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Prepublished online as a Blood First Edition Paper on May 8, 2003; DOI 10.1182/blood-2003-02-0449.
Blood, 1 September 2003, Vol. 102, No. 5, pp. 1654-1660 Hair follicles serve as local reservoirs of skin mast cell precursorsFrom the Departments of Dermatology and Ophthalmology, University of Texas Southwestern Medical Center, Dallas.
Several leukocyte populations normally reside in mouse skin, including Langerhans cells and   T cells in the epidermis and macrophage
and mast cells in the dermis. Interestingly, these skin resident leukocytes
are frequently identified within or around hair follicles (HFs), which are
known to contain stem cell populations that can generate the epidermal
architecture or give rise to the melanocyte lineage. Thus, we reasoned that
HFs might serve as a local reservoir of the resident leukocyte populations in
the skin. When vibrissal follicles of adult mice were cultured in the presence
of stem cell factor (SCF), interleukin 3 (IL-3), IL-7, granulocyte-macrophage
colony-stimulating factor, and Flt3 ligand,
CD45+/lineage–/c-kit+/Fc RI+
cells became detectable on the outgrowing fibroblasts in 10 days and expanded
progressively thereafter. These HF-derived leukocytes showed characteristic
features of connective tissue-type mast cells, including proliferative
responsiveness to SCF, metachromatic granules, mRNA expression for mast cell
proteases-1, -4, -5, and -6, and histamine release on ligation of surface IgE
or stimulation with substance P or compound 48/80. These results, together
with our findings that HFs contain c-kit+ cells and produce SCF
mRNA and protein, suggest that HFs provide a unique microenvironment for local
development of mast cells.
Two leukocyte populations reside in the epidermal compartment of normal skin in adult mice: Langerhans cells (LCs), which are a skin-specific member of the dendritic cell family of antigen-presenting cells,1 and dendritic epidermal T cells (DETCs), which are tissue resident
T cells.2 The dermal
compartment also contains several resident leukocyte populations, including
dermal dendritic cells, macrophages, and mast cells. Keratinocytes are the
major cellular component forming the interfollicular epidermis and the
intrafollicular epidermis known as the outer root sheath (ORS). Keratinocytes
have been reported to sustain the survival and promote the local growth of LCs
and DETCs by producing granulocyte-macrophage colony-stimulating factor
(GM-CSF) and interleukin-7 (IL-7),
respectively.3-5
Fibroblasts play similar roles by producing macrophage colony-stimulating
factor (M-CSF) and stem cell factor (SCF), both of which act as pleiotropic
growth factors in
hematopoiesis.6-9
Interestingly, LCs and DETCs are frequently identified in the ORS of hair
follicles
(HFs),2,10-13
and macrophages and mast cells often show perifollicular distributions in the
dermal extracellular
matrix.10,14,15
In this regard, HFs are now recognized as not only serving as an appendage
specialized for hair shaft production, but also as a "niche" for
tissue regeneration. The "bulge" region of the ORS in adult mice
contains multipotent stem cells that can generate the interfollicular
epidermis, HF structures, and sebaceous
glands.16,17
Stem cells that can give rise to the melanocyte lineage have been recently
identified in the bulge and sub-bulge ORS regions in adult
mice.18 Thus, we
reasoned that HFs might also serve as local reservoirs of precursors for one
or more of the skin resident leukocyte populations. To test this concept, we
isolated vibrissal (whisker) follicles from adult mice and cultured them in
the presence of SCF, IL-3, IL-7, GM-CSF, and Flt3 ligand (Flt3L), which are
known to promote the growth and differentiation of LCs, DETCs, macrophages,
and mast
cells.3,5,19-26
Here we report that relatively large numbers of
