only after 11 weeks EGA (Foster et al., 1986; Schuster et
1
Department of Dermatology, Division of Immunology, Allergy and Infectious
Diseases (DIAID), Laboratory of Cellular and Molecular Immunobiology of the
Skin, Medical University of Vienna, Vienna, Austria. 2Department of Dermatology,
Research Division of Biology and Pathobiology of the Skin, Medical University of
Vienna, Vienna, Austria. 3Department of Obstetrics and Gynaecology, Medical
University of Vienna, Vienna, Austria. 4Gynmed-Ambulatorium, Vienna, Austria.
*Author for correspondence ([email protected])
This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted
use, distribution and reproduction in any medium provided that the original work is properly
attributed.
Received 16 August 2013; Accepted 10 December 2013
al., 2009). Our observation that not all HLA-DR-positive (HLADR+) epidermal cells in embryonic human skin express the LC
markers CD1a, CD1c or CD207/langerin, prompted us to
hypothesize that during ontogeny the LC phenotype is acquired in
the epidermis (Schuster et al., 2009), a notion that was recently
validated in the murine system (Seré et al., 2012). Owing to this
stepwise development of the LC phenotype, it is conceivable that
other molecular structures required for the initiation of immune
responses including MHC-II molecules (e.g. HLA-DR) may also be
acquired in the epidermis. Thus, by looking at early stages of
development the original phenotype of LC precursors could be
unravelled. Indeed, an earlier study identified dendritic or round
epidermal cells in the first trimester that expressed the scavenger
receptor CD36 (Fujita et al., 1991). Concomitant with HLA-DR+
epidermal cells becoming more frequent, CD36+ cells in the human
epidermis decrease in numbers and are – as in healthy adult skin –
not detectable at the end of the first trimester. Their origin, precise
phenotype and ultimate fate have yet to be elucidated. At 7 weeks
EGA, the earliest time point where epidermal LC precursors have
been identified (Foster et al., 1986), haematopoiesis in humans is
active in the yolk sac, the aorta-gonad-mesonephros region and the
foetal liver, representing potential sites of origin for these precursors,
but inactive in the bone marrow (Tavian and Péault, 2005).
Although local proliferation has been described in epidermal
leukocytes during the first trimester (Schuster et al., 2009), the
type(s) of progenitors that colonize the epidermis and their
relationship to haematopoietic stem cells remain unknown. The aim
of this study was to further characterize the phenotype of LC
precursors in first trimester human epidermis, in particular to
analyse co-expression of HLA-DR with various molecules often
thought to be present on LC precursors such as CD14, CD36 and
CD45, thereby broadening our knowledge about their ontogeny in
vivo. Moreover, to extend and confirm our in vivo observations we
used a human three-dimensional full-thickness skin model that
evaluated the LC differentiation potential of various cord blood- or
peripheral blood-derived cells phenotypically resembling the
precursor cells found during ontogeny.
RESULTS
Human embryonic skin harbours leukocytes with distinct
phenotypes
In healthy adult human skin, CD36 is found on a multitude of cells
including immune cells such as dermal dendritic cells and
macrophages as well as non-immune cells including endothelial
cells and melanocytes (Foster, 1993). To explore the expression of
CD45+CD36+ cells in developing human skin, we compared whole
prenatal with adult skin cell suspensions (Fig. 1A). Using flow
cytometry, we found CD45+CD36+ cells in first trimester skin,
though at lower frequencies than in adult skin (0.71±0.33%, n=8
versus 4.06±1.56%, n=6; P=0.003; Fig. 1C). However,
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Development
ABSTRACT
Despite intense efforts, the exact phenotype of the epidermal
Langerhans cell (LC) precursors during human ontogeny has not
been determined yet. These elusive precursors are believed to
migrate into the embryonic skin and to express primitive surface
markers, including CD36, but not typical LC markers such as CD1a,
CD1c and CD207. The aim of this study was to further characterize
the phenotype of LC precursors in human embryonic epidermis and
to compare it with that of LCs in healthy adult skin. We found that
epidermal leukocytes in first trimester human skin are negative for
CD34 and heterogeneous with regard to the expression of CD1c,
CD14 and CD36, thus contrasting the phenotypic uniformity of
epidermal LCs in adult skin. These data indicate that LC precursors
colonize the developing epidermis in an undifferentiated state, where
they acquire the definitive LC marker profile with time. Using a human
three-dimensional full-thickness skin model to mimic in vivo LC
development, we found that FACS-sorted, CD207– cord bloodderived haematopoietic precursor cells resembling foetal LC
precursors but not CD14+CD16– blood monocytes integrate into skin
equivalents, and without additional exogenous cytokines give rise to
cells that morphologically and phenotypically resemble LCs. Overall,
it appears that CD14– haematopoietic precursors possess a much
higher differentiation potential than CD14+ precursor cells.
