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Research article
The Journal of Clinical Investigation http://www.jci.org Volume 117 Number 9 September 2007 2517
Normal human dermis contains distinct
populations of CD11c
+
BDCA-1
+
dendritic
cells and CD163
+
FXIIIA
+
macrophages
Lisa C. Zaba,
1
Judilyn Fuentes-Duculan,
1
Ralph M. Steinman,
2
James G. Krueger,
1
and Michelle A. Lowes
1
1
Laboratory for Investigative Dermatology and
2
Laboratory of Cellular Physiology and Immunology, The Rockefeller University, New York, New York, USA.
We used a panel of monoclonal antibodies to characterize DCs in the dermis of normal human skin. Staining
for the CD11c integrin, which is abundant on many kinds of DCs, revealed cells in the upper dermis. These
cells were positive for blood DC antigen–1 (BDCA-1; also known as CD1c), HLA-DR, and CD45, markers that
are also expressed by circulating myeloid DCs. A small subset of CD11c
+
dermal cells expressed DEC-205/
CD205 and DC-lysosomal–associated membrane glycoprotein/CD208 (DC-LAMP/CD208), suggesting some
differentiation or maturation. When BDCA-1
+
cells were selected from collagenase digests of normal dermis,
they proved to be strong stimulators for T cells in a mixed leukocyte reaction. A second major population of
cells located throughout the dermis was positive for factor XIIIA (FXIIIA), but lacked CD11c and BDCA-1.
They expressed the macrophage scavenger receptor CD163 and stained weakly for HLA-DR and CD45. Isolat-
ed CD163
+
dermal cells were inactive in stimulating T cell proliferation, but in biopsies of tattoos, these cells
were selectively laden with granular pigments. Plasmacytoid DCs were also present in the dermis, marked
by CD123 and BDCA-2. In summary, the normal dermis contains typical immunostimulatory myeloid DCs
identified by CD11c and BDCA-1, as well as an additional population of poorly stimulatory macrophages
marked by CD163 and FXIIIA.
Introduction
DCs represent a major resident leukocyte population in human
skin. Two main types of DCs are found in noninflamed skin: epi
-
dermal Langerhans cells (LCs) and dermal DCs (1, 2). LCs express
Langerin/CD207, an endocytic receptor that localizes to and forms
Birbeck granules, as well as the CD1a class I–like molecule that
presents glycolipids (3). Dermal DCs have long been defined on the
basis of expression of a clotting factor, the transglutaminase fac
-
tor XIIIA (FXIIIA) (4). Studies that define dermal DCs as FXIIIA
+
have often relied on fluorescence-activated cell sorting (FACS)
analysis and functional studies using bulk tissue or enzymatically
manipulated émigrés that “crawl out” of the dermis over a variable
incubation period, which may increase FXIIIA expression (5, 6).
Currently, so-called myeloid or conventional DCs in many tis
-
sues are often identified on the basis of high expression of HLA-DR
antigen-presenting molecules and the CD11c integrin. DCs are
negative for Lin, a cocktail of antibodies to other cell lineages,
including T cells (CD3), B cells (CD19 and CD20), monocytes
(CD14), and granulocytes and NK cells (CD16 and CD56) (7, 8).
Circulating myeloid DCs can be further classified as 3 mutually
exclusive subsets — in order of immunostimulatory capacity, blood
DC antigen–1–positive (BDCA-1
+
), CD16
+
, and BDCA-3
+
(9) — but
these markers have seen very little use in studies of the skin.
Because a large number of prevalent dermatologic conditions
from atopic dermatitis to psoriasis are characterized by extensive
dermal T cell infiltration, and as DCs are pivotal antigen-pre
-
senting cells for T cells, it is important to pursue these distinct
phenotypic definitions of DCs in normal skin and peripheral
blood. Here we report that CD11c and FXIIIA marked mutually
exclusive populations, the former coexpressing BDCA-1 (also
known as CD1c; ref. 10), and the latter uniformly positively
for CD163, a scavenger receptor for hemoglobin/haptoglobin
complexes. CD11c
+
cells were more typical of DCs, with higher
HLA-DR expression and T cell–stimulating activity. Surpris
-
ingly, FXIIIA
+
cells behaved more like macrophages since they
were weak initiators of T cell responses in the mixed leukocyte
reaction (MLR) and had numerous phagocytosed pigment–con
-
taining vacuoles in a tattoo.
Results
FXIIIA
+
and CD11c
+
cells are discrete dermal populations. Immuno-
histochemistry of normal human dermis showed distinct
staining patterns for FXIIIA
+
and CD11c
+
cells (Figure 1A).
The larger FXIIIA
+
cells were located throughout the dermis.
