The role of SHIP in the development and activation of mouse mucosal and connective tissue mast cells.
ABSTRACT Although SHIP is a well-established suppressor of IgE plus Ag-induced degranulation and cytokine production in bone marrow-derived mast cells (BMMCs), little is known about its role in connective tissue (CTMCs) or mucosal (MMCs) mast cells. In this study, we compared SHIP's role in the development as well as the IgE plus Ag and TLR-induced activation of CTMCs, MMCs, and BMMCs and found that SHIP delays the maturation of all three mast cell subsets and, surprisingly, that it is a positive regulator of IgE-induced BMMC survival. We also found that SHIP represses IgE plus Ag-induced degranulation of all three mast cell subsets and that TLR agonists do not trigger their degranulation, whether SHIP is present or not, nor do they enhance IgE plus Ag-induced degranulation. In terms of cytokine production, we found that in MMCs and BMMCs, which are poor producers of TLR-induced cytokines, SHIP is a potent negative regulator of IgE plus Ag-induced IL-6 and TNF-α production. Surprisingly, however, in splenic or peritoneal derived CTMCs, which are poor producers of IgE plus Ag-induced cytokines, SHIP is a potent positive regulator of TLR-induced cytokine production. Lastly, cell signaling and cytokine production studies with and without LY294002, wortmannin, and PI3Kα inhibitor-2, as well as with PI3K p85α(-/-) BMMCs and CTMCs, are consistent with SHIP positively regulating TLR-induced cytokine production via an adaptor-mediated pathway while negatively regulating IgE plus Ag-induced cytokine production by repressing the PI3K pathway.
- SourceAvailable from: jleukbio.org[show abstract] [hide abstract]
ABSTRACT: Mast cells are one of the major effector cells in the pathogenesis of the immediate-type hypersensitivity reaction in a number of non-allergic immune disorders as well as in normal physiological processes. In addition, it has been shown recently that mast cells also play a significant role in a life-saving host response to bacterial reactions. But as much as the immunopathological role of mast cells has been acknowledged, these cells have also aroused much controversy and confusion. By now it is clear that one explanation for the sometimes even contradictory opinions on mast cell function arise from mast cell heterogeneity. This heterogeneity can express itself as differences in histochemical, biochemical, and functional characteristics. In vitro systems provided a powerful tool for the investigation of the basic mechanisms for mast cell development and differentiation and helped to demonstrate that mast cell heterogeneity can be traced back to certain cytokine patterns that are present in different microenvironments. In this context it has also been shown that the growth factors required for human mast cell differentiation are somewhat different than those for rodents. In rodents, the atypical, T cell-dependent mucosal type mast cell can be distinguished from the T cell-independent connective tissue-type mast cell. In humans, the strict classification into mucosal and connective tissue-type mast cells is not possible and the content of mast cell-specific proteases chymase and tryptase is the main criterion for mast cell subtypes in humans. The large quantities of tryptase and chymase that are synthesized by mast cells suggest and emphasize the significance of these proteinases in mast cell function and stimulated investigations about the biological properties of these mast cell-specific proteases. Comparing their biological activities it becomes clear that they share some activities. On the other hand, tryptase seems to participate in proinflammatory mast cell function, whereas chymase seems to be more involved in inflammatory reactions. This review provides a short overview of the discovery, origin, development, and biological significance of mast cells and will then concentrate on mast cell heterogeneity in rodents and humans with respect to the mast cell proteases tryptase and chymase and their function.Journal of Leukocyte Biology 04/1997; 61(3):233-45. · 4.57 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Mast cells (MCs) are found principally in peripheral tissues yet are of bone marrow origin. Recent studies in mice trace the MC lineage from the common myeloid progenitor through the granulocyte-macrophage progenitor in the bone marrow to a committed MC progenitor (MCP). Additionally, at least in the mouse, a bipotent basophil-MC progenitor has been identified in the spleen, suggesting a physiologic role for this organ in MC development. MCPs are especially abundant in the mouse intestine, likely ensuring the capacity for a rapid expansion of MCs in the intestinal epithelium during the effector response to helminth infection and perhaps providing a pool of committed cells capable of redistribution to other tissues. Migration of MCPs to the intestine is constitutive and controlled by alpha chemokine receptor 2 and alpha4beta7 integrins expressed on the MCPs, with the latter integrin interacting with endothelial vascular cell adhesion molecule 1 and mucosal addressin cell adhesion molecule 1. In contrast, normal mouse lung tissue contains few MCPs and MCs, and these cellular reservoirs are not affected by the lack of alpha chemokine receptor 2 or alpha4beta7 integrin. Nonetheless, robust recruitment of MCPs to the lung occurs during experimentally induced allergic pulmonary inflammation and requires alpha4beta7 and alpha4beta1 integrins interacting with vascular cell adhesion molecule 1 but not with mucosal addressin cell adhesion molecule 1. Thus although MCs are present in all organs, the pathways responsible for the trafficking of MCPs from the circulation are organ specific and include both constitutive and inducible systems, ensuring both resident MCs and the potential for incremental recruitment in accord with the requirements of the immune response. These findings in mice await confirmation in human subjects.Journal of Allergy and Clinical Immunology 07/2006; 117(6):1285-91. · 12.05 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: There is an accumulation of evidence to suggest that mast cells may play a key role in gastrointestinal inflammation. We have investigated the numbers and heterogeneity in staining properties of mast cells in biopsies of the duodenum of normal subjects (n = 10), and of normal duodenum from patients with Crohn's disease of the ileum and/or colon (n = 7) or with Helicobacter-associated gastritis of the antrum/corpus (n = 6). In normal donors, two subsets of mast cells, one located in the duodenal mucosa and the other in the submucosa, were clearly distinguished by their morphology and dye-binding properties. Whereas submucosal mast cells stained metachromatically with Toluidine Blue after neutral formalin fixation and emitted a yellow fluorescence after staining with Berberine sulphate, those in the mucosa were invisible using these stains. In patients with gastritis or Crohn's disease, there were marked changes in the numbers of mucosal mast cells compared with control subjects even though the duodenal biopsies were from apparently uninvolved tissue. Gastritis was associated with increased mucosal mast cell numbers (controls: 187 +/- 23 cells mm-2; gastritis: 413 +/- 139 cells mm-2; p = 0.0004), but mean mucosal mast cell counts in the uninvolved duodenum of Crohn's patients were actually decreased (34 +/- 30 cells mm-2, p = 0.0147). The clear differentiation between mucosal and submucosal mast cells on the basis of metachromasia with Toluidine Blue was not seen in biopsies from the patients with gastritis or Crohn's disease. Previous studies which have suggested that there are no distinct mucosal and submucosal mast cell subsets in the human intestine may, therefore, have been affected by the use of tissue from diseased subjects. Heterogeneity in the expression of mast cell tryptase and chymase was seen by immunohistochemistry using specific antibodies, but the relative numbers of mast cell subsets were critically dependent on the methods used. Using a sensitive staining procedure, the majority of mucosal mast cells stained positively for chymase as well as for tryptase, an observation confirmed by immunoelectron microscopy and immunoabsorption studies. Our findings suggest that early stages in intestinal inflammation may be reflected in changes in mast cell numbers and in their staining properties, and call for a reappraisal of mast cell heterogeneity in the human intestinal tract.The Histochemical Journal 11/1997; 29(10):759-73.