RESEARCH ARTICLE
Development (2014) doi:10.1242/dev.102699
CD45–CD36+ cells were significantly more frequent in first trimester
than adult skin (20.06±5.93%, n=8 versus 2.14% ± 1.17, n=6;
P<0.001). These cells most likely represent undifferentiated
mesenchymal, endothelial and periderm cells. Unexpectedly, we also
observed that the relative expression of the monocyte marker CD14
on CD36+ leukocytes did not differ significantly between prenatal
and adult skin (Fig. 1A,B, upper panel). Strikingly, significantly
fewer CD45+CD36+ cells in prenatal skin expressed HLA-DR
compared with adult skin (Fig. 1A,B, lower panel). Expression of
CD14 on CD45+CD36– and CD45–CD36+ cells between the first
trimester and adult skin was comparable, whereas HLA-DR was
found significantly less frequently. The notion that first trimester
skin is devoid of HLA-DR, an indicator of antigen-presenting
capacity, was corroborated by the fact that in first trimester skin
CD36+ leukocytes outnumbered HLA-DR+ as well as CD14+
leukocytes (Fig. 1C). By contrast, HLA-DR+ leukocytes were
significantly more frequent than CD36- or CD14-expressing cells in
adult skin (Fig. 1C).
Fig. 1. CD45+CD36+ cells are the predominant leukocyte population in
human prenatal skin. (A) Multiparameter flow cytometry of freshly isolated
single cell suspensions of prenatal and adult human skin was performed by
incubation with monoclonal antibodies against the cell surface markers
indicated (n=5). Gates in dot plots were set according to isotype-matched
control staining. Dead cells (based on 7-AAD uptake) were excluded.
Histograms show CD14 and HLA-DR expression of indicated regions (black
line; grey line, isotype control). (B) Dot graphs of the percentage expression
of CD14 and HLA-DR in CD45+CD36+ (R1), CD45+CD36– (R2) and
CD45–CD36+ (R3) cells. Bars indicate the mean of investigated groups.
(C) Flow cytometric analysis of the percentage of HLA-DR-, CD14- and
CD36-expressing cells relative to total skin cells. Empty symbols represent
adult skin, filled symbols prenatal (9-14 weeks EGA) skin.
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To further characterize the phenotype and the localization of CD36+
cells in prenatal skin, triple immunofluorescence staining was
performed on cryostat sections. As the moderate thickness of the
embryonic epidermis precludes the enzymatic separation from the
underlying dermis (and consequently a comparative assessment of
epidermal and dermal subsets by flow cytometry),
immunofluorescence remains the only technique available to study
the compartmental distribution of epidermal leukocyte subsets. We
found that epidermal CD36+ cells are primarily round or moderately
dendritic in shape (Fig. 2A, arrowhead) and always co-express the
haematopoietic cell marker CD45+, thus confirming and extending
previous findings (Fujita et al., 1991). The frequency of CD45+
epidermal cells between 9-11 and 12-14 weeks EGA did not differ
but was significantly lower than in adult skin (Fig. 2B).
CD45+CD36+ epidermal cells could be readily identified up to the
end of the first trimester but not beyond 18 weeks EGA. CD45+
epidermal cells in first trimester human skin displayed striking
surface marker heterogeneity. Of note, at 9-11 weeks EGA,
significantly fewer CD45+ epidermal cells stained for HLA-DR than
at 12-14 weeks EGA, confirming our flow cytometric data of HLADR depletion also for the epidermal leukocyte compartment (Fig.
2C, left). When HLA-DR expression became more pronounced on
epidermal leukocytes, the frequency of CD45+CD36+ cells
concomitantly declined (Fig. 2C, middle), although CD36
expression was occasionally found until the end of the first trimester.
Surprisingly, a small percentage of CD45+ epidermal cells expressed
neither CD36 nor HLA-DR (Fig. 2A, double arrow and inset; Fig.
2C, right graph).
Owing to the broad expression of CD36 on haematopoietic cells
(e.g. myeloid precursor cells and their progeny, red blood cells,
monocytes, dendritic cells) and non-haematopoietic cells (e.g.
keratinocytes under inflammatory conditions, a subset of
melanocytes), it was not possible to determine whether it was
associated with one specific cell type more than the others. To better
define the phenotype of embryonic CD36+ skin cells and in
particular CD36+ epidermal cells, we performed co-immunostaining
experiments with other markers. Similar to the distribution of CD36,
CD14 was predominately expressed on mainly round dermal cells
and occasionally in the epidermis (Fig. 3A; supplementary material
Fig. S1). In contrast to CD36, CD14 was exclusively found on
CD45+ cells and appeared to become less frequent on epidermal
Development
CD45+CD36+ cells are the predominant leukocyte population
in human embryonic epidermis
RESEARCH ARTICLE
Development (2014) doi:10.1242/dev.102699
Fig. 2. Human embryonic epidermis contains
various leukocyte subsets with regard to
CD36 and HLA-DR expression.