In contrast, CD11c
+
myeloid cells were located in the papillary
and upper reticular dermis. We counted the absolute number
of FXIIIA
+
and CD11c
+
cells using matched tissue sections
from 15 normal volunteers (Figure 1B). In the epidermis there
were no FXIIIA
+
cells and a mean of 4 CD11c
+
cells per mm
(P = 0.04). In the dermis, there was a mean of 83 FXIIIA
+
cells
compared with 61 CD11c
+
cells per mm (P = 0.19). Double-
labeled immunofluorescence using FXIIIA and CD11c con
-
firmed that these 2 antigens were not expressed on the same
cell (Figure 1C). A polyclonal FXIIIA antibody has typically
been used for staining dermal DCs in the past. We used FXIIIA
affinity-purified sheep antibody for all of our double-label
immunofluorescence studies, as we found it to yield the best
Nonstandard abbreviations used: BDCA, blood DC antigen; DC-LAMP, DC-lyso-
somal–associated membrane glycoprotein; DC-SIGN, DC-specific ICAM-3–grabbing
nonintegrin; FACS, fluorescence-activated cell sorting; FXIIIA, factor XIIIA; LC,
Langerhans cell; MFI, median fluorescence intensity; MLR, mixed leukocyte reaction;
MMR, macrophage mannose receptor; PDC, plasmacytoid DC.
Conflict of interest: The authors have declared that no conflict of interest exists.
Citation for this article:
J. Clin. Invest. 117:2517–2525 (2007). doi:10.1172/JCI32282.
Related Commentary, page 2382
research article
2518 The Journal of Clinical Investigation http://www.jci.org Volume 117 Number 9 September 2007
staining. There was almost complete coexpression of the sheep
FXIIIA antibody and a commonly used mouse monoclonal
(clone AC-1A1), while other mouse monoclonal antibodies to
FXIIIA produced weaker and less consistent staining (data not
shown). These data indicate that 2 commonly used markers for
dermal DCs are actually expressed by comparably abundant but
different populations.
FXIIIA
+
and CD11c
+
populations are not LCs or plasmacytoid DCs.
To further characterize these 2 dermal cell populations, we first
assessed expression of markers used to identify LCs and/or plas
-
macytoid DCs (PDCs), each applied in combination with antibod
-
ies to either FXIIIA or CD11c. Neither FXIIIA nor CD11c colocal
-
ized with the LC markers CD1a (Figure 2, A and B) and Langerin
(Supplemental Figure 1, A and B; supplemental material available
online with this article; doi:10.1172/JCI32282DS1), nor the PDC
markers CD123 (Figure 2, C and D) and BDCA-2 (Supplemental
Figure 1, C and D). Therefore, FXIIIA
+
and CD11c
+
cells are dis-
tinct from additional LC and PDC populations in normal skin.
Table 1 summarizes the expression of various leukocyte markers
on the FXIIIA
+
and CD11c
+
cells.
CD11c
+
cells coexpress the blood DC marker BDCA-1, and a small
fraction also express DC-lysosomal–associated membrane glycoprotein/
CD208 and DEC-205/CD205. BDCA-1 also marks a population
of CD11c
+
myeloid DCs in blood, and we noted coexpression of
these 2 markers in the dermis as well (Figure 3, A and B). Two
other markers that are expressed by tissue DCs are lysosomal
marker DC-lysosomal–associated membrane glycoprotein/
CD208 (DC-LAMP/CD208) and endocytic receptor DEC-205/
CD205. A small fraction of the CD11c
+
cells expressed these 2
markers (Figure 3, C–F). The fraction of CD11c
+
DC-LAMP
+
cells
was qualitatively smaller than the fraction of CD11c
+
DEC-205
+
cells. DC-LAMP/CD208 is expressed during DC maturation,
and we noted that cells positive for this marker were often in
dermal aggregates with other CD11c
+
DC-LAMP
–
cells. These
results indicate that CD11c
+
cells in the dermis share features
with the myeloid DCs in blood, but some are in a more mature
state of differentiation (11).
FXIIIA
+
cells express the macrophage marker and scavenger receptor
CD163. CD163, a hemoglobin/haptoglobin complex scavenger
receptor, identifies tissue resident macrophages, and it was
the only marker we studied that was uniformly coexpressed by
FXIIIA
+
cells and not by CD11c
+
cells (Figure 4, A and B). Sev-
eral other markers were expressed on a fraction of FXIIIA
+
and
CD11c
+
cells. These included the uptake receptors macrophage
mannose receptor/CD206 (MMR/CD206; Figure 4, C and D),
DC-specific ICAM-3–grabbing nonintegrin/CD209 (DC-SIGN/
CD209; Figure 4, E and F), CD45, and HLA-DR (Figure 5). In nor
-
mal skin, both MMR/CD206 and DC-SIGN/CD209 were more
abundant on macrophages (FXIIIA
+
cells) than DCs (CD11c
+
cells), which is consistent with recent studies in inflamed skin
and lymph nodes (11, 12). Reciprocally, CD45 and HLA-DR
were more abundant on CD11c
+
than on FXIIIA
+
cells. All these
observations are consistent with the interpretation that CD11c
+
cells in the dermis are part of the DC pathway of differentiation,
while FXIIIA
+
cells are more macrophage-like.
