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*
Division of Cancer Research, Department of Pathology, University Hospital Zürich, Zürich, Switzerland; and
Basel Institute for Immunology, Basel, Switzerland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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RI), T1 expression precedes expression of FcRI in MC
ontogeny. The finding that the T1 Ag is selectively expressed at
several stages during development of the MC lineage suggests that this
cell surface molecule, in combination with the well-established markers
c-Kit and FcRI, should be valuable for studying the MC
lineage. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, we have analyzed the expression of the T1 Ag in the
hemopoietic system. For this, we utilized the T1-specific mAb DJ8,
which we have recently generated (47). We found that MCs were
the only identifiable cell lineage that expressed T1 at high levels.
MCs are critical effectors in the pathogenesis of IgE-dependent
allergic disorders (15, 16), but are also implicated in host defense
against bacterial infections (17, 18, 19) and parasites (20, 21). MCs can
be classified into two subtypes: the mucosal MC (MMC), which is widely
distributed in mucosal tissues of the respiratory tract and the
intestinal lamina propria, and the connective tissue-type MC, which is
found throughout the skin, musculature, in perivascular tissues, and in
the peritoneal cavity (16). MCs contain characteristic secretory
granules loaded with mediators such as histamine, heparin, proteases,
and others, which can be released upon stimulation. MCs also produce
and secrete a number of proinflammatory cytokines such as IL-1
and
TNF- (22). Ag-mediated engagement of FcRI triggers MC
degranulation and mediator release (23, 24). A second surface receptor,
c-Kit, is also characteristic for MCs. The c-kit
proto-oncogene encodes a protein tyrosine kinase receptor that is
activated by binding to its ligand, stem cell factor (SCF). Activation
of c-Kit results in essential signals for MC differentiation and
function (25).
MCs derived from multipotent hemopoietic stem cells (HSC) (26, 27). In
contrast to other hemopoietic cells that leave the BM after having
undergone partial or complete differentiation, it is thought that
putative, undifferentiated, and as yet unidentified MC precursors leave
the adult BM, migrate via the peripheral blood, and invade mucosal and
connective tissues, where they differentiate into morphologically
distinct mature MCs. In this study, we report that the T1 receptor is
expressed during different developmental stages of the MC lineage. T1
was detected on both immature BM-derived cultured MCs (BMCMCs), as well
as on mature i.p. MCs (IPMCs). Importantly, the earliest committed
progenitor of the MC lineage, which has recently been purified from
fetal blood (FB) (28), but not circulating progenitors containing HSC,
expresses T1 on the cell surface. Our results indicate that the T1 Ag,
along with the FcRI and c-Kit surface receptors, is within the
hemopoietic system selectively expressed on MCs, and that it might be a
useful novel surface Ag marker for studying the MC lineage.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Nonhemopoietic cell lines (NIH3T3, F-2, s.End.1, TME-3H3, L cells, HC11, B16-F10, and 3LL) were maintained in DMEM (Life Technologies, Gaithersburg, MD) supplemented with 10% heat-inactivated FCS (Life Technologies), 2 mM L-glutamine, penicillin (100 U/ml), and streptomycin (100 µg/ml). All hemopoietic cell lines (P815, BW5147, P388, X63.Ag8, SciET27/F) were grown in lymphoid cell growth medium: Iscove’s modified Dulbecco’s medium (IMDM; Life Technologies), supplemented with 10% heat-inactivated FCS (Life Technologies), 50 µM 2-ME, 2 mM L-glutamine, penicillin (100 U/ml), and streptomycin (100 µg/ml).
T1 expression in established cell lines
A total of 1 x 106 viable cultured cells was stained with a 1/1000 pretitrated dilution of an anti-T1 polyclonal rat antiserum (47) or with a 1/1000 dilution of the preimmune serum of the same animal for 30 min at 4°C. After washing the cells in FACS buffer (PBS, 10% FCS, 0.1% sodium azide), 1 µg of a FITC-conjugated goat anti-rat Ig (Southern Biotechnology, Birmingham, AL) was added to the cells and incubated for 30 min at 4°C. Cells were washed and resuspended in FACS buffer, and 10,000 forward scatter/side scatter-gated viable cells were acquired and analyzed on a Becton Dickinson (San Jose, CA) FACSCalibur flow cytometer.