(A) Immunofluorescence triple labelling identified
CD45+CD36+HLA-DR+ (arrowhead),
CD45+CD36–HLA-DR+ (arrow) and
CD45+CD36–HLA-DR– (double arrow) epidermal
cells in first trimester human skin. LCs in healthy
adult skin do not express CD36. Alexa Fluor 488labelled laminin 5 monoclonal antibody visualizes
the dermo-epidermal junction. (B) Dot graph of the
increase of CD45+ epidermal cells/linear
centimetre of epidermis. Bars indicate the mean of
the investigated groups. (C) The percentages of
the indicated surface molecules among total
CD45+ epidermal cells. Bars indicate the mean of
investigated groups. Scale bars: 50 μm.
arrowhead, middle panel), the nature of which remains to be
determined but perhaps they are macrophages, dendritic cells or
mast cell precursors (Tavian and Péault, 2005).
We discovered that some epidermal leukocytes in first trimester
human skin did not express any commonly used monocyte or
dendritic cell marker and could only be identified as haematopoietic
cells on the basis of their staining for CD45. In addition, they did
not express CD34 but partially expressed markers found on
monocytes, namely CD14, CD36 and HLA-DR.
Cord blood cells with phenotypes resembling embryonic
resident epidermal leukocytes develop into CD207+ LCs in a
human skin equivalent model
Human skin equivalents are a highly valuable tool to study the
origin and development of LCs, given that potential precursors can
be mixed with keratinocytes and allowed to integrate in the
differentiating epithelium. These skin equivalents are generated with
primary human keratinocytes and fibroblasts that are seeded into a
collagen matrix. Lifting of keratinocytes to the air-liquid surface
induces terminal differentiation, resulting in a stratified and cornified
epidermis (Fig. 4A) (Mildner et al., 2010). In such a model, LC
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Development
leukocytes towards the end of the first trimester (Fig. 3B). Similar
to HLA-DR expression on CD36+ cells, CD14 was observed on
both CD45+CD36+ and CD45+CD36– cells (data not shown). CD45+
epidermal cells expressing neither CD36 nor CD14 were also found
(Fig. 3A, double arrow; supplementary material Fig. S1). Of note,
the dendritic cell marker CD1c also showed this diversity of
expression (Fig. 3C, arrow and arrowhead; supplementary material
Fig. S2, arrow and arrowhead).
Given that CD14 and CD36 can also be detected on early CD34+
myeloid precursor cells (Thoma et al., 1994), we then analysed
whether epidermal leukocytes express the haematopoietic stem cell
marker CD34. No co-expression of CD34 and CD45 was detectable
in the epidermis at any time point investigated (Fig. 3D, arrow;
supplementary material Fig. S3, arrow), whereas CD34 was evident
on dermal vessels in first trimester human skin (Fig. 3D, arrowhead;
supplementary material Fig. S3, arrowhead). Intriguingly,
CD45+CD34+ dermal cells were rarely detectable and located
predominantly within vessels (Fig. 3D, arrowhead; supplementary
material Fig. S3, arrowhead). Solitary CD45+CD34+ cells were
present at a very low frequency in the developing dermis (Fig. 3D,
arrowhead, middle panel; supplementary material Fig. S3,
RESEARCH ARTICLE
Development (2014) doi:10.1242/dev.102699
precursors should ideally develop from precursors without
additional exogenous cytokines because all important stimuli are
supposed to be provided by the artificial epidermis and dermis. To
examine this, skin equivalents were tested for the production of key
cytokines required for LC development, such as TGFβ1 (Borkowski
et al., 1996; Geissmann et al., 1998; Kaplan et al., 2007; Strobl et
al., 1996), GM-CSF (Geissmann et al., 1998; van de Laar et al.,
2012) and the evolutionarily highly conserved IL-34 (IL-34 –
HGNC) (Greter et al., 2012; Wang et al., 2012). Using
immunohistochemistry, we found that skin equivalents express high
levels of TGFβ1, although in a different pattern compared with adult
skin. In healthy adult skin, TGFβ1 is located exclusively
suprabasally (Schuster et al., 2009), whereas in skin equivalents
TGFβ1 is also expressed in cells of the basal layer. Interestingly, the
basal layer showed varied expression, perhaps reflecting
stratification of pre-existing cells in the formation of the epidermis
of the skin equivalent rather than development through proliferation
attained in normal skin. Moreover, TGFβ1high keratinocytes are
scattered throughout the whole epidermis (Fig. 4A, inset, arrows).
When analysing supernatants and lysates with ELISA, all three
cytokines can be detected in skin equivalents (Fig. 4B). Kinetic
experiments during days 1-8 revealed that the levels of TGFβ1 in
the supernatants remain constant throughout the whole observation
period, whereas its total amount in epidermal lysates gradually
increased until day 5 and then decreased (Fig. 4B, upper and lower
panel). The GM-CSF level in supernatants steadily increased until
day 5 but thereafter reduced, so that on day 8 the level was the same
as at day 3. IL-34 was not detectable in supernatants but showed
constant levels in the lysates. Taken together, these data show that
skin equivalents are endowed with the molecular prerequisites to
promote the development of LC.
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Next, we tested the differentiation capacity of LC precursor
candidates in skin equivalents. For this purpose, CD34+ cord bloodderived cells obtained by fluorescence-activated cell sorting (FACS)
were cultured (Caux et al., 1992; Strobl et al., 1996). After 10 days,
specific subsets (e.g. CD1a+CD14–, CD1a–CD14–, CD1a–CD14+),
and in parallel CD14+CD16– blood monocytes, were analysed (Fig.