The Journal of Immunology
The Role of SHIP in the Development and Activation of
Mouse Mucosal and Connective Tissue Mast Cells
Jens Ruschmann,* Frann Antignano,†Vivian Lam,* Kim Snyder,* Connie Kim,*
Martha Essak,* Angela Zhang,* Ann Hsu-An Lin,* Raghuveer Singh Mali,‡
Reuben Kapur,‡and Gerald Krystal*
Although SHIP is a well-established suppressor of IgE plus Ag-induced degranulation and cytokine production in bone marrow-
we compared SHIP’s role in the development as well as the IgE plus Ag and TLR-induced activation of CTMCs, MMCs, and
BMMCs and found that SHIP delays the maturation of all three mast cell subsets and, surprisingly, that it is a positive regulator of
IgE-induced BMMC survival. We also found that SHIP represses IgE plus Ag-induced degranulation of all three mast cell subsets
and that TLR agonists do not trigger their degranulation, whether SHIP is present or not, nor do they enhance IgE plus Ag-
induced degranulation. In terms of cytokine production, we found that in MMCs and BMMCs, which are poor producers of TLR-
induced cytokines, SHIP is a potent negative regulator of IgE plus Ag-induced IL-6 and TNF-a production. Surprisingly, however,
in splenic or peritoneal derived CTMCs, which are poor producers of IgE plus Ag-induced cytokines, SHIP is a potent positive
regulator of TLR-induced cytokine production. Lastly, cell signaling and cytokine production studies with and without LY294002,
wortmannin, and PI3Ka inhibitor-2, as well as with PI3K p85a2/2BMMCs and CTMCs, are consistent with SHIP positively
regulating TLR-induced cytokine production via an adaptor-mediated pathway while negatively regulating IgE plus Ag-induced
cytokine production by repressing the PI3K pathway.The Journal of Immunology, 2012, 188: 3839–3850.
and granule contents in mice into long-lived connective tissue MCs
(CTMCs) and short-lived mucosal MCs (MMCs) (1, 2). In terms
of distribution, CTMCs are located in sterile tissues such as
the connective tissues of the skin and peritoneum as well as the
submucosa of the gastrointestinal tract and the lung, whereas
MMCs are present in nonsterile tissues, such as the mucosal lin-
ings of the gastrointestinal tract and the lungs. In terms of granule
contents, CTMC granules contain serotonin and the proteoglycan,
heparin (which is stained by safranin), whereas MMCs do not
contain serotonin and possess the proteoglycan, chondrotin sulfate
ast cells (MCs), which play critical roles in both aller-
gic inflammation and host defense against microbial
infections, can be subclassified based on distribution
E, instead of heparin, and so do not stain with safranin (1, 3). As
well, CTMC granules contain higher levels of histamine and
different proteases than that present in MMC granules. Because it
is difficult to get sufficient numbers of these CTMCs and MMCs
for biochemical studies, most of what we know about MCs comes
from experiments with in vitro derived bone marrow MCs
(BMMCs), which are typically generated by culturing mouse bone
marrow (BM) with IL-3. This culturing regimen results, after 4–6
wk, in a large number of uniform mature MCs, as assessed by the
cell surface expression of FcεRI and c-kit. These cells stain with
alcian blue, but not with safranin, and are thought to resemble
immature MMCs (4, 5). Interestingly, though, not only does there
appear to be considerable plasticity between CTMCs and MMCs
(6, 7), but introduction of BMMCs into MC-deficient mice results
in the generation of both CTMCs and MMCs, depending on the
tissues where these BMMCs lodge (7, 8).
MCs play a central role in type I hypersensitivity reactions,
which occur following multivalent allergen-induced cross-linking
of MC-bound IgE, and this mechanism of MC activation has
learned about the intracellular signaling pathways involved (6, 9–
11). However, because MCs are located at the portals between self
and nonself and because of the more recent discovery that they
express pathogen recognition receptors, like TLRs, they are now
thought to be one of the first cells to recognize invading micro-
organisms and trigger an appropriate immune response. In keeping
with this, MC-deficient mice have been shown to be far more sus-
ceptible to septic peritonitis and other bacterial infections than their
MC-reconstituted counterparts or wild-type (WT) mice (12, 13).
negative regulator of FcεR1-mediated BMMC activation (14, 15).
This is attributed to SHIP’s ability to hydrolyze the 59-phosphate of
the PI3K-generated second messenger phosphatidylinositol-3,4,5-
trisphosphate, making SHIP a negative regulator of the PI3K
*Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, British Colum-
bia, Canada V5Z 1L3;†Biomedical Research Centre, University of British Columbia,
Vancouver, British Columbia, Canada V6T 1Z3; and‡Wells Center for Pediatric
Research, Indiana University, Indianapolis, IN 46202
Received for publication November 24, 2010. Accepted for publication February 9,
This work was supported by the Terry Fox Foundation and the Canadian Cancer
Society, with core support from the British Columbia Cancer Foundation and the
British Columbia Cancer Agency. F.A. was supported in this work by Michael Smith
Foundation for Health Research and Natural Sciences & Engineering Research Coun-
Address correspondence and reprint requests to Dr. Gerald Krystal, British Columbia
Cancer Agency, 675 West 10th Avenue, Vancouver, BC V5Z 1L3, Canada. E-mail
The online version of this article contains supplemental material.
Abbreviations used in this article: BM, bone marrow; BMMC, BM-derived mast cell;
CT-like BMMC, BM-derived connective tissue-like MC; CTMC, connective tissue
mast cell; HF, HBSS-modified medium containing 2% FCS; HSA, human serum
albumin; LY, LY294002; MC, mast cell; MMC, mucosal MC; N/S, nonstimulated;
pCTMC, peritoneal CTMC; PGN, peptidoglycan; SCF, stem cell factor; W, wortmannin;
pathway. However, very little is known about its role in CTMCs or
MMCs. We therefore compared SHIP’s role in the maturation,
asinIgE plusAg-inducedandTLR-inducedactivation oftheseMC
subsets, and that is the focus of this study. Our findings are con-
sistent with SHIP’s established role as a negative regulator of the
PI3K pathway. In addition to that, however, our results using PI3K
inhibitors and p85a2/2MCs also suggest that SHIP’s reported
adaptor function(s) may be responsible for SHIP being a positive
regulator of TLR-induced cytokine production.