mAbs for flow cytometry
The following primary mAbs were utilized in this study:
biotinylated or FITC-labeled DJ8 (anti-T1 mAb) (47); biotinylated
ACK-4 (anti-c-Kit) (29); R-phycoerythrin (PE)-conjugated 3C1
(anti-c-Kit); PE-conjugated CT-TH1 (anti-Thy-1); PE-conjugated
RA3-6B2 (anti-B220); PE-conjugated M1/70.15 (anti-Mac-1);
PE-conjugated F4/80 (anti-mouse macrophage; all from Caltag
Laboratories, San Francisco, CA); PE-conjugated H129.19 (anti-CD4);
biotinylated 53-6.7 (anti-CD8); PE-conjugated TER-119
(antierythroid BM cells); IgE-3 (antitrinitrophenol mouse IgE, 
);
and unlabeled rat IgG1, isotype control (all from PharMingen, San
Diego, CA). PE-conjugated isotype control Abs IgG2a-PE and IgG2b-PE
were from Caltag. Second-step reagents were:
streptavidin-allophycocyanin (Sav-APC; PharMingen) and FITC-labeled
goat anti-mouse Ab (Southern Biotechnology, Birmingham, AL). The
anti-T1 DJ8 mAb was purified from hybridoma supernatants on protein
G-Sepharose (Pharmacia, Uppsala, Sweden). DJ8 was biotinylated with
NHS-LC-biotin (Pierce, Rockford, IL), following the instructions of the
manufacturer. DJ8 and rat IgG1 isotype control mAbs were identically
treated for labeling with FITC (isomer I on celite; Sigma, St. Louis,
MO) following standard procedures (30). After the labeling procedure,
all labeled Abs were carefully titrated by FACS analysis, and the
determined optimal concentration was used in subsequent experiments.
BM cultures
BM cells were obtained from 8- to 10-wk-old C57BL/6 mice by flushing femora and tibiae with HBSS buffer (Life Technologies). BM cells were cultured in lymphoid culture medium (see above) supplemented with 3% conditioned culture supernatant from murine IL-3-secreting X63/IL-3 cells (31). BM cultures were enriched for MCs by repetitively transferring the suspension cell fraction into fresh culture flasks every 5 days in the presence of IL-3. MCs were identified by double staining for IgE receptors and c-Kit (32). To this end, BM suspension cells were first incubated with IgE mAb at 10 µg/ml and anti-c-Kit-PE (10 µg/ml), followed by a FITC-labeled goat anti-mouse Ab (1:100). After about 4 wk in culture, >98% of the cells displayed typical MC-like IgE+c-Kit+ phenotype as well as metachromatic staining with Giemsa. BMCMCs were analyzed for T1 expression by incubating cells with IgE, c-Kit-PE, and DJ8-biotin (0.5 µg/ml), followed by the secondary step reagents goat anti-mouse Ig-FITC and Sav-APC (1:200), respectively.
To follow the appearance of BMCMCs in BM cultures, BM cells were prepared and cultured as outlined above. At different time points, suspension cells were removed from the culture flasks and stained with c-Kit-PE and DJ8-FITC.
For analysis of T1 expression in the c-Kit+ cells present in freshly isolated BM suspensions, cells were incubated with DJ8-FITC. To visualize c-Kit+ BM cells, they were additionally stained with biotinylated anti-c-Kit, followed by Sav-APC and a mixture of PE-conjugated lineage-specific Abs (Thy-1-PE, B220-PE, TER-119-PE, and Mac-1-PE).
In the presence of IL-3, BM-derived monocytes also differentiate into macrophages that are present in the adherent fraction of the BM culture (30, 33). To assay the expression of T1 on BM-derived macrophages, adherent cell fractions of 6-day-old BM cultures were trypsinized and simultaneously stained with DJ8-FITC (0.5 µg/ml) and the macrophage-specific Abs Mac-1-PE or F4/80-PE (both at 10 µg/ml).
Flow cytometry of primary lymphoid cells
The spleen, axillary lymph nodes, and the thymus were removed surgically from 8-wk-old C57BL/6 mice, and the organs were mechanically homogenized to obtain a single-cell suspension. From the same animals, BM cells were prepared as described above. PBMCs were obtained from whole heparinized peripheral blood after removal of RBC through osmotic lysis. Fetal livers were derived from day 13 and day 15 timed-pregnant mice. For the analysis of T1 expression, single-cell suspensions were directly stained with DJ8-FITC in the presence of 10% FCS. Background staining was determined with IgG1-FITC, an isotype-matched control Ab.
T1 expression in spleen-derived T cells was assessed by double staining with either CD4-PE, Thy-1-PE, and CD8-biotin, followed by avidin-PE vs T1-FITC or IgG1-FITC. For the analysis of B cells, splenocytes were double stained with B220-PE and T1-FITC. All stainings were performed in PBS, 10% FCS, and 0.05% sodium azide at 4°C for 30 min.
Immunoprecipitation of T1 M in BMCMCs
BMCMCs were grown as described above. For each immunoprecipitation, 1 x 107 viable BMCMCs were solubilized in lysis buffer (1% Nonidet P-40, 150 mM NaCl, 50 mM Tris/HCl, pH 8, 5 mM EDTA, 10 mM iodoacetamide), supplemented with a mixture of protease inhibitors (Complete; Boehringer Mannheim, Mannheim, Germany). Postnuclear cell lysates were incubated with 30 µl protein G-Sepharose (Pharmacia) that had been coated with 3 µg anti-T1 mAbs DJ4 and DJ8 (47) or IgG1 isotype control mAb for 4 h at 4°C. After washing the resin three times with PBS, the beads were boiled in reducing Laemmli sample buffer, and the eluted proteins were electrophoresed through 10% SDS-PAGE gels. Proteins were transferred onto a polyvinylidene difluoride membrane (Millipore), and unspecific binding sites were blocked with PBS-T (PBS, 0.4% Tween-20) containing 5% skim milk powder (Fluka, Buchs, Switzerland). The immunoblot was probed with a rabbit polyclonal antiserum directed against bacterially produced murine T1 protein (34), followed by an AP-conjugated goat anti-rabbit Ab (Promega, Madison, WI). After extensively washing with PBS-T, the blot was developed using the Western Blue AP substrate according to the manufacturer’s recommendations (Promega).