4C) and sorted to high purity (Fig. 5C). Basically, these populations
lacked the expression of the LC marker CD207 and variably
expressed CD36. Although almost all CD1a+CD14– and
CD1a–CD14+ cells co-express CD36, this marker is only found on
a subset of CD1a–CD14– haematopoietic precursors. All populations
expressed the pan-leukocyte marker CD45, the myeloid cell marker
CD33 and to a varying degree HLA-DR. CD34 had been lost in all
populations, whereas a small CD117+ population was identified
exclusively in the CD1a–CD14– subset, most probably representing
mast cells. The dendritic cell marker CD1c was only detectable on
CD1a+CD14– cells. CD14+CD16– monocytes invariably expressed
CD36 and HLA-DR and lacked expression of the dendritic cell
markers CD1a, CD1c and CD207. Cord blood-derived subsets, as
shown in Fig. 5A, were co-cultured with keratinocytes for 8 days
and equivalents were then analysed by immunohistochemistry (Fig.
5B). Unexpectedly, all cord blood-derived precursor subsets gave
rise to CD1a+ and CD207+ LC in skin equivalents (Fig. 5C). These
cells were dendritic in shape and principally evenly distributed with
occasional clusters. Although the difference of integrated CD1a or
CD207 cells within one cord blood sample was rather low, a high
specimen-specific variability in cell number was observed (Fig. 4D).
CD1a+CD14– and CD1a–CD14– cells had a higher differentiation
potential than CD1a–CD14+ cells (Fig. 4D) to develop into CD1a+
or CD207+ dendritic cells. Although CD207+ cells slightly
outnumbered CD1a+ cells in most samples, the difference was not
Development
Fig. 3. Human embryonic epidermis contains leukocytes
with distinct phenotypes. (A,C) Immunofluorescence triple
labelling identified (A) CD45+CD36+CD14+ (arrowhead) and
CD45+CD36–CD14– (double arrow) as well as (C)
CD45+CD36+CD1c+ (arrow) and CD45+CD36–CD1c+ (arrowhead)
epidermal cells in first trimester human skin. (B) Dot graph of the
percentage of CD14+ leukocytes relative to all epidermal CD45+
cells. Bars indicate the mean of investigated groups. Scale bars:
50 μm. (D) Immunofluorescence double labelling identified solitary
(middle; arrowhead) and vessel-associated (right; inset,
arrowhead) CD45+CD34+ dermal cells. Epidermal leukocytes do
not express CD34 (left; arrow). Scale bars: 50 μm.
RESEARCH ARTICLE
Development (2014) doi:10.1242/dev.102699
statistically significant. CD14+CD16– blood monocytes never
matured into LCs under these conditions (Fig. 5C,D).
DISCUSSION
This study, which focused on the development of epidermal
leukocytes in the first trimester, showed that human epidermis
contains a multitude of phenotypically distinct LC precursors, in
contrast to the uniformity of LCs in adult skin. Using a human threedimensional full-thickness skin model, we also found that cord
blood-derived CD14+ progenitor cells possess the potential to give
rise to CD207+ LCs (Schaerli et al., 2005), although to a
significantly lesser extent than CD1a+CD14– and CD1a–CD14–
progenitors under similar conditions.
LC precursors in human first trimester skin are
heterogeneous
HLA-DR+ leukocytes in embryonic epidermis can be subdivided
according to the expression of the scavenger receptor CD36, as has
been described previously (Fujita et al., 1991). Interestingly, we
found that 23.8% of epidermal leukocytes between 9 and 11 weeks
EGA express neither CD36 nor HLA-DR and that HLA-DR
expression on CD45+ cells roughly equals expression of CD36, thus
confirming and extending previous results (Fujita et al., 1991). In
short, these epidermal leukocytes are CD45+CD36+HLA-DR+,
CD45+CD36–HLA-DR+
or
CD45+CD36+HLA-DR–,
CD45+CD36–HLA-DR–. At the end of the first trimester, HLA-DR
becomes dominant whereas CD36 expression abates and cannot be
found at later stages of development. CD14 on epidermal leukocytes
is found less frequently than CD36, yet it is almost invariably coexpressed with CD36. Like CD36, CD14 is also not detectable on
epidermal cells after 18 weeks EGA. It should be emphasized that
the lower frequency of CD14+ compared with CD36+ epidermal
cells could be explained by the intricate detection of CD14 by
immunofluorescence. In mice, it has been shown that during skin
inflammation Gr1+ monocytes can give rise to epidermal LCs
(Ginhoux et al., 2006). CD36 and CD14 are also found on a subset
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Development
Fig. 4. Skin equivalents fulfil the requirements
for LC development. (A) Immunohistochemical
staining was performed on cryostat sections of skin
equivalents at day 8. TGFβ1 is found throughout
the epidermis with occasional cells expressing it at
high levels (inset, arrows). Data are representative
of five experiments. Scale bar: 50 μm. (B) TGFβ1,
GM-CSF and IL-34 levels were analysed in cell
culture supernatants of skin equivalents or lysed
epidermal equivalents at the indicated time points.