Materials and Methods
All cytokines and tissue culture media were purchased from StemCell Tech-
from Calbiochem (San Diego, CA), whereas the PI3Ka inhibitor-2 was from
Aldrich (St. Louis, MO). CpG, with the sequence 59-TCCATGACGTTCCT-
GACGTT-39, was synthesized and HPLC purified by Invitrogen (Burlington,
from Sigma-Aldrich. Peptidoglycan (PGN) from Staphylococcus aureus was
human serum albumin (HSA) were from Sigma-Aldrich.
SHIP+/+and SHIP2/2F2mice, on a mixed C57BL/6 3 129Sv background,
and p85a+/+and p85a2/2C57BL/6 mice were used between 6 and 12 wk
of age and housed in the Animal Resource Centre of the British Columbia
Cancer Research Centre under specific pathogen-free conditions and
according to approved and ethical treatment of animal standards of the
University of British Columbia. Animals were euthanized by CO2as-
Generation of different MC subsets
BMMCs were generated, as described previously (16). To generate MMCs,
mouse spleens were cut into small pieces and pressed through a 100-mm
nylon cell strainer, and the resulting single-cell suspension was set up at
1 3 106cells/ml in MMC medium (IMDM, 10% FCS, 10 ng/ml IL-3). After
7 d, nonadherent cells were kept between 3 3 105and 1 3 106cells/ml in
MMC medium. To generate CTMCs, spleen cells were cultured at 1 3 106
cells/ml in CTMC medium (IMDM, 10% FCS, 50 ng/ml stem cell factor
[SCF]) and, after 7 d, nonadherent cells were kept between 3 3 105and
1 3 106cells/ml in CTMC medium. Alternatively, IMDM was injected into
the peritoneal cavity, and 1 3 106nucleated cells/ml were plated in Opti-
MEM I (Invitrogen), 10% FCS, 100 IU/ml penicillin, 100 mg/ml strepto-
mycin, and 4% conditioned medium from murine SCF-secreting Chinese
hamster ovary cells (CHO-KL). After 24 h, nonadherent cells were dis-
carded, and fresh culture medium was added to the adherent cells. After
3 d, nonadherent and adherent cells, detached with trypsin-EDTA, were
resuspended at 3 3 105cells/ml in fresh culture medium. This was re-
peated twice per week. The resulting peritoneal CTMCs (pCTMCs) were
used for experiments at 3–9 wk of culture (17). BM-derived connective
tissue-like MCs (CT-like BMMCs) were also derived, in a fashion similar
to BMMCs (16), but with the addition of 25 ng/ml SCF plus 1 ng/ml IL-4
MC survival assay
For MC survival studies, MCs werewashed, resuspended at 1.5 3 106cells/
ml in IMDM plus 0.1% BSA 6 10 mg/ml SPE-7 IgE, and 100 ml cells
were seeded in a 96-well plate. Viable cell counts were determined by
trypan blue exclusion.
MC degranulation assay
Degranulation assays were carried out, as previously described (18), in
Tyrode’s buffer at 6.25 3 106cells/ml. Cells were seeded at 10 ml/well in
96-well V-bottom plates, and degranulation was triggered with 10 ml 23
concentrated stimulus, prepared in Tyrode’s buffer. After 1 h at 37˚C, cells
were centrifuged for 5 min at 335 3 g and 10 ml cell-free supernatant, and
the 0.5% Triton X-100 lysed cell pellet was analyzed for b-hexosamini-
dase levels (19). Percentage of degranulation was calculated by dividing
the absorbance of the supernatant by the sum of the absorbencies of the
supernatant and cell lysate.
Stimulation of MCs for cytokine production
To stimulate with TLR agonists 6 IgE, MCs were incubated in MC
starvation medium (IMDM, 10% FCS) at 1 3 106cells/ml for 18 h (for
BMMCs and MMCs) or 3 h (CTMCs and pCTMCs) at 37˚C and then
washed and resuspended in MC starvation medium at 1 3 106cells/ml.
The cells (750 ml) were then seeded in 12-well flat-bottom plates. A total
of 750 ml MC starvation medium 6 IgE (for nonstimulated [N/S] samples)
or TLR ligands 6 SPE-7 IgE, in MC starvation medium at twice the in-
dicated final concentration, was added to the MCs for 24 h at 37˚C. To
stimulate with TLR ligands plus IgE plus Ag, MCs were washed and in-
cubated at 1 3 106cells/ml in MC starvation medium plus 0.1 mg/ml SPE-
7 for 18 h (for BMMCs and MMCs) or 3 h (CTMCs and pCTMCs) at 37˚C.
MCs were then washed and resuspended at 1 3 106cells/ml with MC
starvation medium plus 2 ng/ml DNP-HSA, and 750 ml cells were seeded
in 12-well flat-bottom plates. TLR ligands were made up in MC starvation
medium at twice the indicated final concentration, and 750 ml was added to
the MCs. For N/S samples, 750 ml MC starvation medium was added. For
samples containing PI3K inhibitors, it was important to keep the final
DMSO concentration #0.05% because of differential effects of higher
DMSO concentrations on SHIP+/+and SHIP2/2MCs. MCs were stimu-
lated for 24 h at 37˚C. Tissue culture supernatants were assayed for the
concentration of IL-12p40, IL-6, IL-10, TNF-a, IFN-g, and IL-4 by
ELISA, according to the manufacturer’s instructions (BD Biosciences,
Mississauga, ON, Canada).
Stimulation of MCs for SDS-PAGE and Western analysis
CTMCs, starved for 3 h, and MMCs, or BMMCs starved overnight, at 37˚C
in IMDM plus 10% FCS were washed, resuspended in IMDM, and pre-
treated or not for 20 min at 37˚C with LY, W, or PI3K p110a inhibitor 2.