Intraperitoneal MCs
Unfractionated cell suspensions were obtained from the peritoneal cavity by peritoneal lavage of 6- to 8-wk-old C57BL/6 mice. The fraction of i.p. MCs in this preparation was assessed by cytologic staining with Giemsa or Berberine sulfate and found to be 2 to 3%. For flow cytometry, FcR were blocked with FACS buffer (containing 10% FCS) and stained for IgE receptor, c-Kit, and T1, as described above for BMCMCs. In a second experiment, IPMCs were prepared identically and stained with DJ8-FITC and c-Kit-PE. Double-positive T1+c-Kit+ cells were identified and sorted on a FACStar flow cytometer. Cytocentrifuge preparations of the total, unsorted cells, as well as the sorted cell population that was found to be about 94% pure upon reanalysis, were examined with May-Grünwald-Giemsa stain.
FB mast cell progenitors
The preparation of FB progenitor as well as the FACS staining and sorting of pro-MCs was performed as described (28, 35). In short, fetuses of timed-pregnant C57BL/6 mice were obtained at day 15.5 of gestation. The uterus was excised and washed in PBS, and the embryos were removed without injuring the umbilical cord. Fetuses were placed on their backs, and the jugular veins and cervical arteries were cut. About 20 µl of FB was collected from each fetus. The heparinized FB was washed in PBS and 5% FCS, and the fetal leukocytes were purified by discontinuous Percoll (60% v/v) (Pharmacia, Uppsala, Sweden) gradient centrifugation.
The obtained FB leukocytes were blocked with 0.5 mg/ml normal mouse Ig (Jackson ImmunoResearch, West Grove, PA) and stained with Thy-1-PE (1/50 diluted) and biotinylated c-Kit (5 µg/ml) mAb, followed by Sav-APC (1:200). The fetal MC progenitors were identified as the Thy-1lowc-Kithigh population and sorted on a FACStar flow cytometer. As a negative control, the Thy-1-c-Kit+ multipotent progenitor population (35) was also sorted. The two sorted cell populations that were found to be >99% pure upon reanalysis were subsequently stained with the anti-T1 mAb DJ8-FITC (0.6 µg/ml).
A second independent preparation was performed with day 15.5 fetuses of timed-pregnant BALB/c mice. The obtained FB leukocytes from this preparation were directly analyzed without cell sorting after triple staining with Thy-1-PE, c-Kit-biotin, and DJ8-FITC or IgG1-FITC, followed by Sav-APC.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The expression levels of the T1 Ag were analyzed in a number of
hemopoietic and nonhemopoietic established cell lines by flow
cytometry. Lymphoid cell lines such as the BW5147 T cell hybridoma, the
X63.Ag8 B cell myeloma, and the P388 macrophage cell line were all
shown to express low but significant levels of the T1 receptor (Fig. 1
). In agreement with previous results
(7, 10, 47), substantial amounts of T1 were also detectable on NIH3T3
fibroblasts. However, the highest levels of T1 expression were observed
in primary bone BMCMCs and the P815 mastocytoma cell line. T1
expression was undetectable in three endothelial cell lines (F-2,
s.End.1, amd TME-3H3), another fibroblast-like cell line (L cells), in
HC11 breast epithelial cells, in a pro-T cell line, and in the two
tumor cell lines B16-F10 (melanoma) and 3LL (Lewis lung
carcinoma).
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On the basis of the results obtained with established cell lines,
we next analyzed T1 expression in freshly isolated cells that were
prepared from a number of murine lymphoid organs (Fig. 2). Weak staining with the anti-T1
DJ8-FITC mAb was observed consistently in a subpopulation of BM cells
comprising 
5 to 7% of all cells (Fig. 2; see also Fig. 5, first panel, and Fig. 8B). Similar weak staining
intensities were detected on fetal liver cells of about days 13 and 15
of gestation. In contrast, all other lymphoid organs analyzed, such as
lymph nodes, spleen, thymus, and PBMCs, were negative for T1
expression.
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), and T1 mRNA expression has been found in the same cell types
(6, 8), we analyzed primary lymphocytes and macrophages in greater
detail (Fig. 3). For this, splenocytes
were double stained for T or B cell markers vs T1. Thy-1+,
CD4+, and CD8+ T cells were clearly negative
for T1 expression, as were B220+ B cells. A very small
fraction of cells that did not express any of the tested lymphoid
lineage markers did express low levels of T1. Next, T1 expression was
measured in BM-derived macrophages (BMM
) that grew out of primary BM
cultures in the presence of IL-3 (33) and on freshly isolated i.p.
macrophages (IPM). All macrophages were identified by staining with
F4/80 or anti-Mac-1 macrophage-specific Abs. Both BM-derived and
i.p. macrophages did not express significant levels of T1 (Fig. 3). The
small population of cells that express T1, but not the
macrophage-specific markers, represents BMCMCs and IPMCs, as will be
shown below.