Values are means ± s.d. of three experiments.
(C) Multiparameter flow cytometry of propagated
and cultured CD34+ cord blood-derived
haematopoietic stem cells and blood monocytes
was performed by incubation with monoclonal
antibodies against the cell surface markers
indicated (black line, indicated antibody; grey line,
isotype control). Gates in dot plots were set
according to isotype-matched control staining.
Dead cells (based on 7-AAD uptake) were
excluded.
RESEARCH ARTICLE
Development (2014) doi:10.1242/dev.102699
of human blood monocytes (Zamora et al., 2012), hence it cannot
be excluded that these cells represent monocytic LC precursors. Of
note, a subset of CD45+CD36+ LC precursors in first trimester
epidermis expresses CD1c, indicating that committed dendritic cells
can also co-express CD36 (Schuster et al., 2009).
The lack of CD34 on epidermal leukocytes suggests that the
initial precursor does not have haematopoietic stem cell potential.
Given that the expression of CD34 is highest on uncommitted stem
cells and is increasingly lost as the progenitors differentiate, it
cannot be excluded that these epidermal leukocytes in first trimester
skin show low-level expression of CD34, which is not detectable
using immunofluorescence (Ordóñez, 2012). Owing to the lack of
appropriate immunohistochemical markers for different
differentiation stages of the progeny of haematopoietic stem cells, it
is, at present, not possible to investigate whether these early
precursors possess at least some regenerative potential. However,
we and others have previously shown that leukocytes in developing
mouse and human skin show a higher proliferation capacity than
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those in adult skin (Chang-Rodriguez et al., 2005; Schuster et al.,
2009; Chorro et al., 2009).
The phenotypic heterogeneity of LC precursors during skin
development results from the presence and absence of molecules
representative of LCs in healthy adult skin. The principal cause of
this diversity remains unexplained to date and different scenarios, or
a combination thereof, are possible: (1) gain and/or loss of antigens,
(2) asynchronous maturation of precursors, or (3) colonization by
different precursor cells. In humans, blood formation starts in the
yolk sac, and later shifts to the aorta-gonad-mesonephros region, the
foetal liver, and finally, around 11 weeks EGA, to the bone marrow
(Péault and Tavian, 2003). It is possible that LC precursors differ
depending on their origin (Hoeffel et al., 2012). Evidence for this
comes from the most recent fate-mapping experiments in mice
showing that the primitive epidermis is colonized by a first wave of
yolk sac-derived precursors and macrophages (Hoeffel et al., 2012).
Subsequently, these early precursors are largely replaced by
precursors that originate in the foetal liver. Based on these data we
Development
Fig. 5. Cord blood-derived cells develop into
CD207+ LCs in a human skin equivalent model.
(A) Representative dot plot of FACS-sorted
haematopoietic precursors and blood monocytes
(n=6). (B) Schematic view of LC development from cord
blood-derived precursors or blood monocytes in skin
equivalents composed of primary human
keratinocytes and fibroblasts in a collagen matrix.
(C) Immunohistochemical analysis of CD1a and CD207
expression in the epidermis of a skin equivalent at day 8.
Primary antibodies were detected using secondary horse
anti-mouse biotin-conjugated antibodies, followed by
incubation with streptavidin-HRP. Staining was visualized
by AEC. Scale bars: 50 μm. (D) Statistical analyses of
CD1a+ and CD207+ cell frequency in the indicated
populations; data are from six independent experiments.
RESEARCH ARTICLE
CD207– precursors integrate in artificial skin
To directly test whether CD14+CD36+ epidermal leukocytes give
rise to LCs, we used a human three-dimensional full-thickness skin
model (Mildner et al., 2010) and studied the integration of distinct
precursor cells in the newly forming epidermis. To date, only a few
groups have been able to achieve integration of LC precursors or
LCs into skin equivalents (Bechetoille et al., 2007; Hubert et al.,
2005; Laubach et al., 2011; Ouwehand et al., 2011; Régnier et al.,
1997; Schaerli et al., 2005). To our knowledge, we are the first to
have succeeded in comparatively assessing the differentiation
capacity of distinct progenitor subsets using artificial skin. Our
finding that cord blood-derived precursor cells but not CD14+CD16–
monocytes from peripheral blood were able to mature into
CD1a+CD207+ LCs might indicate that CD36+ or CD14+ leukocytes
in first trimester human epidermis are not monocytes but
phenotypically similar precursors or progenitors (Ziegler-Heitbrock
et al., 2010). Of note, although various functional differences
between foetal or neonatal and adult monocytes have been described
(Adkins et al., 2004; Fadel and Sarzotti, 2000; Levy, 2005), it is still
unknown whether monocytes possess a higher differentiation
capacity during the first trimester than in adult life. Surprisingly,
CD1a+ precursors, which are generally regarded to be dendritic
cells, do not possess a higher potential than CD1a–CD14–
haematopoietic precursors. It is conceivable that – as has been
postulated by various in vitro studies – CD14+ cord-blood derived
precursors represent an intermediate stage between CD34+
haematopoietic stem cells and CD1a+ precursors (Caux et al., 1992;
Jaksits et al., 1999). Of note, CD36 is found to a varying degree on
all these cord blood-derived subsets as well as on CD14+ monocytes.