Other markers of potential interest. Other markers used for char-
acterization of FXIIIA
+
and CD11c
+
cells were CD14 (identifies
monocytes); CD68, CD11b, and RFD7 (commonly used macro
-
phage markers); and CD63 (present on macrophages and DCs).
These markers were not helpful in discriminating these 2 popula
-
tions in normal skin (Supplemental Figure 2). CD14, a compo
-
nent of the LPS receptor on monocytes (13), was present on a few
FXIIIA
+
cells and CD11c
+
cells. We have previously shown that
CD68 identifies a subset of CD11c
+
cells that produce IL-20 in
psoriasis (14), so this is not an exclusive macrophage marker. In
keeping with this, CD68 also stained both CD11c
+
and FXIIIA
+
Figure 1
FXIIIA
+
and CD11c
+
cells are unique dermal populations. (A) Immunohistochemistry on normal human skin using FXIIIA (left panel) and CD11c
(right panel) antibodies (n = 15). FXIIIA
+
cells were spread throughout the dermis, while CD11c
+
cells were mainly localized to the superficial
dermis. (B) There were similar numbers of CD11c
+
and FXIIIA
+
cells per mm in normal dermis. Error bars indicate SEM. (C) FXIIIA and CD11c
identified 2 discrete populations. White lines denote dermo-epidermal junction. Scale bars: 100 μm.
research article
The Journal of Clinical Investigation http://www.jci.org Volume 117 Number 9 September 2007 2519
cells. CD11b and RFD7, considered to be common macrophage
markers (13, 15), were present on both CD11c
+
and FXIIIA
+
cells.
CD63, an MHC class II internalization antigen, marked occasional
CD11c
+
and FXIIIA
+
cells in normal skin.
Isolated CD11c
+
dermal cells contain nonoverlapping major and
minor populations of BDCA-1
+
and BDCA-3
+
cells. To confirm our in
situ data and assess whether the 3 myeloid DC subsets found
in blood were also found in tissue, we performed 6-color FACS
on single-cell suspensions of normal human skin compared with
peripheral blood, using a custom-made group of monoclonal
antibodies to lineage (Lin) that did not contain monoclonal anti
-
bodies to CD14 or CD16. Peripheral blood FACS dot plots are
shown in Figure 6, A and B, and the parallel analysis from dermal
cells is shown in Figure 6, C and D. There were 3 discrete Lin
–
CD11c
+
HLA-DR
+
DC populations in peripheral blood, BDCA-1
+
,
CD16
hi
, and BDCA-3
+
(Figure 6A), but only 2 clear populations in
skin, BDCA-1
+
CD16
lo
and BDCA-3
+
CD16
lo
(Figure 6C). In blood,
2 discrete CD11c
+
DC populations, BDCA-1
+
and BDCA-3
+
,
were both HLA-DR
+
CD14
–
(Figure 6B). In contrast, BDCA-1
+
cells from skin increased HLA-DR expression, and BDCA-3
+
cells
acquired low-level CD14 expression (Figure 6D). We also con
-
firmed that BDCA-1 and BDCA-3 identified discrete populations
in normal skin in situ (data not shown).
BDCA-1 and CD163 are superior markers to CD11c and FXIIIA,
respectively, for FACS of isolated dermal cells. To begin to test the
functional properties of cell populations isolated from dermis,
we required markers that would be optimal for FACS. While
culture conditions may alter cellular surface phenotype, it was
important to compare DC populations using the new BDCA-1
and CD163 markers with previous studies on dermal DCs. It
was necessary to balance obtaining sufficient cells to study
with as little manipulation as possible. An overnight culture in
dispase/collagenase media (16) yielded insufficient cells to per
-
form our MLR experiments. Enzymic alteration of surface epi
-
topes is also possible, although experiments on PBMCs did not
show loss of FACS staining after culture in dispase collagenase
Figure 2
FXIIIA
+
and CD11c
+
populations are not LCs or plasmacytoid
DCs. Neither FXIIIA nor CD11c showed coexpression with
Langerhans antigen CD1a (A and B) or plasmacytoid antigen
CD123 (C and D). Scale bar: 100 mm.