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In the comparative expression analysis shown in Figure 1, highest
levels of T1 were detected on the surface of BMCMCs. To further
substantiate this finding, we analyzed BMCMCs by triple-color analysis.
The hallmark of mature MCs is the expression of the high affinity IgE
receptor, FcRI, and the c-Kit receptor (16). The FcRI on BMCMCs
was detected by binding of a purified IgE mAb, and the c-Kit receptor
by staining with an anti-c-Kit mAb. Most cells of a 6-wk-old BMCMC
culture that was grown in the presence of IL-3 homogenously expressed
both FcRI and c-Kit (Fig. 4A). Giemsa staining of
metachromatic granules further confirmed the MC nature of those cells
(not shown). All of the IgE+c-Kit+ MCs were
shown to express uniformly high levels of T1 when stained with
biotinylated DJ8 mAb followed by Sav-APC. In contrast, incubation with
the secondary staining reagent alone resulted in background staining.
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B).
We next analyzed the emergence of T1+c-Kit+
immature MCs in IL-3-induced primary BM cultures (Fig. 5). As shown in a previous experiment
(Fig. 2), about 8% of freshly isolated BM cells expressed low amounts
of T1 (T1low). Importantly, no
T1+c-Kit+ double-positive cells were detectable
in freshly isolated BM cells. However, when the BM cells were
cultivated in the presence of IL-3, T1+c-Kit+
double-positive immature MCs were readily detectable at day 6,
comprising about 2% of all cells. The expression level of T1 observed
in the T1+c-Kit+ BMCMCs was at least 10 times
higher than the level measured on the T1low cells at the
onset of the culture. Over time, the T1+c-Kit+
BMCMC population constantly expanded and comprised about 99% of all
cells after 4 wk of culture.
Mature IPMCs express high levels of T1 in vivo
After having demonstrated that immature BMCMCs express high levels
of T1, we were interested to investigate whether mature in vivo MCs do
also express T1. Hence, we isolated cells from the peritoneal cavity of
C57BL/6 mice and analyzed them by flow cytometry. For this, the
peritoneal cells were triple stained with IgE, anti-c-Kit, and
anti-T1 mAbs. Mature IPMCs were clearly identifiable as a
IgE+c-Kit+ double-positive population (Fig. 6A). The
IgE+c-Kit+ IPMC population comprised about 2%
of the total cell number. When only analyzing the
IgE+c-Kit+ MC population, it was revealed that
all of them homogeneously expressed high levels of T1. In contrast,
IgE-c-Kit- non-MCs in the peritoneal lavage
did not express significant levels of T1 (not shown). Our result was
confirmed by quantification of the T1+ cells present in the
total peritoneal cell preparation. As shown in the histogram of
DJ8-stained ungated cells (Fig. 6A), about 2% of the total
cell population expressed high levels of T1. Since this percentage of
T1+ cells is identical to the percentage of
IgE+c-Kit+ MCs present in the total cell
population, we conclude that all and only MCs express T1.
Interestingly, the T1-staining intensities detected on IPMCs and on
BMCMCs were almost identical (compare histograms in Figs. 4 and 6A).
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B). It is
important to note that a single staining of peritoneal cells with the
DJ8 mAb alone (see Fig. 6A, upper left panel,
"ungated cells") will suffice to identify and sort-purify the IPMC
population. T1 is expressed on the earliest detectable Thy-1lowc-Kithigh MC precursor
Stimulated by the fact that both mature IPMCs as well as immature
BMCMCs express high levels of T1, we investigated T1 expression in MC
precursors. The earliest committed precursor of the MC lineage has been
identified recently in FB of day 15.5 of gestation (28). It is defined
as a population that expresses low levels of the Thy-1 surface
glycoprotein and high levels of c-Kit
(Thy-1lowc-Kithigh). To isolate the MC
progenitors, we collected FB from fetuses at day 15.5 of gestation. FB
leukocytes were enriched by discontinuous Percoll gradient
centrifugation. MC-committed precursors were identified by staining the
FB leukocytes with anti-Thy-1 and anti-c-Kit mAbs. This
staining resolved three typical cell populations that differ in their
respective expression of Thy-1 and c-Kit. The
Thy-1+c-Kitlow population of FB cells has been
found to contain prothymocytes, whereas
Thy-1-c-Kit+ cells were shown to be HSC (35, 28). The rarest population, defined by the
Thy-1lowc-Kithigh phenotype, represents MC
lineage-committed progenitors. In the first experiment (Fig. 7A), the
Thy-1lowc-Kithigh pro-MCs and the
Thy-1-c-Kit+ HSC population were purified by
flow-cytometric cell sorting. The two sorted populations were shown to
be >99% pure upon reanalysis. Staining of the highly purified cells
with the T1-specific Ab DJ8 revealed that all of the
Thy-1lowc-Kithigh pro-MCs expressed high levels
of T1. In contrast, the Thy-1-c-Kit+ HSC and
the Thy-1+c-Kitlow pro-T cell (not shown)
populations did not stain with the anti-T1 Ab. These results were
confirmed in a second, slightly modified experiment. Percoll
gradient-separated FB leukocytes of 15.5-day-old BALB/c fetuses were
directly triple stained with Thy-1, c-Kit, and T1 Abs without cell
sorting. Analysis of the Thy-1lowc-Kithigh
pro-MC population revealed again that all cells stained brightly for T1
(Fig. 7B). In contrast, the
Thy-1-c-Kit+ HSC population did not exhibit
significant levels of T1. Negative control stainings using an Ab that
matches the isotype of the anti-T1 DJ8 mAb resulted in background
staining.