The cells were then treated with LPS, IL-3, or IgE plus Ag for the indi-
cated times, lysed with SDS sample buffer, boiled for 2 min, and loaded
onto 10% polyacrylamide gels, as described previously (20). The following
Abs were used for Western blotting: anti–phospho-IKKa/b (Ser176/180,
catalog 2697), anti–phospho-p38 MAPK (Thr180/Tyr182, catalog 9211),
anti–phospho-JNK (Thr183/Tyr185, catalog 9251), and anti–phospho-
ERK1/2 (Thr202/Tyr204, catalog 9106) all from Cell Signaling Technol-
ogy (Pickering, ON, Canada); anti–phospho-Akt (S473, catalog 44-621G)
from Invitrogen (Burlington, ON, Canada); and anti–Grb-2 (catalog sc-
255) from Santa Cruz Biotechnology (Santa Cruz, CA).
To assess maturity, MCs at 1 3 106cells/ml in HBSS-modified medium
containing 2% FCS (HF) were incubated for 30 min at 4˚C with anti-
CD16/32 (2.4G2) (StemCell Technologies) to block FcRs, and then in-
cubated for 45 min in the dark with FcεR1a-FITC and c-kit allophyco-
cyanin Abs, added at 1:1000 and 1:200, respectively. Then they were
washed with HF and subjected to FACS at 5 3 105cells/ml. To assess
TLR4 and CD14 levels, unconjugated Abs to TLR4 (Santa Cruz Bio-
technology, Santa Cruz, CA) and CD14 (Pharmingen, Mississauga, ON,
Canada) were used in conjunction with anti-rat PE secondary Ab. Data
were collected using a FACSCalibur flow cytometer using CellQuest Pro
software, and data were analyzed using FlowJo software.
Cytospin and granule staining of MCs
MCs (5 3 104cells) in 0.5 ml PBS were centrifuged onto a microscope
slide at 500 rpm for 5 min in a Cytospin 3 centrifuge and stained for 15
min in 0.5% alcian blue/0.3% acetic acid solution, followed by 20 min in
0.1% safranin/0.1% acetic acid. Cells were air dried, and pictures were
taken using an Axioplan 2 imaging microscope.
Analysis of TLR expression by RT-PCR
TotalRNAwaspreparedfrom MCswithTRIzolreagent,and genomicDNA
contaminants were removed using the TURBO DNA-free kit (Ambion),
according to each supplier’s recommendations. To RNA samples obtained
from CTMCs, heparinase 1 (Sigma-Aldrich) was added according to the
supplier’s recommendations. First-strand synthesis was performed using
Moloney murine leukemia virus reverse transcription. The reactions were
performed as per the manufacturer’s instructions, except the reactions were
linearly upscaled to 25 ml. An oligo(dT)18primer was used. PCRs were
performed using the Phusion High-Fidelity DNA Polymerase kit. Reac-
tions were performed in a 25 ml total volume consisting of 1 ml template,
16.5 ml PCR-H2O, 5 ml 53 Phusion HF buffer (containing 7.5 mM
MgCl2), 0.75 ml DMSO, 0.25 ml Phusion High-Fidelity DNA Polymerase
(2 U/ml), 0.5 ml 10 mM forward primer solution, 0.5 ml 10 mM reverse
primer solution, and 0.5 ml dNTP (10 mM each) solution. PCRs with
3840SHIP’S ROLE IN CTMCs, MMCs, AND BMMCs
specific primers for GAPDH were carried out with an annealing temper-
ature of 63˚C and with the oligonucleotide primers 59-TTAGCCCCCC-
TGGCCAAGG-39 and 59-CTTACTCCTTGGAGGCCATG-39, amplifying
a 541-bp fragment. PCRs for mouse TLRs were performed with primers
from Invivogen (version 07H09-SV) and carried out with an annealing
temperature of 58˚C. PCR amplifications were performed at 98˚C for 75 s
(initial denaturing step), followed by 35 cycles at 98˚C for 15 s, 58˚C or
63˚C for 20 s, and 72˚C for 30 s, followed by a final step at 72˚C for 10
min. All reactions were run in a GeneAmp PCR System 9700 thermo
SHIP is a negative regulator of MC maturation
To compare SHIP’s role in the maturation and proliferation of
CTMCs and MMCs, SHIP+/+and SHIP2/2mouse spleens were
cultured in CTMC medium (containing SCF) or MMC medium
(containing IL-3), respectively, as described previously (21).
SHIP+/+and SHIP2/2BM cultures were also initiated for com-
parison (16). As shown in Fig. 1A, SHIP2/2MCs matured faster
than WT MCs, as assessed by cell surface expression of c-kit and
FcεR1a, regardless of the subtype examined, and this was most
pronounced with CTMCs and BMMCs. Whereas the SHIP2/2
MMC maturation rate was only slightly faster than WT MMCs, it
was consistently observed in five independent experiments. Of
note, both SHIP+/+and SHIP2/2CTMCs matured faster than
MMCs or BMMCs, with .80% of SHIP+/+and .95% of SHIP2/2
spleen-derived cells becoming mature CTMCs by 3 wk of culture.
By week 5, .90% of the cells in all cultures were mature MCs.
Also of note, mature CTMCs were substantially smaller than
MMCs or BMMCs, as assessed by flow cytometry, and the absence
of SHIP resulted in slightly larger MMCs and BMMCs (Supple-
mental Fig. 1A).
Interestingly, SHIP’s influence on cell proliferation depended
on the MC subtype (Fig. 1B). Whereas SHIP2/2CTMCs prolif-
erated substantially faster than WT CTMCs, there was no sig-
nificant difference in the growth of SHIP+/+and SHIP2/2MMCs,
and SHIP2/2BMMCs actually proliferated far slower than WT
BMMCs. Of note, staining of the different SHIP+/+and SHIP2/2
mature MC subtypes with safranin, which stains heparin red (1, 3),
and with alcian blue, which stains chondroitin sulfate blue (1, 3),
confirmed that the derived CTMCs were indeed CTMCs, that the
MMCs and BMMCs were mucosal-like MCs. These staining
studies also showed that the absence of SHIP did not prevent the
appearance of these granular components (Supplemental Fig. 1B).
We also compared the cell surface expression of c-kit and FcεR1a
on week 5 cultures of SHIP+/+and SHIP2/2CTMCs, MMCs, and
BMMCs by flow cytometry and found they all expressed similar
levels of these two maturation markers (Supplemental Fig. 1C).
SHIP enhances IgE-induced BMMC survival
Because IgE, in the absence of Ag, has been shown to enhance
BMMC survival [with some IgEs, e.g., SPE-7, being much more
potent (cytokinergic) than others (22–24)], we asked whether IgE
was also capable of enhancing the survival of MMCs and CTMCs
when deprived of their growth factors. As shown in the left panel
of Fig. 1C, IgE substantially enhanced the survival of CTMCs,
with no difference being observed between SHIP+/+and SHIP2/2
MCs. The survival of SHIP+/+and SHIP2/2MMCs, however, was
only slightly increased with IgE (middle panel, Fig. 1C), although
maturation, but positively regulates IgE-in-
duced BMMC survival. SHIP+/+and SHIP2/2
CTMC, MMC, and BMMC cultures were
initiated, as described in Materials and
Methods, and (A) their maturity was assessed
by surface expression of c-kit and FcεR1a.