Table 1
Characterization of CD11c
+
and FXIIIA
+
cells in normal human
dermis
Antigen Main cell location FXIIIA CD11c
CD1a LCs, IDECs – –
Langerin/CD207 LCs – –
CD123 PDCs – –
BDCA-2 PDCs – –
BDCA-1 Myeloid DCs – +++
DC-LAMP/CD208 Mature DCs – +
DEC-205/CD205 Mature DCs – +
CD163 Macrophages +++ –
MMR/CD206 Macrophages, DCs +++ ++
DC-SIGN/CD209 Macrophages, immature DCs +++ ++
CD45 Bone marrow–derived cells + +++
HLA-DR Antigen-presenting cells + ++
CD14 Monocyte-derived cells + +
CD68 Macrophages, DCs + +
CD11b Myeloid cells + ++
RFD7 Macrophages, DCs ++ +
CD63 Macrophages, DCs + +
Qualitative coexpression of FXIIIA and CD11c with additional antibody
is denoted as follows: –, no coexpression; +, some cells show coexpres-
sion; ++, a moderate number of cells show coexpression; +++, most
cells show coexpression. IDEC, inflammatory dendritic epidermal cell.
research article
2520 The Journal of Clinical Investigation http://www.jci.org Volume 117 Number 9 September 2007
mix (data not shown). This method was modified to allow 1–2
days of culture so any lost surface markers could be reexpressed
and to allow DCs and macrophages to move out of the scored
upper and lower dermal surfaces. Comparison of BDCA-1 ver
-
sus CD163 on the first day of culture (after overnight culture
in dispase/collagenase) and after 24 and 48 hours of culture
showed a similar clear distinction of the 2 populations (data
not shown). Culture of bulk dermal cells from normal skin
using this method with and without DC maturation cytokines
(IL-1β, IL-6, TNF, and PGE
2
) showed that there was surpris-
ingly little maturation of myeloid DCs (BDCA-1
+
) as measured
by HLA-DR and CD83 (data not shown).
We found that BDCA-1, which colabeled more than 90% of
CD11c
+
cells by in situ immunofluorescence (Figure 3B), had a
much higher median fluorescence intensity (MFI) than did
CD11c (data not shown). Likewise, CD163, which showed
100% coexpression with FXIIIA in situ (Figure 4A), proved to
be a much better FACS marker. FXIIIA antibodies bound non
-
specifically to dermal cells in suspension, perhaps as a result
of adherence of platelet fragments rich in FXIIIA, which is
upregulated during cell culture (5, 6).
Double-label immunofluorescence of BDCA-1 and CD163
confirmed that they were distinct populations in situ
(Figure 7A), which supports their use as alternative markers
for CD11c and FXIIIA, respectively, in FACS studies. Bulk
dermal single-cell suspensions showed distinct BDCA-1
+
and
CD163
hi
populations by FACS (Figure 7B). BDCA-1
+
gated
cells had a higher MFI for the CD11c than the CD163
hi
popu-
lation (Figure 7C), most likely because of increased sensitiv
-
ity of FACS compared with immunohistochemistry. CD163
hi
cells had a higher MFI for FXIIIA than did BDCA-1
+
cells
(Figure 7C). BDCA-1
+
gated cells also had a higher MFI for
HLA-DR and CD45 than did CD163
hi
gated cells (Figure 7C).
A subset of BDCA-1
+
cells was positive for DC maturation
markers CD86, CD83, and DC-LAMP/CD208 (Figure 7D).
Because B cells may also express BDCA-1, we confirmed that
none of the BDCA-1
+
cells were CD19
+
by FACS (data not
shown). Thus the dermal cells have a pattern of expression
similar to in situ characterization.
BDCA-1
+
cells are a major immunostimulatory population from
normal human skin. To test for the immunostimulatory prop-
erties of dermal leukocytes, we focused on FACS-sorted popu
-
lations of BDCA-1
+
and CD163
hi
cells released from the der-
mis with collagenase. Data are summarized in Supplemental
Table 3, and Figure 8 shows representative FACS plots of
the MLR. The sorted cells were 99% pure compared with iso
-
type (Figure 8A). In the MLR induced by in vitro monocyte-
derived mature DCs, 63% of the surviving T cells had under
-
gone extensive proliferation at a stimulator/responder ratio
of 1:100 on day 8 after sorting (Figure 8B). In parallel cultures
stimulated by BDCA-1
+
cells, 9.1% of the T cells had prolifer-
ated (1:10 ratio), compared with 2.1% of CD163
+
cells and
1.0% background T cell proliferation (Figure 8C). When these
sorted BDCA-1
+
and CD163
+
populations were cultured for 2
days in a DC-maturing cytokine cocktail before setting up the
MLR, the immunostimulatory capability of BDCA-1
+
cells was
increased to 25.2% (1:100 ratio), but the capacity of CD163
+
cells was unchanged (2.2%; 1:250 ratio, low because of low
cell survival during the culture period; Figure 8D). BDCA-1
+
sorted cells were cultured for 2 days without cytokines, and
the supernatant from these cells after culture also increased T
cell proliferation (data not shown).