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The in vitro formation of MC colonies in the presence of SCF (the
c-Kit ligand) and IL-3 suggests the existence of MC precursors in the
BM (36). Inspired by this, we attempted to identify a phenotypic
equivalent of the FB Thy-1lowc-Kit+
promastocyte in the BM. To this end, unfractionated preparations of
freshly prepared BM cells from 4-wk-old C57BL/6 mice were double
stained with Thy-1 and c-Kit mAbs. This staining resolved two distinct
populations, a Thy-1+c-Kit- and a
Thy-1-c-Kit+ population (Fig. 8A). The first population
probably consists of Thy-1+ T cell lineage cells, whereas
the latter probably represents c-Kit+ early progenitors
including HSC. However, a population corresponding to the FB
Thy-1lowc-Kithigh pro-MCs was clearly not
detectable. We reasoned that the Thy-1 expression level of the putative
BM MC precursors might be lower than of the FB progenitors, and
consequently considered the existence of
Thy-1-c-Kithigh MC precursors. Based on this
assumption and on our previous results, we investigated the possibility
to employ T1 as a marker for pro-MCs in the BM. To better distinguish
the c-Kit+ population from cells of the mature hemopoietic
lineage (lin) such as B cells, T cells, macrophages, and erythroid
cells, we stained BM cells with a combination of different
lineage-specific markers. This resulted in two clearly separable
populations, the lin+c-Kit- and the
lin-c-Kit+ cells. Triple-color analysis and
gating on the lin-c-Kit+ cells clearly
revealed that T1 is not expressed in this population at levels
comparable with FB promastocytes. From this result, we conclude that
the adult BM might not contain a pro-MC precursor that can be defined
on the basis of the simultaneous expression of c-Kit and T1.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and 2).
Our results are consistent with the presence of T1 mRNA in
BMCMCs (10) and in several established MC lines (8, 10). In primary BM
cultures, T1 expression, in combination with c-Kit, was shown to be
instrumental for studying the kinetics of BMCMC appearance (Fig. 5).
Interestingly, we detected no T1+c-Kit+ cells,
i.e., a phenotype consistent with that of immature BMCMCs, in freshly
isolated BM cells (see below). However, 6 days after cultivation in
IL-3-containing medium, we observed a small population of
T1+c-Kit+ cells that were identifiable as
BMCMCs by morphology. This population continuously expanded over time
until, at 4 wk, the culture homogeneously consisted of
T1+c-Kit+ BMCMCs. To address the in vivo
relevance of this finding, we tested T1 expression in freshly isolated
IPMCs and found that all mature c-Kit+IgE+
IPMCs also uniformly express high levels of T1 in vivo. BMCMCs resemble immature MCs that exhibit some characteristics of MMCs. However, it is important to note that cultured BMCMCs have the potential to differentiate into both MC types, depending on the local microenvironment (38). Whereas T cell-derived factors such as IL-3 and IL-4 promote the development of MMCs, the presence of fibroblast-derived SCF induces the maturation of connective tissue-type MCs (38). IPMCs represent an example of the latter cell type (39). Our finding that T1 is expressed in both mature IPMCs as well as immature BMCMCs precursors suggests that T1 expression is a general characteristic of MCs.