Cells that were doubly positive were consid-
ered mature. (B) Proliferation was assessed by
counting trypan blue excluding cells. Shown
is the absolute cell number divided by the
starting number of cells at week 0. (A and B)
SHIP+/+MCs = : with solid lines; SHIP2/2
MCs = Δ with dashed lines. (C) SHIP+/+and
SHIP2/2MC subsets were incubated in 0.1%
BSA medium (SHIP+/+= :; SHIP2/2= Δ,
solid lines) or +5 mg/ml SPE-7 IgE (SHIP+/+=
▼; SHIP2/2= ,, dashed lines), and viable
cells were counted. Values are the mean 6
SD from two independent experiments in
triplicate. Significant differences (p , 0.05)
are indicated (*).
SHIP negatively regulates MC
The Journal of Immunology3841
this was mainly because MMCs were substantially more capable
of surviving in the absence of IgE than CTMCs. Once again, the
presence of SHIP made no difference to survival, with or without
IgE. Lastly, as shown in the right panel of Fig. 1C, SHIP+/+and
SHIP2/2BMMCs survived equally well, and the best of the three
MC subtypes, in the absence of IgE. Interestingly, however,
whereas IgE increased the survival of both SHIP+/+and SHIP2/2
BMMCs over the 4 d of the study, it was far more effective at
promoting the proliferation/survival of SHIP+/+BMMCs. Thus,
surprisingly, SHIP appears to be a positive regulator of BMMC
proliferation/survival in the presence of a highly cytokinergic IgE.
SHIP is a negative regulator of CTMC, MMC, and BMMC
To explore SHIP’s role following the activation of mature CTMCs
and MMCs, we first asked whether SHIP, which is an established
negative regulator of IgE plus Ag-induced BMMC degranulation
(18), also negatively regulated CTMC and MMC degranulation.
Specifically, we compared the ability of SHIP+/+and SHIP2/2
CTMCs, MMCs, and BMMCs to degranulate in response to IgE
plus Ag by preloading the MCs with 0.1 mg/ml SPE-7 IgE and
then stimulating with increasing concentrations of DNP-HSA.
As shown in Fig. 2A, all subsets of SHIP2/2MCs degranulated
significantly more than SHIP+/+MCs at all Ag concentrations
tested, establishing that SHIP negatively regulates IgE plus Ag-
induced MMC and CTMC degranulation. However, the SHIP2/2
BMMCs and MMCs were more sensitive to IgE plus Ag-induced
degranulation than SHIP2/2CTMCs because they degranulated to
a significant extent with as little as 1 ng/ml Ag.
TLR agonists neither trigger nor synergize with IgE plus Ag in
triggering SHIP+/+or SHIP2/2CTMC, MMC, or BMMC
Because MCs are among the first cells to recognize invading
microorganisms, and they do so, in large part, via their TLRs, we
asked whether TLR agonists triggered degranulation of any of the
three MC subsets and, if so, whether SHIP influenced the extent
of degranulation. Specifically, SHIP+/+and SHIP2/2MMCs,
CTMCs, and BMMCs were stimulated with concentrations of
TLR agonists that gave maximal cytokine production from
BMMCs in preliminary studies, that is, 100 ng/ml Escherichia coli
LPS, 50 mg/ml dsRNA, 0.3 mM phosphorothioate-modified CpG-
containing oligodeoxynucleotide (CpG), and 10 mg/ml PGN.
However, there was no degranulation in any MC subset with these
TLR agonists whether SHIP was present or not (Fig. 2B). This is
consistent with other reports showing TLR agonists do not induce
BMMC degranulation (25, 26), with the exception of PGN, which
has been reported to induce BMMC degranulation under certain
Because synergistic interactions between TLR agonists and IgE
plus Ag have been reported for cytokine production from BMMCs
(26, 28, 29), we next asked whether the same TLR agonists could
augment IgE plus Ag-induced degranulation, that is, SHIP+/+or
SHIP2/2MMCs, CTMCs, and BMMCs were preloaded with 0.1
mg/ml SPE-7 IgE and then stimulated with 2 ng/ml Ag in the
presence or absence of TLR ligands for 1 h. As shown in Fig. 2C,
none of the TLR agonists increased degranulation. These studies
were repeated with the SHIP2/2MMCs and BMMCs at a lower Ag
concentration (0.2 ng/ml) because these cells maximally degranu-
lated with 2 ng/ml Ag. However, the addition of TLR ligands once
again did not increase degranulation (Fig. 2D), confirming that
TLR stimulation did not affect IgE plus Ag-induced degranulation.
Because we showed in previous studies that SHIP2/2BMMCs,
unlike WT BMMCs, degranulated in the presence of SCF (30) or
following addition of highly cytokinergic IgEs [without subse-
quent Ag-induced cross-linking (18)], we asked whether this held
true for SHIP2/2MMCs and CTMCs as well. As shown in Fig.
2E, SHIP2/2MMCs did indeed degranulate to some extent in the
presence of SPE-7 IgE or with SCF, but SHIP2/2CTMCs did not,
perhaps reflecting their lower responsiveness to IgE plus Ag-
SHIP positively regulates TLR-induced, but negatively
regulates IgE plus Ag-induced cytokine production from MCs
Activated MCs not only degranulate, releasing preformed medi-
ators of inflammation, but also secrete de novo synthesized
mediators, and this release can occur in the absence of degranu-
lation (31, 32). Because TLR stimulation has been shown to be
a major trigger of this de novo mediator release from BMMCs (13,
33), we compared the ability of SHIP+/+and SHIP2/2CTMCs,
MMCs, and BMMCs to secrete the cytokines IL-6, TNF-a, IL-12,
and IL-10 in response to dsRNA, which binds TLR3 and acts
through a MyD88-independent pathway; CpG, which binds TLR9
and signals via the MyD88-dependent pathway; and LPS, which
binds TLR4 and stimulates both MyD88-dependent and inde-
pendent pathways (34). As shown in Fig. 3, when stimulated with
TLR agonists alone, WT CTMCs were by far the most robust
producers of IL-6, TNF-a, IL-12, and IL-10. In marked contrast,
SHIP2/2CTMCs produced little or no cytokines in response to
these TLR agonists. These results suggest, quite surprisingly, that
SHIP is a potent positive regulator of TLR-induced cytokine
production from CTMCs. Both SHIP+/+and SHIP2/2MMCs, in
contrast, produced little to no cytokines in response to TLR
stimulation. Lastly, BMMCs, like MMCs, were poor responders,
but WT BMMCs were slightly better than their SHIP2/2coun-
terparts at producing IL-6, again suggesting SHIP is a positive
regulator of TLR-induced cytokine production, but that MMCs
and BMMCs are far less responsive to TLR ligands than CTMCs.