CD163
+
cells phagocytose large particles in a tattoo and have the struc-
tural features of macrophages. Ultrathin sections of a green dye tat-
too were cut from tattoo-bearing normal skin. These sections
confirmed that the dye was intracytoplasmic and located mostly
in cells clustered around blood vessels (Figure 9A). Electron
microscopy of the tattoo revealed membrane-bound (Figure 9B,
blue arrow) tattoo dye particles (red arrow) and microvillous pro
-
jections (green arrow), confirming the identity of these cells as
macrophages. Immunohistochemistry using BDCA-1 (Figure 9C)
and CD163 (Figure 9D) showed that dye-laden cells were CD163
+
and not BDCA-1
+
, confirming that typical macrophages rather
than DCs had ingested the pigment.
Figure 3
CD11c
+
cells are defined by BDCA-1, DC-LAMP/CD208, and DEC-205/
CD205. FXIIIA did not overlap with BDCA-1 (A), DC-LAMP/CD208 (C),
or DEC-205/CD205 (E). Most CD11c
+
cells coexpressed BDCA-1 (B).
Small subsets of CD11c
+
cells coexpressed DC-LAMP/CD208 (D) and
DEC-205/CD205 (F). Scale bar: 100 μm.
research article
The Journal of Clinical Investigation http://www.jci.org Volume 117 Number 9 September 2007 2521
Discussion
DCs are important sentinels of the cutaneous immune system,
performing central roles in both the innate and the acquired
immune systems. Previous characterization of DC subsets in
human dermis has been influenced by results with a rabbit
polyclonal antibody to FXIIIA, which identifies dermal cells with
a dendritic morphology (4, 17–20). Here we have characterized
populations of cells within the normal dermis and found that,
surprisingly, cells expressing the CD11c integrin and the BDCA-1
antigen-presenting molecule represent a distinct population func
-
tionally differentiated along the DC pathway, whereas FXIIIA
+
cells are differentiated along the macrophage pathway. Whereas
mouse skin contains approximately 1 CD11c
+
DC for every 5 mac-
rophages (21), the concentration of CD11c
+
DCs in human skin is
much higher, closer to a 1:1 ratio.
CD11c colocalized with several well-recognized DC markers:
BDCA-1, DC-LAMP/CD208, and DEC-205/CD205. BDCA-1,
also known as CD1c (10), is an invariant MHC class I–like
antigen receptor molecule that recognizes lipids in mycobac
-
terial cell walls. BDCA-1 is found on immature and mature
DCs, and also on a subset of B cells. BDCA-1, in the absence of
the B cell markers CD19 and CD20, would therefore seem to
be a valuable marker to compare dermal DCs in both normal
skin (22) and inflammatory skin diseases (23). DC-LAMP/
CD208 is a lysosomal protein that specifically marks mature
DCs (24). DEC-205/CD205 is a surface receptor that partici
-
pates in DC antigen endocytosis; its expression increases dur
-
ing maturation, and it has been previously demonstrated in
normal human skin (25). The low frequency of CD11c
+
DCs
expressing these 2 antigens observed in normal skin is con
-
sistent with their expected immature DC status. CD11c
+
cells
also stained brightly with HLA-DR and CD45, confirming
their antigen-presenting potential and bone marrow origin,
respectively. The phenotype of BDCA-1
+
cells from skin indi-
cates that they are all CD45
hi
HLA-DR
hi
and that a subset of
these cells is CD86
+
and CD83
+
.
Our studies show that FXIIIA identifies a population of
tissue-resident macrophages in normal skin. The only anti
-
body that overlapped completely with FXIIIA in situ was the
scavenger receptor CD163, which is selectively expressed on
monocytes and macrophages (reviewed in ref. 26). The best-
characterized function of CD163 is to bind hemoglobin/
haptoglobin complexes, which may be important in homeo
-
stasis. There was low-level CD45 and HLA-DR expression,
consistent with other tissue macrophages, and limited anti
-
gen-presenting capacity (27). MMR/CD206 and DC-SIGN/
CD209 are C-type lectin receptors that are found on both
macrophages and DCs (11, 28), so it was not surprising that
there was expression of both lectin receptors on both FXIIIA
+
and CD11c
+
cells in normal human skin.