An interesting aspect of our study is the expression of T1 in the
earliest committed MC progenitors, which have recently been identified
in FB as cells that express low levels of Thy-1 and high levels of
c-Kit (Thy-1lowc-Kithigh). FB pro-MCs already
contain metachromatic granules, but do not yet express the FcRI
(28). From this, we conclude that T1 precedes FcRI expression in MC
ontogeny. This fact makes T1 an interesting novel surface Ag for the
identification of MC progenitors. It is thought that MC precursors
exist in the BM of adult mice, peripheral blood, and fetal liver (26, 40, 41). However, a morphologically defined MC precursor has not yet
been purified from these sites (28, 36, 42). Our attempts to utilize T1
expression as a surrogate marker for the identification of a putative
T1+ MC precursor population in the adult BM were
unsuccessful. Neither freshly isolated, unfractionated BM cells nor the
c-Kit-expressing population that was devoid of cells belonging to
mature hemopoietic lineages (c-Kit+lin-) did
contain a cell population expressing substantial amounts of T1. In
agreement with our findings, other authors were also unsuccessful in
isolating committed MC progenitors in the BM. Lantz and Huff (43)
reported the immunomagnetic purification of
c-Kit+lin- BM cells and showed that this
population did not contain cells that either expressed FcRI or
showed metachromatic granules. This
c-Kit+lin-FcRI- metachromatic
granule- population was shown to be highly enriched for
CFU-mast, i.e., cells that proliferated in response to SCF/IL-3 and
gave rise to immature granulated MCs. However, since
c-Kit+lin- BM cells include HSC, these
experiments could not resolve the question as to whether the MC
colony-forming progenitor in the BM is a multipotent cell type, or a MC
lineage-committed cell, or a mixture of both. Within the CFU
mast-enriched, c-Kit+lin- population, a
subpopulation of granulated cells, a phenotype consistent with that of
the MC precursors found in FB (28), has not yet been identified.
These results, in combination with our own data, suggest that a granulated FB-like MC precursor with a c-Kit+T1+ phenotype may not be found in the BM. To explain this finding, we consider the following possibilities: 1) T1 might only be expressed on fetal, but not adult MC progenitors; 2) T1+c-Kit+ promastocytes might exist in the BM, but at frequencies too low to allow detection by flow cytometry; 3) the CFU-mast activity found in the BM (26) does not result from a FB-like committed pro-MC, but rather from a more primitive pluripotent HSC; and 4) a FB-like promastocyte does not reside in the BM, but circulates in the blood or is localized in other tissues. Taken together, we have established T1 as a novel marker for the MC lineage that is expressed during all stages of its ontogeny. This raises the question of the biologic function of T1 in MCs. Since T1 is expressed throughout MC development, we consider a role of T1 in the induction of MC differentiation unlikely. T1, as c-Kit (44), might function as a survival factor for MCs. We also consider a role of T1 as a modulator of MC effector functions. Interestingly, the cytoplasmic domain of T1 bears an amino acid sequence (IIYPRV) (7) that fits the consensus sequence I/VxYxxL/V (D.R.M., unpublished observation) of an immunoreceptor tyrosine-based inhibition motif that is found in the cytoplasmic portions of NK cell receptors (killer cell inhibitory receptors, KIRs) and members of the gp49 receptor family (45, 46). However, the possible roles of T1 discussed in this work remain speculative. Future studies, such as the targeted inactivation of the T1 gene in mice, will have to clarify the physiologic role of T1 in MC biology.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Cardiovascular Research, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080.
3 Address correspondence and reprint requests to Dr. Roman Klemenz, Division of Cancer Research, Department of Pathology, University Hospital Zürich, Schmelzbergstrasse 12, CH-8091 Zürich, Switzerland. E-mail address:
4 Abbreviations used in this paper: MC, mast cell; AP, alkaline phosphatase; BM, bone marrow; BMCMB, bone marrow-derived cultured mast cell; BMM, bone marrow-derived macrophage; FB, fetal blood; HSC, hemopoietic stem cell; IPM, intraperitoneal macrophage; IPMC, intraperitoneal mast cell; MMC, mucosal mast cell; PE, R-phycoerythrin; Sav-APC, streptavidin-allophycocyanin; SCF, stem cell factor.
Received for publication April 3, 1998. Accepted for publication June 24, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI case. Immunol. Today 14:222.[Medline]
RIneg mast cells in mouse bone marrow cell cultures: use of a monoclonal anti-p161 antibody. J. Exp. Med. 182:575.