Consistent with this, TNF-a, IL-12, and IL-10 were not detectable
from TLR-stimulated SHIP+/+or SHIP2/2MMCs or BMMCs.
When MCs were stimulated for 24 h with TLR ligands in the
presence of 0.1 mg/ml SPE-7 IgE, there was no change in the level
of cytokine production from either SHIP+/+or SHIP2/2CTMCs
(compare the top panels of Fig. 4 with those of Fig. 3). Also, IgE
alone (the N/S columns) was incapable of stimulating cytokine
secretion from WT or SHIP2/2CTMCs. Interestingly, however,
SHIP2/2MMCs and SHIP2/2BMMCs now produced higher
levels of IL-6 and TNF-a than their WT counterparts (compare
middle and bottom panels of Fig. 4 with those of Fig. 3), and IgE
alone triggered a substantial amount of IL-6 and TNF-a from
SHIP2/2MMCs and BMMCs. However, IgE plus TLR ligands,
like TLR ligands alone, were incapable of triggering the secretion
of detectable levels of IL-12 or IL-10 from these cells. These
results suggest that SHIP is a negative regulator of IgE-induced
cytokine secretion from MMCs and BMMCs, consistent with our
previous studies showing that IgE stimulates more IL-6 from
SHIP2/2than from WT BMMCs (35).
When the different MC subsets were stimulated with TLR ago-
nists in the presence of IgE plus Ag, there was a slight increase in
cytokine production from WT CTMCs, but still no detectable pro-
duction of cytokines from SHIP2/2CTMCs (top panels of Fig. 5).
Interestingly, of the TLR ligands tested, only CpG was capable of
Figs.3–5).Importantly,therewas adramaticincreaseinthe levelsof
IL-6 and TNF-a produced by SHIP2/2MMCs and BMMCs, even
without TLR ligand stimulation (N/S indicates IgE plus Ag without
TLR stimulation inFig. 5),and thelevels increasedwiththeaddition
3842SHIP’S ROLE IN CTMCs, MMCs, AND BMMCs
of TLR agonists. However, IgE plus Ag 6 TLR ligands stimulated
far less IL-6 and TNF-a from WT MMCs and WT BMMCs, con-
sistent with SHIP being a potent negative regulator of IgE plus Ag-
induced cytokine production. Interestingly, however, the absence of
SHIP did not increase IL-10 or IL-12 secretion to detectable levels.
To confirm the unexpected finding that SHIP was a potent
positiveregulator of TLR-induced cytokines from CTMCs, we also
derived CTMCs from SHIP+/+and SHIP2/2peritoneal cavity cells
(pCTMCs), according to Malbec et al. (17), and found, as shown
in Fig. 6A, that LPS once again triggered far more IL-6 and TNF-
a from these SHIP+/+than SHIP2/2CTMCs.
SHIP influences TLR expression in MCs
To gain some insight into why TLR-stimulated WT CTMCs se-
creted far higher cytokine levels than either SHIP2/2CTMCs or
SHIP+/+and SHIP2/2MMCs or BMMCs, we first examined the
expression levels of their TLRs. RT-PCR analysis of the SHIP+/+
and SHIP2/2CTMCs, MMCs, and BMMCs revealed that the
levels of TLR3 and TLR9 in SHIP2/2CTMCs were actually
higher than in their WT counterparts, whereas TLR4 levels were
about the same in the presence or absence of SHIP (Fig. 6B).
Because we found TLR2 expression was also far higher in SHIP2/2
than SHIP+/+CTMCs (Fig. 6B, top left panel), we compared
cytokine production following PGN (a TLR2 agonist) stimulation
of these cells and found that, even though WT CTMCs expressed
far lower TLR2 levels, they secreted substantially higher levels of
IL-6, TNF-a, IL-12, and IL-10 (Fig. 6C). Thus, because the
SHIP+/+CTMCs produced substantially higher levels of IL-6,
TNF-a, IL-12, and IL-10, rather than lower levels, when TLR2,
3, 4, and 9 were stimulated, it was unlikely that this could be
CTMCs, MMCs, and BMMCs were preloaded with 0.1 mg/ml IgE for 18 h at 37˚C and exposed to the indicated concentration of Ag for 1 h at 37˚C, and
50 mg/ml dsRNA, 0.3 mM CpG, or 10 mg/ml PGN for 1 h, and degranulation was assessed. (C and D) MCs were preloaded with IgE, as in (A), and then either
N/S or treated with TLR ligands [same concentrations as in (B)] plus 2 ng/ml Ag (B) or 0.2 ng/ml Ag (C) for 1 h. (E) SHIP+/+(black bars) and SHIP2/2(white
bars) BMMCs, MMCs, and CTMCs were treated with 10 mg/ml SPE-7 IgE or 400 ng/ml SCF for 1 h at 37˚C, and degranulation was assessed. As well, as
a positivecontrol,SHIP+/+(blackbars)and SHIP2/2(whitebars)CTMCswere preloadedwith 0.1mg/mlSPE-7IgEfor18 h at37˚Candexposedto 20 ng/ml
(A–C) or one experiment in triplicate (D) or duplicate (E). Similar results were obtained in at least two independent experiments.