To evaluate the function of these 2 dominant dermal popu
-
lations, we used 2 approaches. By comparing BDCA-1 and
CD163 cells selected from collagenase digests of normal skin
as inducers of an allogeneic MLR, we found that BDCA-1
+
cells were the main immunostimulatory population. These
BDCA-1
+
cells induced increased T cell proliferation when
cultured before setting up the MLR, but were not as immuno
-
stimulatory as in vitro–matured DCs, suggesting that there
were few mature DCs in normal skin compared with skin
under inflammatory conditions such as psoriasis. This result
reflects studies by Nestle et al. showing that bulk tissue émigrés
from normal skin are not as stimulatory as those from patients
with psoriasis (20). In comparison, CD163
hi
cells were not immu-
nostimulatory in an allo-MLR, nor were they induced to be stimu
-
latory, although they may possess some antigen-presenting capac
-
ity with upregulation of MHC class II molecules (29).
Skin tattoos provided a second functional study: the phagocytic
activity of tissue macrophages. Pigment granules were found in
lysosomal-like cellular structures, and cells containing pigment
stained uniformly with CD163. The fate of tattoo pigment inject
-
ed into dermal tissues has been studied in the past, and fibroblasts
were considered to be the primary long-term reservoir of the pig
-
ment granules, with pigment in occasional macrophages (30, 31).
However, our data suggest that macrophages are indeed a signif
-
icant store of the dermal pigment. The cells with pigment were
Figure 4
The macrophage marker CD163 defines FXIIIA
+
cells. FXIIIA
+
cells
expressed macrophage marker CD163 (A), and a subset overlapped with
MMR/CD206 (C) and DC-SIGN/CD209 (E). CD11c did not overlap with
CD163 (B), but a subset overlapped with MMR/CD206 (D) and DC-SIGN/
CD209 (F). Scale bar: 100 μm.
research article
2522 The Journal of Clinical Investigation http://www.jci.org Volume 117 Number 9 September 2007
CD163
+
and BDCA-1
–
, were round, had numerous microvillous
projections, and the pigment was contained within membrane-
bound structures (lysosomes). These characteristics are consistent
with macrophages (31, 32).
In summary, we have identified CD11c
+
cells as immature myeloid
DCs in normal human skin and FXIIIA
+
cells as tissue-resident mac-
rophages, not DCs, as they were previously classified. In future stud
-
ies of cutaneous DCs, we recommend considering the use of
CD11c/BDCA-1 and CD163 as alternative markers to identify
dermal DCs and macrophages, respectively, because they are
more specific and more useful in flow cytometry applications.
Methods
Skin samples. Skin punch biopsies (6 mm diameter) were obtained
from 15 normal volunteers under a protocol approved by the Rock-
efeller University Institutional Review Board. Informed consent
was obtained. Biopsies were frozen in OTC (Sakura) and stored at
–80°C for immunohistochemistry and immunofluorescence. Nor-
mal samples from abdominoplasty were processed within 4 hours
after surgery. These abdominoplasty samples were also the source
of tattoo material (n = 2).
Immunohistochemistry. Normal skin sections (n = 15) were stained
with purified mouse anti-human CD11c (diluted 1:100) and
sheep affinity-purified anti-human FXIIIA (diluted 1:100; Enzyme
Research Laboratories). Biotin-labeled horse anti-mouse and biotin-
labeled rabbit anti-sheep antibodies (Vector Laboratories) were used
to detect CD11c and FXIIIA antibodies, respectively. The staining
signal was amplified with avidin-biotin complex (Vector Labora-
tories) and developed using chromogen 3-amino-9-ethylcarbazole
(Sigma-Aldrich). The number of positive cells per mm was counted
manually using computer-assisted image analysis (NIH Image 6.1;
http://rsb.info.nih.gov/nih-image). For FXIIIA immunostaining,
several additional antibodies were screened, including AC-1A1 (Lab-
vision), E5014 (Spring Bioscience), and E980.1 (Biogeniex). We chose
the sheep affinity-purified antibody because it gave the most specific
cellular staining with the least background epidermal staining.
Antibodies. All antibodies used for immunofluorescence and FACS
of normal tissue are listed in Supplemental Tables 1 and 2.
Immunofluorescence. Normal skin sections (n = 6) were fixed with
acetone and blocked in 10% normal goat serum (Vector Laborato-
ries) for 30 minutes. Primary antibodies CD11c and FXIIIA were incu-
bated overnight at 4°C and amplified with the appropriate secondary
antibody: goat anti-mouse IgG1 conjugated with Alexa Fluor 488 or 568,
or donkey anti-sheep conjugated with Alexa Fluor 488 or 568, respective-
ly. For colocalization with CD11c or FXIIIA, sections were then costained
overnight with a second antibody, as listed in Supplemental Table 1, and
amplified with the appropriate goat-anti mouse secondary antibody
Figure 5
HLA-DR and CD45 mark both CD11c
+
and FXIIIA
+
cells. FXIIIA
+
cells had a
lower expression level of CD45 (A) than did CD11c
+
cells (B). HLA-DR was
also expressed to a lower extent on FXIIIA
+
cells (C) than on CD11c
+
cells
(D). Scale bar: 100 μm.