CD45+/lineage-negative
(Lin–)/c-kit+/Fc
Animals and HF cultures
C57BL/6 mice and the transgenic mice expressing the enhanced green
fluorescence protein (EGFP) under the control of the chicken Flow cytometric and immunohistochemical analyses
Cells were harvested from HF cultures by vigorous pipetting for phenotypic
analyses. Samples were stained with phycoerythrin (PE)–conjugated Lin
markers (anti-CD3, anti-CD4, anti-CD8, anti-CD11b, anti-CD11c, anti-IA/IE,
B220, Gr-1, and Ter-119 monoclonal antibodies [mAbs]), PE–anti-CD45 mAb,
allophycocyanin (APC)–anti–c-kit mAb, and fluorescein
isothiocyanate (FITC)– or PE–anti-Sca-1 mAb (BD PharMingen, San
Diego, CA). To examine Fc Histamine release and 3H-thymidine uptake assays To test histamine release, cells harvested from HF cultures were incubated for 60 minutes with 2 µg/mL mouse IgE mAb against dinitrophenol (DNP; Sigma, St Louis, MO) and then challenged with 10 ng/mL DNP-conjugated human serum albumin (HSA; Sigma) or HSA alone for 45 minutes.30 Some samples were treated for 30 minutes with compound 48/80 (10 µg/mL) or substance P (100 µM). Supernatants were then examined for histamine using the Histamine Research Kit (Neogen, Lexington, KY). To test proliferative responses, loosely adherent cells were carefully released from underlying fibroblast layers by gentle pipetting, cultured in 96-well plates (3 x 104 cells/well) in the presence of various growth factors, and examined for 3H-thymidine uptake on day 3. SCF mRNA and protein expression in HF samples were tested by reverse transcription–polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA; R&D Systems), respectively. In some experiments, cells recovered from HF cultures were fractionated into CD45+ and CD45– populations by magnetic beads or fluorescence-activated cell sorting (FACS) before functional analyses. RT-PCR analyses The following primer sets were used to examine mouse mast cell protease (MCP) mRNA expression profiles: 5'-ACCACACTCCCGTCCTTACAT-3' and 5'-GGGCCACACCAGCACAC-3' for MCP-1; 5'-GACCACATTCTCGCCCTTACA-3' and 5'-ATTCTCAGTTTCACCTCCCTCAGT-3' for MCP-4; 5'-CTTCATCTGCTGCTCCTTCTCCTG-3' and 5'-GGCTGGCTCATTCACGTTTGTTCT-3' for MCP-5; and 5'-TGCACCCCCACTATTACACG-3' and 5'-CACCCCAGCTGACCACTCCT-3' for MCP-6. PCR products after 30 cycles of amplification were examined after ethidium bromide staining. SCF mRNA expression was examined by real-time RT-PCR as before31 using the following primer sets: 5'CCAGAGTCAGTGTCACAAAACC-3' and 5'-CTTCCAGTATAAGGCTCCAAAGC-3'.
Surface phenotype and growth potential of HF-derived leukocytes Vibrissal follicles isolated from adult C57BL/6 mice were dissected into 3 fragments, specifically, an upper fragment containing the sebaceous gland, an intermediate fragment containing the bulge region, and a lower fragment containing the follicular papilla (Figure 1A-B). Each fragment was placed underneath a cover glass and cultured in the presence of SCF, IL-3, IL-7, GM-CSF, and Flt3L. Fibroblast outgrowth was observed in 4 to 7 days in virtually all cultures, and round-shaped cells became detectable on the top of fibroblast layers in 10 days and expanded progressively thereafter (Figure 1C-D). About 40% of the HF cultures from the intermediate fragments (containing the bulge regions) showed apparent growth of the round-shaped cells in 2 weeks, and this frequency reached 70% in 4 weeks (Figure 1E, closed triangles). By contrast, the round cells were observed only in 20% of the HF cultures from the upper or the lower fragments even at 7 weeks (Figure 1E, closed circles and squares). Neither fibroblasts nor round-shaped cells emerged when peripheral blood samples in the amount (20 µL) much greater than an estimated volume (0.5-1 mm3) of a vibrissal follicle were placed under the cover glass and cultured in the presence of the same 5 growth factors (Figure 1E, open circles).