Therefore, in contrast to CD14, CD36 cannot be used to further
define the phenotype of potential LC precursors.
Similarities between LC development in mice and humans
The development of LCs in mice and humans differs in various
aspects, exemplified by the postnatal acquisition of MHC class II
and subsequently langerin in mice (Chorro et al., 2009; Elbe et al.,
1989; Romani et al., 1986; Tripp et al., 2004). In humans, by
contrast, 54.2% of epidermal leukocytes express HLA-DR by 11
weeks EGA. This percentage increases in utero to 88.0% after 12
weeks EGA. Although the chronological developmental steps of LC
precursors in mice and humans differ (Chorro and Geissmann, 2010;
Elbe-Bürger and Schuster, 2010; Ginhoux and Merad, 2010), recent
experiments reveal amazing similarities. After injection of wild-type
bone marrow into IdD2–/– mice entirely lacking LCs, an LC network
was generated over a period of 3 weeks (Romani et al., 2012b; Seré
et al., 2012). Intriguingly, CD45+ and less numerous HLA-DR+ LC
precursors were present 1 week after bone marrow transplantation,
whereas prominent CD11c and CD207 expression was not seen until
2-3 weeks. Thus, the sequential acquisition of surface markers not
only resembled the murine but also human ontogeny and
corroborates the notion that CD45+ precursors colonized the
epidermis, where they sequentially acquire their phenotype. Given
that: (1) T cells are not present in prenatal skin before 18 weeks
EGA (Di Nuzzo et al., 2009; Schuster et al., 2012); (2) no other
immune cells exist with a tropism for the epidermis; and (3)
apoptosis is not detectable in first trimester epidermal leukocytes,
we assume that CD45+CD36–HLA-DR– cells in embryonic skin are
LC precursors. CD45+HLA-DR– cells were also identified in the
epidermis of healthy adult skin, but these cells most probably are
lymphocytes as previously reported (Foster and Elbe, 1997).
We identified phenotypically heterogeneous LC precursors in
human embryonic epidermis, whereas leukocytes resembling
phenotypically adult LC can only be found by the end of the second
trimester. Together with our finding that defined cord blood-derived
subsets, phenotypically similar to those described in embryonic
epidermis, can give rise to LCs in a human three-dimensional fullthickness skin model, our data are an important step in our
understanding of LC biology in human skin.
MATERIALS AND METHODS
Skin samples
After legal termination of pregnancy, 42 specimens of human embryonic (914 weeks EGA, first trimester) and foetal (18-24 weeks EGA) trunk skin
were studied. The age was estimated by crown-rump length and maternal
history. Healthy adult skin was collected after plastic surgery. The study was
approved by the local ethics committee of the Medical University of Vienna
and conducted in accordance with the declaration of Helsinki Principles.
Parents or participants gave their written informed consent.
Preparation of skin cell suspensions
Prenatal and adult skin was incubated on 1.2 U/ml Dispase II (Roche
Diagnostics, Indianapolis, IN) in PBS overnight at 4°C. Because it was
impossible to efficiently separate dermis and epidermis in embryonic skin,
unseparated skin, regardless of age, was vigorously agitated in a shaking
bath in 0.53 U/ml Liberase 3 (Roche Diagnostics) in PBS at 37°C for 60-90
minutes. The resulting single-cell suspensions were washed with RPMI
1640 medium (Life Technologies) supplemented with 10% heat-inactivated
FCS (PromoCell, Heidelberg, Germany), 25 mM HEPES, 10 μg/ml
gentamicin, 2 mM L-glutamine, 0.1 mM non-essential amino acids, 1 mM
sodium pyruvate, 50 μM 2-mercaptoethanol and 0.002% antibioticantimycotic solution (all Invitrogen) and analysed by flow cytometry.
Generation of cord blood-derived CD34+ haematopoietic
precursors and isolation of CD14+ blood monocytes
Samples of cord blood from healthy human donors were collected from fullterm deliveries. The collection was approved by the local ethics committee
and conducted in accordance with the declaration of Helsinki Principles.