Figure 6
Cutaneous DCs compared with blood
DCs. (A) There were 3 nonoverlapping
DC populations in peripheral blood,
gating on Lin
–
CD11c
+
HLA-DR
+
cells:
BDCA-1
+
, CD16
hi
, and BDCA-3
+
. (B)
Blood BDCA-1
+
(left) and BDCA-3
+
(right) cells were both HLA-DR
+
CD14
–
.
(C) In dermal single-cell suspensions,
BDCA-1
+
cells acquired CD16. (D)
BDCA-1
+
(left) dermal cells increased
HLA-DR expression, and BDCA-3
+
(right) dermal cells acquired low-level
CD14 expression.
research article
The Journal of Clinical Investigation http://www.jci.org Volume 117 Number 9 September 2007 2523
(Supplemental Table 1). Images were acquired using appropriate filters
of a Zeiss Axioplan 2I microscope with Plan Apochromat 20 × 0.7 numer-
ical aperture lens and a Hagamatsu orca ER-cooled charge-coupled device
camera, controlled by METAVUE software (Universal Imaging). Dermal
collagen fibers gave green autofluorescence. Antibodies conjugated with
a fluorochrome often gave background epidermal fluorescence. Images
in each figure are presented as single-color stains of green and red to
make apparent the localization of 2 markers on similar or different cells.
Merged images are shown below the single-color stains. Cells that coex-
press the 2 markers in a similar location are often yellow in color.
Figure 7
BDCA-1 and CD163 are alternative markers for
CD11c and FXIIIA, respectively. (A) BDCA-1 and
CD163 identified discrete populations of dermal
cells. (B) BDCA-1
+
cells (red circle) and CD163
+
cells
(blue circle) were also discrete populations in dermal
single-cell suspensions. (C) FACS histograms gated
on BDCA-1
+
cells (red line), CD163
+
cells (blue line),
or isotype (green line). BDCA-1
+
cells were CD11c
hi
,
FXIIIA
lo
, HLA-DR
hi
, and CD45
hi
. CD163
+
cells were
CD11c
mid
, FXIIIA
hi
, HLA-DR
mid
, and CD45
lo
. (D) A
subset of BDCA-1
+
cells was CD86
hi
, CD83
+
, and
DC-LAMP
hi
. Representative graphs from 3 experi-
ments. Scale bar: 100 μm.
Figure 8
BDCA-1
+
cells are more immunostimulatory. (A) Post-
sort dot plot of dermal cells from normal skin into BDCA-1
+
and CD163
+
populations (red, left and right panels,
respectively) compared with isotype (blue). (B) Posi-
tive control (monocyte-derived mature DC) for MLR on
day 8 after sorting at a 1:100 stimulator/responder ratio.
Gate contains CD3
+
proliferating T cells with left-shifted
CFSE. (C) Using BDCA-1
+
sorted cells as stimula-
tors (1:10 ratio), 9.1% of the T cells proliferated; using
CD163
+
cells (1:10 ratio), 2.1% of live T cells prolifer-
ated. Background proliferation of T cells alone without
stimulation was 1.0%. (D) After cells had been sorted
and cultured for 2 days with cytokines to induce matura-
tion, there was a marked increase in the T cell stimula-
tory capacity of BDCA-1
+
cells (25.2%, 1:100 ratio) ver-
sus CD163 (2.2%, 1:250 ratio). Representative graphs
from 3 experiments.
research article
2524 The Journal of Clinical Investigation http://www.jci.org Volume 117 Number 9 September 2007
Electron microscopy. Skin was fixed in 2.5% glutaraldehyde and processed
by routine transmission electron microscopy procedure. Semithin plastic
sections were stained with toluidine blue for light microscopic evaluation.
Ultrathin (65-nm) sections were cut with a diamond knife, collected on
copper grids, and stained with both uranyl acetate and lead citrate before
viewing in a Tecnaispirit electron microscope (FEI Company) equipped
with an Ultrascan 895 charge-coupled device camera (Gatan).
Skin sample processing. Dermal single-cell suspensions from normal skin
were obtained using a modified collagenase/dispase method (16). Subcu-
taneous fat was excised, and remaining tissue was washed with PBS. The
dermal layer was heavily scored with a scalpel and incubated in 1 mg/ml
type 1 collagenase (Invitrogen), 1 mg/ml dispase (Invitrogen), and 1%
penicillin-streptomycin solution (Sigma-Aldrich) overnight at 37°C.