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 L. Guo, G. Wei, J. Zhu, W. Liao, W. J. Leonard, K. Zhao, and W. Paul IL-1 family members and STAT activators induce cytokine production by Th2, Th17, and Th1 cells PNAS, August 11, 2009; 106(32): 13463 - 13468. [Abstract] [Full Text] [PDF]
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Q. Espinassous, E. Garcia-de-Paco, I. Garcia-Verdugo, M. Synguelakis, S. von Aulock, J.-M. Sallenave, A. N. J. McKenzie, and J. Kanellopoulos IL-33 Enhances Lipopolysaccharide-Induced Inflammatory Cytokine Production from Mouse Macrophages by Regulating Lipopolysaccharide Receptor Complex J. Immunol., July 15, 2009; 183(2): 1446 - 1455. [Abstract] [Full Text] [PDF]
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P. N. Pushparaj, H. K. Tay, S. C. H'ng, N. Pitman, D. Xu, A. McKenzie, F. Y. Liew, and A. J. Melendez The cytokine interleukin-33 mediates anaphylactic shock PNAS, June 16, 2009; 106(24): 9773 - 9778. [Abstract] [Full Text] [PDF]
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 J. Kearley, K. F. Buckland, S. A. Mathie, and C. M. Lloyd Resolution of Allergic Inflammation and Airway Hyperreactivity Is Dependent upon Disruption of the T1/ST2-IL-33 Pathway Am. J. Respir. Crit. Care Med., May 1, 2009; 179(9): 772 - 781. [Abstract] [Full Text] [PDF]
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M. Suzukawa, M. Iikura, R. Koketsu, H. Nagase, C. Tamura, A. Komiya, S. Nakae, K. Matsushima, K. Ohta, K. Yamamoto, et al. An IL-1 Cytokine Member, IL-33, Induces Human Basophil Activation via Its ST2 Receptor J. Immunol., November 1, 2008; 181(9): 5981 - 5989. [Abstract] [Full Text] [PDF]
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D. Xu, H.-R. Jiang, P. Kewin, Y. Li, R. Mu, A. R. Fraser, N. Pitman, M. Kurowska-Stolarska, A. N. J. McKenzie, I. B. McInnes, et al. IL-33 exacerbates antigen-induced arthritis by activating mast cells PNAS, August 5, 2008; 105(31): 10913 - 10918. [Abstract] [Full Text] [PDF]
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 M. D. Smithgall, M. R. Comeau, B.-R. Park Yoon, D. Kaufman, R. Armitage, and D. E. Smith IL-33 amplifies both Th1- and Th2-type responses through its activity on human basophils, allergen-reactive Th2 cells, iNKT and NK Cells Int. Immunol., August 1, 2008; 20(8): 1019 - 1030. [Abstract] [Full Text] [PDF]
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Y. Kondo, T. Yoshimoto, K. Yasuda, S. Futatsugi-Yumikura, M. Morimoto, N. Hayashi, T. Hoshino, J. Fujimoto, and K. Nakanishi Administration of IL-33 induces airway hyperresponsiveness and goblet cell hyperplasia in the lungs in the absence of adaptive immune system Int. Immunol., June 1, 2008; 20(6): 791 - 800. [Abstract] [Full Text] [PDF]
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 L. H. Ho, T. Ohno, K. Oboki, N. Kajiwara, H. Suto, M. Iikura, Y. Okayama, S. Akira, H. Saito, S. J. Galli, et al. IL-33 induces IL-13 production by mouse mast cells independently of IgE-Fc{epsilon}RI signals J. Leukoc. Biol., December 1, 2007; 82(6): 1481 - 1490. [Abstract] [Full Text] [PDF]
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S. Ali, M. Huber, C. Kollewe, S. C. Bischoff, W. Falk, and M. U. Martin IL-1 receptor accessory protein is essential for IL-33-induced activation of T lymphocytes and mast cells PNAS, November 20, 2007; 104(47): 18660 - 18665. [Abstract] [Full Text] [PDF]
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 X. Huang, W. Du, R. P. Barrett, and L. D. Hazlett ST2 Is Essential for Th2 Responsiveness and Resistance to Pseudomonas aeruginosa Keratitis Invest. Ophthalmol. Vis. Sci., October 1, 2007; 48(10): 4626 - 4633. [Abstract] [Full Text] [PDF]
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Z. Allakhverdi, D. E. Smith, M. R. Comeau, and G. Delespesse Cutting Edge: The ST2 Ligand IL-33 Potently Activates and Drives Maturation of Human Mast Cells J. Immunol., August 15, 2007; 179(4): 2051 - 2054. [Abstract] [Full Text] [PDF]
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 C. Waskow, S. Bartels, S. M. Schlenner, C. Costa, and H.-R. Rodewald Kit is essential for PMA-inflammation-induced mast-cell accumulation in the skin Blood, June 15, 2007; 109(12): 5363 - 5370. [Abstract] [Full Text] [PDF]
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C. T. Fagundes, F. A. Amaral, A. L. S. Souza, A. T. Vieira, D. Xu, F. Y. Liew, D. G. Souza, and M. M. Teixeira ST2, an IL-1R family member, attenuates inflammation and lethality after intestinal ischemia and reperfusion J. Leukoc. Biol., February 1, 2007; 81(2): 492 - 499. [Abstract] [Full Text] [PDF]
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S. Nakae, H. Suto, M. Iikura, M. Kakurai, J. D. Sedgwick, M. Tsai, and S. J. Galli Mast Cells Enhance T Cell Activation: Importance of Mast Cell Costimulatory Molecules and Secreted TNF J. Immunol., February 15, 2006; 176(4): 2238 - 2248. [Abstract] [Full Text] [PDF]