Mononuclear cells were isolated as described and stored in liquid nitrogen
(Mairhofer et al., 2013). After thawing, CD34+ haematopoietic precursors
were separated from cord blood mononuclear cells by positive
immunoselection (CD34+ MicroBead Kit; Miltenyi Biotec GmbH, Bergisch
Gladbach, Germany) and the number of isolated, viable CD34+ cells was
determined using a modified ISHAGE gating strategy (Mairhofer et al.,
2013). Dead cells were excluded with 7-AAD (BD Biosciences). The
staining protocol was calibrated with the BD Stem Cell Control Kit (BD
Biosciences). Isolated CD34+ haematopoietic precursors were cultured for
3 days under expansion conditions before being induced to differentiate into
CD14+ and CD1a+ cells (Caux et al., 1996; Jaksits et al., 1999; Strobl et al.,
1996). Briefly, CD34+ haematopoietic precursors were expanded using XVivo 15 (Lonza, Basel, Switzerland) containing penicillin (100 U/l), and
streptomycin (100 μg/ml) and supplemented with thrombopoetin (50 ng/ml),
SCF (50 ng/ml) and Flt3L (50 ng/ml; all Peprotech, Rocky Hill, NJ) for 3
days. Subsequently, cells were differentiated using the above mentioned
medium supplemented with heat-inactivated 10% FCS (PAA, NJ), GM-CSF
813
Development
speculate that the heterogeneity of human epidermal leukocytes
might also reflect different precursors. To date, this concept has not
been validated in humans, first and foremost because the immune
cells of the human yolk sac remain poorly characterized (Janossy et
al., 1986). However, given that the haematopoiesis in these organs
in the human embryo and foetus is overlapping (Péault and Tavian,
2003), a dissection of the precise origin without concomitantly
looking at the haematopoietic sites may be futile. In addition, further
analysis is hindered because it is not currently possible to analyse
embryonic skin before 9 weeks EGA, given the extremely low
numbers of epidermal leukocytes at this developmental time point,
which precludes a meaningful statistical analysis.
Development (2014) doi:10.1242/dev.102699
RESEARCH ARTICLE
Generation of organotypic skin cultures
In vitro organotypic skin cultures were generated as described previously
with minor modifications (Mildner et al., 2010). Briefly, a suspension of
collagen type I (Biochrom, Berlin, Germany) containing human primary
fibroblasts (1×105 cells/ml) was poured into cell-culture inserts (3 μm pore
size; BD Biosciences) and allowed to form a gel for 2 hours at 37°C. The
gels were then equilibrated with keratinocyte growth medium (KGM;
Lonza) for 2 hours. Subsequently, primary human neonatal keratinocytes
(1.5´106 cells; Lonza) together with FACS-sorted cord blood-derived
CD1a+CD14–, CD1a–CD14– or CD1a–CD14+ (8×104 cells) precursors or
CD14+CD16– blood monocytes (8×104 cells) were placed onto the collagen
matrix. After overnight incubation the medium was removed from both the
inserts and external wells, and placed in the external wells with 10 ml
serum-free keratinocyte defined medium (SKDM), consisting of KGM but
without bovine pituitary extract and supplemented with 1.3 mM calcium
chloride (Sigma, Vienna, Austria), 10 μg/ml transferrin (Sigma), 50 μg/ml
ascorbic acid (Sigma) and 0.1% BSA (Sigma). Medium was changed every
second day. After 8 days of co-culture, the newly formed epidermis
containing integrated LCs was mechanically peeled off, fixed in ice-cold
acetone for 10 minutes and subjected to immunohistochemical staining.
Cytokine determination in cell culture supernatants and lysates
of organotypic skin cultures
To assess the production and secretion of TGFβ1, GM-CSF and IL-34,
culture media from organotypic skin cultures devoid of haematopoietic cells
were collected at days 1, 3, 5 and 8 after a 24-hour incubation period in 1
ml SKDM. Supernatants were collected and cell lysates were prepared by
incubation of the epidermal sheets in a 0.1% NP40 lysis buffer for 30
minutes on ice. Cell culture supernatants and lysates of epidermal sheets
were stored at –80°C until use. Cytokine levels were determined by ELISA
(R&D Systems) according to the manufacturer’s instructions. Experiments
were performed in duplicate using cells from three different donors.
Flow cytometry
Single-cell suspensions were stained with the following monoclonal
antibodies: PE anti-CD1c (AD5-8E7, Miltenyi), PE anti-CD33 (WM53, BD
Biosciences), PE anti-CD34 (581, BD Biosciences), PE anti-CD36 (TR9,
Biolegend, San Diego, CA, USA), PE anti-CD45 (HI30, BD Biosciences),
PE anti CD117 (YB5.B8, BD Biosciences), PE anti-CD207 (DCGM4,
Beckman Coulter), PE and APC anti-HLA-DR (L243; BD Biosciences),
APC anti-CD14 (TuK4, Invitrogen), FITC anti-CD1a (HI149, BD
Biosciences) and PE/Cy7 anti-CD45 (J.33; Beckman Coulter). Appropriate
isotype controls were included. Dead cells were excluded with 7-AAD
(Calbiochem, Darmstadt, Germany). Four-colour flow cytometry analyses
were performed on an LSR-II (Becton Dickinson) and on a FACSAria
(Becton Dickinson). Data were analysed using FlowJo software (Tree Star,
Ashland, OR).