The epidermis was peeled off and discarded, and the dermis was trans-
ferred to fresh RPMI 1640 supplemented with 10% pooled human serum
(Mediatech Inc.), 0.1% gentamicin reagent solution (Invitrogen), and 1%
1 M HEPES buffer (Sigma-Aldrich). The dermis was incubated 24–48
hours at 37°C, and the supernatant was collected and filtered with 40-μm
cell strainers (BD Biosciences). Cells were then either used immediately
(for MLR; n = 3) or frozen in RPMI 1640 (Invitrogen) and 10% DMSO
(ATCC) for FACS (n = 3).
FACS. Cells were stained with the antibodies listed in Supplemental
Table 2. Briefly, cells were stained for 20 minutes at 4°C, washed with
FACSwash (PBS 0.1% sodium azide and 2% FBS), and resuspended in 1.3%
formaldehyde (Fisher Scientific) in FACSwash. To detect DC-LAMP, cells
were first stained for surface markers, then permeabilized (FacsPerm; BD)
before intracellular staining. Samples were acquired using FACSCanto or
LSR-II (both from BD Biosciences) and analyzed with FlowJo (Treestar).
Appropriate isotypes were used.
FACS and MLR. Dermal cells from single-cell suspensions of normal skin
were stained with BDCA-1, CD19, and CD163 antibody and sorted on
a FACSAria (BD Biosciences) using a low-pressure setting. Two popula-
tions were obtained: BDCA-1
+
CD19
–
and CD163
+
. A post-sort collection
was performed to confirm the purity of each stimulating population. For
some experiments, sorted cells were cultured for 2 days with and without
cytokines for maturing DCs ex-vivo (IL-1β, IL-6, TNF-α, and PGE
2
), then
washed and prepared for the MLR.
Responding T cells were obtained from a normal volunteer by density
centrifugation over Ficoll-Paque Plus (Amersham Biosciences), followed
by T cell purification using a T cell–negative selection kit (Dynal). T cells
were labeled with 10 μm CFSE and cocultured with either BDCA-1
+
or
CD163
+
sorted cells at ratios of 1:10, 1:50, and 1:100 (depending on cell
yield). T cells without a stimulator population were used as a negative
control, and monocyte-derived mature DCs were added to T cells as
a positive control. The process for making mature DCs was previously
described (33). T cell proliferation was analyzed on day 8 after sorting. The
cultures were harvested, stained with 250 ng/ml propidium iodide (PI) to
label dead cells, and CD3-APC (BD) for 15 minutes at room temperature.
PI-negative cells were gated and then plotted as CFSE versus CD3
+
cells,
where proliferating cells diluted their content of CFSE and moved to the
left of the nonproliferating cells. The CFSE-low cells were quantified as a
percentage of live cells in the culture (34).
Statistics. A 2-tailed paired Student’s t test was used to compare CD11c and
FXIIIA
+
cells in normal skin sections. Results are shown as mean ± SEM.
A P value less than 0.05 was considered significant.
Acknowledgments
This research was supported by NIH grants R01 AI-49572, AI-49832,
UL1 RR024143, and AI40045 (R.M. Steinman). M.A. Lowes is sup
-
ported by NIH grant 1 K23 AR052404-01A1, and L. Zaba is sup
-
ported by NIH MSTP grant GM07739. We thank plastic surgeons
A.N. LaBruna and D.M. Senderoff for their generous donation
of abdominoplasty surgical waste, H. Shio and A. Khatcherian
for their technical assistance on electron microscopy and
immunohistochemistry of the tattoo, respectively, and A. Piperno
for the kind gift of monoclonal antibody DEC-205/CD205. We
also appreciate the assistance and advice of the Flow Cytometry
Core Facility (S. Mazel) and Bio-imaging Resource Center (A.
North) at Rockefeller University. We thank Patricia Gilleaudea and
Mary Whalen-Sullivan for excellent care of our patients.
Received for publication March 7, 2007, and accepted in revised
form June 6, 2007.
Address correspondence to: Michelle A. Lowes, Laboratory for
Investigative Dermatology, The Rockefeller University, 1230 York
Avenue, New York, New York 10021, USA. Phone: (212) 327-7576;
Fax: (212) 327-8353; E-mail: lowesm@rockefeller.edu.
Figure 9
CD163
+
cells phagocytose large particles and have the structural fea-
tures of macrophages. (A) Tattoo skin section (0.5 μm) stained with
toludine blue. Cells containing green tattoo dye in their cytoplasm
(black arrow) surrounded a blood vessel. (B) Electron microscopy of
a tattoo showed that dye particles (red arrow) were membrane bound
(blue arrow) within the cytoplasm of a cell with multiple microvillus pro-
trusions (green arrow). (C and D) Cells containing green tattoo dye
particles stained for CD163 (D) but not BDCA-1 (C). Scale bar: 10 μm
(A, C, and D); 200 nm (B).
research article
The Journal of Clinical Investigation http://www.jci.org Volume 117 Number 9 September 2007 2525
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