Round-shaped cells were loosely adhered to fibroblast layers on tissue-culture plates (Figure 1C-D), and some of them could be released by gentle pipetting. For phenotypic analyses, we forcefully expelled the ice-cold medium over the culture plates and repeated this vigorous pipetting procedure to recover as many round cells as possible without enzymatic digestion. Cells harvested after various culture periods (3-8 weeks) were examined for surface expression of CD45, c-kit (SCF receptor), Lin markers, and Sca-1. CD45 expression was detected in 40% to 90% of the recovered cells, and this variation correlated to the extent of fibroblast contamination in the test samples, but not the culture period. A representative phenotype of 5-week-old HF cultures is shown in Figure 2A-B. Virtually all the CD45+ cells expressed c-kit at high levels, but not any conventional Lin markers, including those for dendritic cells (CD11c and IA/IE), macrophages (CD11b), granulocytes (Gr-1), T cells (CD3, CD4, and CD8), B cells (B220), or erythroid cells (Ter-119). A very early hematopoietic lineage marker Sca-132 was detected on some, but not all, the CD45+/Lin–/c-kit+ populations. Although we confirmed that 3-week-old HF cultures exhibited virtually the identical phenotype (Figure 2C), we were unable to harvest sufficient numbers of cells for phenotypic analysis at earlier time points. Thus, it remains unclear whether one or more short-living leukocyte populations with distinct surface phenotypes may have emerged transiently in our HF cultures.
To study time-kinetics for ex vivo expansion of HF-derived leukocytes, we established large numbers of HF cultures in parallel and harvested them by vigorous pipetting at different time points (3, 4, 5, and 7 weeks). These samples were then examined for the expression of c-kit, Lin markers, and Sca-1 (Figure 3A). Consistent with our microscopic observations, the number of Lin–/c-kit+ leukocytes increased progressively over the 7-week culture periods (Figure 3A, open squares). Cells harvested at different time points were phenotypically indistinguishable from each other. In fact, Sca-1 expression was detected in 30% to 50% of the Lin–/c-kit+ populations throughout the above periods (Figure 3A, closed circles).
Significant BrdU incorporation was detected in the c-kit+ populations, documenting their mitotic potential in the presence of 5 added growth factors (Figure 3B). To determine which cytokines were responsible for this growth, we isolated loosely adherent cells from 5-week-old HF cultures by gentle pipetting to minimize fibroblast contamination. The resulting preparations, which contained more than 90% CD45+ cells, were then cultured in 96-well plates in the presence of each of the 5 cytokines (Figure 3C, upper panels). They showed marked 3H-thymidine uptake in response to SCF, but not to other cytokines that were included in the original growth medium. Conversely, the growth-promoting activity of the original medium was abrogated almost completely by removing SCF, but not other cytokines (Figure 3C, lower panels). Thus, it appeared that SCF was required and sufficient for promoting the ex vivo growth of HF-derived leukocytes.
In an attempt to assess the location for initial emergence of HF-derived
leukocytes, we transplanted bone marrow cells from EGFP transgenic mice
(C57BL/6 background) to
Characterization of HF-derived leukocytes
Despite phenotypic resemblance to hematopoietic stem cells
(CD45+/Lin–/c-kit+/Sca-1+),33-36
HF-derived leukocytes exhibited morphologic features of mast cells, such as
inclusion of metachromatic granules and nonsegmented oval-shaped nuclei
(Figure 5A). Exogenously added
IgE uniformly bound to the c-kit+ populations, indicating
Fc
Unfractionated HF cultures (containing 40%-70% CD45+ cells)
released significant amounts of histamine on ligation of surface IgE
receptors, as well as in response to substance P or compound 48/80
(Figure 6A, left panel). When
cells harvested from different HF cultures were fractionated into
CD45+ and CD45– populations by FACS, the histamine
release activity was observed exclusively within the CD45+
populations (Figure 6A, right
panel). Substantial variations were observed among different HF cultures in
terms of the amounts of histamine detected in the supernatants. This variation
failed to correlate to the culture duration, the purity of CD45+
cells, the expression level of Fc
We next cultured vibrissal follicles (intermediate fragments) in the
presence of SCF alone. As we observed in the presence of 5 cytokines,
round-shaped cells became first detectable over the outgrowing fibroblasts in
10 days and showed progressive expansion thereafter. Cells harvested from
these cultures established with SCF alone expressed the same surface phenotype
of Lin–/c-kit+/Fc
Although most of the HF cultures were terminated after 3 to 8 weeks for phenotypic and functional analyses, several cultures were maintained for longer periods by repeated passages of loosely adherent cells to new culture plates in the presence of 5 cytokines. When tested even after 6 months in culture, these lines maintained the phenotype of short-term HF cultures, except that they contained only negligible numbers of CD45– cells (ie, fibroblasts) and expressed Sca-1 uniformly (Figure 8A). These long-term lines also retained the ability to release histamine on ligation of surface IgE (Figure 8B).