IgE plus Ag-induced degranulation of CTMCs, MMCs, and BMMCs is repressed by SHIP. (A) SHIP+/+(black bars) and SHIP2/2(white bars)
The Journal of Immunology3843
explained by differences in TLR expression. In contrast, the un-
detectable levels of TLR3 and TLR9 observed with SHIP+/+and
SHIP2/2MMCs and BMMCs were consistent with the relatively
low response levels of these cells to dsRNA and CpG, respec-
tively. The similar TLR expression pattern seen with MMCs and
BMMCs reinforces the notion that these two MC subsets are
Because mRNA levels do not necessarily reflect protein levels
and certainly not cell surface protein levels, we then assessed the
cell surface levels of TLR4 and its coreceptor, CD14, on the
different MC subsets. As shown in Supplemental Fig. 2, the levels
were similar on all MC subsets. Thus, the different responses of the
MC subsets to LPS were not due to different cell surface receptor
IgE plus Ag or LPS stimulation of SHIP2/2MCs triggers
higher PI3K pathway activity than in WT MCs
To gain some insight into why SHIP was a positive regulator of
TLR-induced cytokine secretion but a negative regulator of IgE
plus Ag-induced cytokine secretion, we carried out signaling
studies with SHIP+/+and SHIP2/2BMMCs. This was done not
only because it was extremely difficult to obtain reproducible cell
signaling results with CTMCs, but also to ensure that the same
signaling pathways were available (i.e., same cellular context) to
respond to LPS (which, as shown in the bottom panel of Fig. 3,
triggers more IL-6 from WT BMMCs) and to IgE plus Ag (which,
as shown in the N/S lanes of the bottom panel of Fig. 5, triggers
more IL-6 from SHIP2/2BMMCs). As shown in Fig. 7A, acti-
vation of the PI3K pathway (i.e., phosphorylation of Akt) by either
IgE plus Ag (left panel) or by LPS (right panel) was consistently
higher in SHIP2/2than WT BMMCs. This suggested that the
PI3K pathway was a positive regulator of IgE plus Ag-induced
cytokines, consistent with earlier studies with BMMCs (11, 30,
36), and perhaps a negative regulator of TLR-induced cytokine
secretion. Because the JNK and p38 pathways have been impli-
cated in previous MC studies involving TLR-induced cytokines
(26, 37, 38), we examined the activation of these pathways as
well. Interestingly, whereas the phosphorylation of p38 may have
been slightly more prolonged in SHIP2/2BMMCs (Fig. 7A), we
were unable to detect any phospho-JNK in response to IgE plus
Ag or LPS stimulation (data not shown). An examination of the
phosphorylation of the third MAPK, ERK, suggested no differ-
ence with or without SHIP in response to IgE plus Ag, but perhaps
more prolonged phosphorylation in the absence of SHIP following
LPS stimulation (Fig. 7A). Because the NF-kB pathway has also
been shown to play an important positive role in cytokine pro-
duction from MCs (35), we also assessed the activation of its
upstream activator, IKK, and found a similar IgE plus Ag-induced
phosphorylation/activation of this Ik kinase in the presence and
absence of SHIP (Fig. 7A), but could not detect its phosphoryla-
tion in response to LPS (data not shown).
Reducing PI3K activity markedly reduces IgE plus Ag-induced,
but does not increase LPS-induced cytokine production from
Given that SHIP is a negative regulator of the PI3K pathway (39),
our results to this point suggested that the PI3K pathway was
a positive regulator of IgE plus Ag-induced, but a negative regu-
lator of TLR-induced cytokine production from MCs. However,
because SHIP can also act as an adaptor (40–43), we wanted to
rule in or out SHIP adaptor effects on cytokine production. To test
this, we first used the PI3K inhibitors LY and W and PI3Ka
inhibitor-2 (because preliminary studies with different p110
isoform-specific inhibitors established that p110a inhibitors were
the most potent at inhibiting IgE plus Ag-induced IL-6 production
[data not shown]). We employed three different PI3K inhibitors
rather than just one because all PI3K inhibitors possess some off-
target effects. However, because these off-target effects tend to
differ from one inhibitor to another (44–46), we felt we could be
more confident that they were exerting their effects via inhibition
CTMCs secrete the highest levels
of cytokines. SHIP+/+(black bars)
and SHIP2/2(white bars) CTMCs,
MMCs, and BMMCs were incubated
for 24 h with buffer alone (N/S), 100
ng/ml LPS, 50 mg/ml dsRNA, or 0.3
mM CpG, and cell-free supernatants
were assessed for IL-6, TNF-a, IL-
12, and IL-10 levels by ELISA. Val-
ues shown are the mean 6 SD from
one representative experiment, mea-
sured in duplicate. Similar results
were obtained in three independent
3844 SHIP’S ROLE IN CTMCs, MMCs, AND BMMCs
of PI3K if they all elicited the same biological response. As shown
in Fig. 7B, we found that low concentrations of these three PI3K
inhibitors consistently reduced IgE plus Ag-induced pAkt in
SHIP+/+(left panel) and LPS-induced pAkt in SHIP2/2(right
panel) BMMCs, but did not consistently reduce pAkt levels in IgE
plus Ag-stimulated SHIP2/2or LPS-induced SHIP+/+BMMCs,
unless we went to higher concentrations of inhibitors (data not
shown). We then tested these inhibitors on IgE plus Ag-induced
and LPS-induced IL-6 production from SHIP+/+and SHIP2/2
BMMCs and CTMCs, respectively. Of note, LY, W, and PI3Ka
inhibitor-2, at the concentrations used, had no deleterious effects
on cell viability in these cells (data not shown). As shown in the
left panel of Fig. 7C, these three inhibitors inhibited IgE plus Ag-
induced IL-6 production from both SHIP+/+and SHIP2/2BMMCs
(and MMCs; data not shown), and these findings were consistent
with the PI3K pathway being a positive regulator of IgE plus Ag-
induced cytokine production from MCs. However, looking at their
effects on LPS-induced cytokine production, none of the inhibitors
increased IL-6 production above control, LPS-induced levels, and
LY significantly reduced them (Fig. 7C, right panel). Importantly,
we tested LY, W, and PI3Ka inhibitor-2 at various concentrations
and for various times (from 3 to 24 h) for their effects on cytokine
production and found that only W, at very high concentrations
(above 100 nM, where nontarget effects are observed), stimulated
IL-6 productionsignificantlyabove controllevels(data notshown).
Importantly, significant stimulation above control IL-6 levels with
BMMCs. Because an increase in IL-6 production with low levels of
PI3K inhibition would be expected, especially in SHIP2/2BMMCs,
if the PI3K pathway was negatively regulating LPS-induced IL-6
production, we therefore tentatively concluded that SHIP positively
regulates LPS-induced cytokine production from MCs primarily via
an adaptor-mediated pathway.
To confirm this, we made numerous attempts to express WTand
phosphatase-dead forms of SHIP in SHIP2/2CTMCs. However,
293T producer cell lines, we were unsuccessful in achieving de-
tectable expression, using either lipofectamine or retroviral infec-
tion, in mature SHIP2/2CTMCs, BMMCs, or BM progenitors.
While engaged in these studies, Ma et al. (47) reported that IL-3–
stimulated BMMCs that were deficient in PI3K p85a were far less
capable of activating Akt than WT BMMCs. We therefore investi-
gated whether Akt was also less activated in response to LPS in
these p85a2/2BMMCs. As shown in Fig. 8A, this was indeed the
its role as an adaptor using a genetic approach rather than via small
molecule inhibitors, with their associated off-target effects. If SHIP
would secrete more IL-6 than WT cells. As shown in Fig. 8B,
secreted substantially more IL-6. This is consistent with the PI3K
pathwaybeinga positive regulatorofLPS-inducedIL-6 production,
but that SHIP’s adaptor function(s) overrides its negative enzymatic
effects on the PI3K pathway.