Immunofluorescence
Prenatal and adult skin specimens were embedded in Optimum Cutting
Tissue compound (Tissue-Tek, Sakura Finetek, Zoeterwoude, The
Netherlands), snap-frozen in liquid nitrogen, and stored at –80°C until
814
further processing. Six micrometer sections were cut, air dried, fixed in icecold acetone for 10 minutes and washed in PBS. Fixed sections were stained
with unconjugated primary antibodies [anti-CD1c (L161; Serotec,
Kidlington, UK), anti-CD14 (TuK4, Invitrogen), anti-HLA-DR (L243, BD
Biosciences)] overnight at 4°C and subsequently incubated for 2 hours at
room temperature with biotin-conjugated goat anti-mouse IgG (Vector
Laboratories). Biotinylated antibodies were detected with streptavidinconjugated NorthernLight 637 (R&D Systems). Subsequently, sections were
blocked with 10% mouse serum and 2% mouse IgG (BD Biosciences) for
1 hour at room temperature and stained overnight at 4°C with Alexa Fluor
555 anti-CD45 (MEM28, Exbio, Vestec, Czech Republic), FITC anti-CD34
(AC136, Miltenyi), FITC anti-CD36 (TR9, Immunotools, Friesoythe,
Germany) and Alexa Fluor 488 anti-laminin 5 (D4B5; Millipore) to
visualize the dermo-epidermal junction. Finally, the CD36 signal was
amplified using anti-fluorescein Alexa Fluor 488-labelled rabbit
immunoglobulin (Invitrogen). Slides were mounted using Vectashield
(Vector Laboratories) and images were recorded using a confocal laser
scanning microscope (LSM 410; Carl Zeiss) equipped with four lasers
emitting lights at 405, 488, 543 and 633 nm.
Immunohistochemistry
After fixation, the epidermis of skin equivalents was washed in PBS.
Epidermal sheets were then incubated with anti-CD1a (WM35, Peli Cluster,
Amsterdam, The Netherlands), anti-CD207 (DCGM4, Beckman Coulter) or
appropriate isotype antibodies overnight at 4°C, followed by blocking of
endogenous peroxidase activity by incubating the sheets for 10 minutes in
methanol containing 0.03% hydrogen peroxide. Subsequently, sections were
incubated for 2 hours at room temperature with biotin-conjugated goat antimouse IgG using the Elite mouse IgG Vectastain kit (Vector Laboratories).
Biotinylated antibodies were detected with HRP streptavidin and staining
was visualized with AEC (Dako, Glostrup, Denmark). Finally, sections were
mounted with Aquatex (Merck) and examined using an Eclipse 80
microscope (Nikon). Appropriate isotype controls (BD Biosciences) were
included. Staining for TGFβ1 (Santa Cruz Biotechnology) and rabbit control
serum (Dako) on cryostat section was performed as described previously
(Schuster et al., 2009).
Quantification of cells in skin sections and epidermal sheets
Immunoreactive cells were counted in multiple skin sections with a total
epidermal length of 4-27 cm or on epidermal sheets with a total surface area
of 2.2-25.7 mm2 by two independent investigators (×40 object lens; Nikon).
Statistics
Differences between groups were assessed with the Mann-Whitney U-test,
the Wilcoxon matched-pair signed rank test or Student’s t-test (GraphPad
Software). The reported P-values are from two-sided tests. A P-value
smaller than 5% was considered statistically significant.
Acknowledgements
We thank Dr Anton Stift (Department of Surgery, Medical University of Vienna,
Austria) for the provision of adult human skin samples, and Drs Herbert Strobl and
Muzlifah Haniffa for fruitful discussion.
Competing interests
The authors declare no competing financial interests.
Author contributions
C.S., M. Mildner, M. Mairhofer, W.B. and M.P. performed the experiments and
analysed the data. C.F. and W.E. provided study material. C.S., A.K., E.T., G.S.
and A.E.-B. conceived and designed the research. C.S. and A.E.-B. wrote the
manuscript.
Funding
This project was funded by an Austrian Science Fund (FWF) grant [P19474-B13 to
A.E.-B.]. Deposited in PMC for immediate release.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.102699/-/DC1
Development
(100 ng/ml), TNFα (2.5 ng/ml), SCF (20 ng/ml) and Flt3L (50 ng/ml; all
Peprotech) for 7 days. On day 10, cells were harvested, stained and FACSsorted according to the expression of CD14 and CD1a using a FACSAria
(Becton Dickinson). For the isolation of CD14+CD16– human peripheral
blood monocytes from healthy volunteers, PBMCs were prepared by density
gradient centrifugation using BD Vacutainer CPT tubes (BD Biosciences).
T cells, B cells, NK cells and haematopoietic precursors were labelled with
FITC-conjugated monoclonal antibodies against CD3 (UCHT1, An der
Grub, Kaumberg, Austria), CD15 (W6D3, BD Biosciences), CD19 (LT19,
Miltenyi), CD34 (AC136, Miltenyi) and CD56 (C218, Beckman Coulter)
and subsequently depleted using anti-mouse IgG1 magnetic beads
(Miltenyi). Resulting cells were then labelled with PE-Cy7-anti-CD16 (3G8,
Beckman Coulter, Fullerton, CA) and APC-CD14 (TuK4, Invitrogen) and
CD14+CD16– cells were sorted using a FACSAria.
Development (2014) doi:10.1242/dev.102699
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