HFs provide 2 key factors for mast cell development An important question concerned the putative origins of the mast cells that emerged in vitro from the HFs. To address this question, we conducted a series of histologic and immunohistologic analyses using vertical sections and cross-sections of freshly isolated vibrissal follicles. In complete agreement with the previous reports that mast cells are frequently found in the perifollicular areas of the dermal connective tissue, but not within the HFs,10,14,15 we failed to identify mature mast cells showing metachromatic staining in our vibrissal follicle samples. Within the ORSs, however, we observed large numbers of CD45+ cells, the majority of which presumably represented LCs and DETCs known to be distributed to this location.2,10-13 We also identified smaller numbers of c-kit+ cells within the ORSs in both the bulge and sub-bulge regions (Figure 9A-B,E-F), corroborating the report by Grichnik et al, who identified c-kit+ cells within HFs of human skin.37 Although the observed immunoreactivity was rather weak, staining with an isotype-matched control antibody showed no signals, indicating the specificity (Figure 9C-D,G-H). Unfortunately, we were unable to further characterize the intrafollicular c-kit+ populations due to the low expression level of this marker.
A second key question concerned the potential availability of SCF in the follicular microenvironment. In this regard, we observed constitutive SCF mRNA expression in freshly isolated vibrissal follicles (Figure 10). Our finding corroborates the previous report that LacZ reporter gene driven by a 2-kb 5' regulatory sequence of the SCF gene is expressed almost exclusively in the neural tissues and the HFs in fetal and newborn mice.38 We also detected SCF proteins (26.0 ± 0.4 pg/mg protein, n = 3) by ELISA in crude extracts prepared from the vibrissal follicles. Moreover, when vibrissal follicle specimens were placed in culture without prior dissection and in the absence of added cytokines, significant amounts of SCF became detectable in the culture supernatants (Figure 10 closed circles). Cultures of HF-derived, CD45– fibroblasts also secreted SCF into the media (Figure 10B open circles). These results suggest that SCF can be produced locally by one or more populations (eg, fibroblastic stromal cells) in the HFs.