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Y. Arinobu, H. Iwasaki, M. F. Gurish, S.-i. Mizuno, H. Shigematsu, H. Ozawa, D. G. Tenen, K. F. Austen, and K. Akashi Developmental checkpoints of the basophil/mast cell lineages in adult murine hematopoiesis PNAS, December 13, 2005; 102(50): 18105 - 18110. [Abstract] [Full Text] [PDF]
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 M. Shimizu, A. Matsuda, K. Yanagisawa, T. Hirota, M. Akahoshi, N. Inomata, K. Ebe, K. Tanaka, H. Sugiura, K. Nakashima, et al. Functional SNPs in the distal promoter of the ST2 gene are associated with atopic dermatitis Hum. Mol. Genet., October 1, 2005; 14(19): 2919 - 2927. [Abstract] [Full Text] [PDF]
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M. N. Mann-Chandler, M. Kashyap, H. V. Wright, F. Norozian, B. O. Barnstein, S. Gingras, E. Parganas, and J. J. Ryan IFN-{gamma} Induces Apoptosis in Developing Mast Cells J. Immunol., September 1, 2005; 175(5): 3000 - 3005. [Abstract] [Full Text] [PDF]
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C.-C. Chen, M. A. Grimbaldeston, M. Tsai, I. L. Weissman, and S. J. Galli From The Cover: Identification of mast cell progenitors in adult mice PNAS, August 9, 2005; 102(32): 11408 - 11413. [Abstract] [Full Text] [PDF]
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Z. Orinska, E. Bulanova, V. Budagian, M. Metz, M. Maurer, and S. Bulfone-Paus TLR3-induced activation of mast cells modulates CD8+ T-cell recruitment Blood, August 1, 2005; 106(3): 978 - 987. [Abstract] [Full Text] [PDF]
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 T. B. Feyerabend, H. Hausser, A. Tietz, C. Blum, L. Hellman, A. H. Straus, H. K. Takahashi, E. S. Morgan, A. M. Dvorak, H. J. Fehling, et al. Loss of Histochemical Identity in Mast Cells Lacking Carboxypeptidase A Mol. Cell. Biol., July 15, 2005; 25(14): 6199 - 6210. [Abstract] [Full Text] [PDF]
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 C. L. Weller, S. J. Collington, J. K. Brown, H. R.P. Miller, A. Al-Kashi, P. Clark, P. J. Jose, A. Hartnell, and T. J. Williams Leukotriene B4, an activation product of mast cells, is a chemoattractant for their progenitors J. Exp. Med., June 20, 2005; 201(12): 1961 - 1971. [Abstract] [Full Text] [PDF]
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B. P. Leung, D. Xu, S. Culshaw, I. B. McInnes, and F. Y. Liew A Novel Therapy of Murine Collagen-Induced Arthritis with Soluble T1/ST2 J. Immunol., July 1, 2004; 173(1): 145 - 150. [Abstract] [Full Text] [PDF]
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 E. K. Brint, K. A. Fitzgerald, P. Smith, A. J. Coyle, J.-C. Gutierrez-Ramos, P. G. Fallon, and L. A. J. O'Neill Characterization of Signaling Pathways Activated by the Interleukin 1 (IL-1) Receptor Homologue T1/ST2. A ROLE FOR JUN N-TERMINAL KINASE IN IL-4 INDUCTION J. Biol. Chem., December 13, 2002; 277(51): 49205 - 49211. [Abstract] [Full Text] [PDF]
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 P. Kropf, S. Herath, R. Tewari, N. Syed, R. Klemenz, and I. Muller Identification of Two Distinct Subpopulations of Leishmania major-Specific T Helper 2 Cells Infect. Immun., October 1, 2002; 70(10): 5512 - 5520. [Abstract] [Full Text] [PDF]
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Z. Wiener, M. Andrasfalvy, E. Pallinger, P. Kovacs, C. Szalai, A. Erdei, S. Toth, A. Nagy, and A. Falus Bone marrow-derived mast cell differentiation is strongly reduced in histidine decarboxylase knockout, histamine-free mice Int. Immunol., April 1, 2002; 14(4): 381 - 387. [Abstract] [Full Text] [PDF]
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M. J. Sweet, B. P. Leung, D. Kang, M. Sogaard, K. Schulz, V. Trajkovic, C. C. Campbell, D. Xu, and F. Y. Liew A Novel Pathway Regulating Lipopolysaccharide-Induced Shock by ST2/T1 Via Inhibition of Toll-Like Receptor 4 Expression J. Immunol., June 1, 2001; 166(11): 6633 - 6639. [Abstract] [Full Text] [PDF]
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C. Meisel, K. Bonhagen, M. Lohning, A. J. Coyle, J.-C. Gutierrez-Ramos, A. Radbruch, and T. Kamradt Regulation and Function of T1/ST2 Expression on CD4+ T Cells: Induction of Type 2 Cytokine Production by T1/ST2 Cross-Linking J. Immunol., March 1, 2001; 166(5): 3143 - 3150. [Abstract] [Full Text] [PDF]
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M. J. Townsend, P. G. Fallon, D. J. Matthews, H. E. Jolin, and A. N.J. McKenzie T1/St2-Deficient Mice Demonstrate the Importance of T1/St2 in Developing Primary T Helper Cell Type 2 Responses J. Exp. Med., March 20, 2000; 191(6): 1069 - 1076. [Abstract] [Full Text] [PDF]
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K. Hoshino, S.-i. Kashiwamura, K. Kuribayashi, T. Kodama, T. Tsujimura, K. Nakanishi, T. Matsuyama, K. Takeda, and S. Akira The Absence of Interleukin 1 Receptor-Related T1/St2 Does Not Affect T Helper Cell Type 2 Development and Its Effector Function J. Exp. Med., November 15, 1999; 190(10): 1541 - 1548. [Abstract] [Full Text] [PDF]
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