We have demonstrated in this study that relatively large numbers of CD45+/Lin–/c-kit+ leukocytes can be readily propagated ex vivo from HF specimens in the presence of 5 added growth factors (SCF, IL-4, IL-7, GM-CSF, and Flt3L). These HF-derived leukocytes exhibited characteristic features of mast cells, including inclusion of metachromatic granules, surface expression of Fc RI, proliferative
responsiveness to SCF, and histamine release on ligation of surface IgE. Two
types of mast cells have been identified based on their biochemical and
functional
properties.39 For
example, substance P and compound 48/80 induce histamine release from the
connective tissue type, but not from the mucosal type, and they also differ
from each other in their MCP profiles, with MCP-3, -4, -5, and -6 expressed
preferentially by the connective tissue
type.40-43
The overall features of HF-derived leukocytes suggest their resemblance to the
connective tissue-type mast cells, except for MCP-1 mRNA expression, which was
reported to occur predominantly in the mucosal
type.43
Two mouse strains deficient in mast cells, known as SI/SId and
W/Wv, have mutations in the SCF gene and c-kit (SCF receptor) gene,
respectively.44,45
These earlier findings formed the basis of our current understanding that the
SCF/c-kit system plays key roles in mast cell
development.46 Mast
cells have since been expanded in vitro using SCF as a growth factor from
various human tissues, including fetal liver, bone marrow, cord blood,
peripheral blood, and
skin.40,47-51
In circulating blood of fetal mice, Rodewald et al identified a mast
cell–committed,
Lin–/Thy-1low/c-kit+/Fc Two major pitfalls of this study must be noted. First, the cellular identity of HF-associated mast cell precursors remains to be determined. One may argue that our observations simply reflect the in vitro expansion and differentiation of hematopoietic stem cells present as blood contaminants in our HF preparations. This possibility needs to be formally excluded, although no mast cell outgrowth was observed when peripheral blood samples were cultured under the same conditions (except for the absence of HF-associated microenvironment). Alternatively, HFs may contain small numbers of mature, noncycling mast cells that can undergo mitosis only when placed in culture, although mature mast cells have not been detected within the HF structures.10,14,15 It is tempting to speculate that some of the intrafollicular c-kit+ cells identified in human HF37 and mouse HF (Figure 9) may represent a putative progenitor population for mast cell development. Equally attractive is the possibility that mast cells may be derived from the recently identified "melanocyte stem cells" that reside in the bulge and sub-bulge regions and express low or undetectable levels of c-kit18,56 or the "follicular stem cells" in the bulge region that can form interfollicular epidermis, HFs, and sebaceous glands.16,17 Secondly, our experimental system did not allow us to examine a full spectrum of leukocyte lineages that may emerge from HFs. For example, although CD45+ leukocytes harvested from HF cultures after 3 weeks (the earliest time point tested) barely expressed Lin markers for dendritic cells, macrophages, granulocytes, T cells, or B cells, it is conceivable that some of these leukocyte subsets may be generated only transiently or in the presence (or absence) of particular growth factors. It also remains unclear whether HF-derived mast cells emerge from mast cell–committed progenitors that uniquely reside in the HFs. Alternatively, primitive hematopoietic stem cells or multilineage progenitors, which are presumably recruited to the HFs via SCF or other factors, may differentiate primarily into the mast cell lineage due to a unique cytokine milieu in this microenvironment. Further studies (eg, colony-forming assays at earlier time points) are required to clarify this important issue. In summary, we report that relatively large numbers of mast cells, which are virtually indistinguishable from those generated from fetal blood or adult bone marrow, can be readily propagated from HF specimens isolated from adult mice and that the HFs provide 2 critical factors for mast cell development (c-kit+ cells and SCF production). Our results support the current concept that HFs serve as a "niche" for tissue regeneration and provide both technical and conceptual frameworks for studying the role of HFs in local hematopoiesis in the skin.
Submitted February 11, 2003; accepted April 27, 2003.
Prepublished online as Blood First Edition Paper, May 8, 2003; DOI
10.1182/blood-2003-02-0449.
Supported by National Institutes of Health grants RO1-AI46755, RO1-AR35068,
RO1-AR43777, and RO1-AI43232.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked "advertisement" in accordance with 18 U.S.C. section
1734.
Reprints: Akira Takashima, Department of Dermatology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390; e-mail: akira.takashima{at}utsouthwestern.edu.
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Top
Abstract
Introduction
-actin
promoter and cytomegalovirus enhancer (C57BL/6
background)
), intermediate (
), and lower fragments (
) of vibrissal
follicles and peripheral blood samples (
) were cultured for 7 weeks under
the same conditions and examined every week under phase-contrast microscopy
for the emergence of round-shaped cells. The data indicate cumulative
frequencies of the cultures showing apparent expansion of round-shaped cells
(n = 50 for each source). Results shown in this figure are representative of 3
independent experiments.
B-dependent gene transactivation in sun-burn. J Clin
Invest. 2000;105:
1751-1759.