Although BMMCs have been used extensively as a surrogate for
in vivo derived MCs, they are phenotypically distinct from either
TLR ligands to trigger TNF-a and
MMCs and BMMCs, but not from
CTMCs. SHIP+/+(black bars) and
MMCs, and BMMCs were treated for
24 h with 0.1 mg/ml SPE-7 IgE alone
(N/S) or plus 100 ng/ml LPS, plus 50
mg/ml dsRNA, or plus 0.3 mM CpG,
assessed for IL-6, TNF-a, IL-12, and
IL-10 levels by ELISA. Values shown
are the mean 6 SD from one repre-
sentative experiment, measured in
duplicate. Similar results were ob-
tained in two independent experi-
IgE synergizes with
The Journal of Immunology3845
MMCs or CTMCs (48), and we have therefore compared, in this
work, SHIP’s role in the development and activation of CTMCs,
MMCs, and BMMCs. For the majority of our studies, CTMCs
and MMCs were derived from SHIP+/+and SHIP2/2spleen cells
(21), because this resulted in robust, homogeneous populations
of safranin-positive CTMCs and safranin-negative/alcian blue-
positive MMCs. However, we also derived pCTMCs from
SHIP+/+and SHIP2/2peritoneal cells, as described by Malbec
et al. (17), and obtained similar results to those obtained with
spleen-derived CTMCs, both in degranulation (data not shown)
and cytokine production studies. However, our degranulation
results differed from those of Malbec et al. (17), who found that
pCTMCs degranulated more robustly than BMMCs and that the
absence of SHIP did not increase their degranulation. Of note,
whereas we found that spleen cells yielded far more CTMCs upon
in vitro culturing than BM cells, it was the cytokine mixture used
during in vitro expansion that determined the type of mature MC
In terms of SHIP’s role in the maturation, proliferation, and
survival of the three MC subsets, they all matured faster in the
absence of SHIP, in keeping with our previous results with
BMMCs (14), but major differences were observed in prolifera-
tion, with the absence of SHIP enhancing the proliferation of
CTMCs, reducing that of BMMCs and having no effect on MMCs.
We also found differences in IgE-induced survival, with IgE
prolonging the survival of CTMCs and MMCs, but the presence of
SHIP making no difference to this survival. The presence of SHIP
in BMMCs, however, actually promoted their proliferation,
whereas in its absence the BMMCs simply survived. This differ-
ence could be related to the far more rapid maturation of the
SHIP2/2BMMCs, which could explain their subsequent reduced
proliferation rate and, perhaps, reduced proliferation potential
with IgE. However, an important caveat to keep in mind when
comparing the proliferation and/or survival of SHIP+/+and SHIP2/2
BM cells is that SHIP2/2C57BL/6 mice rapidly become sys-
temically inflamed, and this might affect hematopoietic precursor
frequencies and cytokine exposure in vivo. Whereas we used
young mice to try and keep these effects to a minimum, we cannot
rule out that these in vivo conditions could affect the cell types
that grow out in vitro.
Examining SHIP’s role in degranulation, we found that SHIP is
a potent negative regulator of IgE plus Ag-induced degranulation
of CTMCs and MMCs, consistent with our earlier studies with
BMMCs (18). Importantly, TLR ligands alone did not induce
degranulation, in keeping with previous reports (25, 26), nor did
they enhance IgE plus Ag-induced degranulation from any MCs,
even those lacking SHIP. This contrasts with one report claiming
that PGN induces the degranulation of BMMCs (27), but this may
be because they derived their BMMCs with 10% PWM-stimulated
spleen cell-conditioned medium rather than IL-3 and/or because
they derived their BMMCs from C3H/HeN rather than C57BL/6
In contrast to our degranulation results, we found substantial
differences in cytokine production between CTMCs and MMCs/
BMMCs. For example, we found much higher TLR-induced cy-
tokine production from WT CTMCs than from WT BMMCs,
consistent with a recent report showing higher cytokine production
from TLR2-stimulated pCTMCs than from BMMCs (49). Also,
although the responses were weak, we found that WT BMMCs
produced higher levels of IL-6 in response to LPS and dsRNA
cally synergizes with TLR ligands to
trigger TNF-a and IL-6 production
from SHIP2/2MMCs and BMMCs,
but not from WTor SHIP2/2CTMCs.
SHIP+/+(black bars) and SHIP2/2
(white bars) CTMCs, MMCs, and
BMMCs were treated for 24 h with 0.1
mg/ml SPE-7 IgE plus 1 ng/ml Ag
alone (N/S) or plus 100 ng/ml LPS,
plus 50 mg/ml dsRNA, or plus 0.3 mM
CpG, and cell-free supernatants were
assessed for IL-6, TNF-a, IL-12, and
IL-10 levels by ELISA. Values shown
are the means 6 SD from one repre-
sentative experiment, measured in du-
plicate. Similar results were obtained
in three independent experiments.
IgE plus Ag dramati-
3846 SHIP’S ROLE IN CTMCs, MMCs, AND BMMCs
than SHIP2/2BMMCs, suggesting that SHIP is a positive regu-
lator of TLR stimulation in BMMCs as well as in CTMCs (Fig. 3).
In contrast, IgE plus Ag stimulated far higher cytokine production
from SHIP2/2MMCs/BMMCs than SHIP2/2CTMCs, and this
low IgE plus Ag-induced cytokine production from SHIP2/2
CTMCs, relative to SHIP2/2BMMCs, cannot be explained by
a simple lack of response to IgE plus Ag because SHIP2/2
CTMCs degranulate in response to this stimulus (Fig. 2A). Con-
sistent with our results using IgE+/2TLR ligands (Fig. 4), highly
cytokinergic IgE alone, in conjunction with TLR2 or TLR4
stimulation, has been reported to increase inflammatory cytokines
from BMMCs (50). As well, TLR2 and TLR4 agonists have been
reported to synergize with IgE plus Ag in triggering cytokine
secretion from BMMCs (26, 28, 51). In contrast, however, TLR2
activation has also been reported to reduce both IgE plus Ag-
induced BMMC degranulation and TNF-a production (52).
To explorewhy WT MMCs and BMMCs responded so poorly to
so weakly, in the absence of SHIP, to IgE plus Ag, we first
compared TLR mRNA levels by RT-PCR. We found the expression
4, 6) than in WT CTMCs (TLR1, 2, 4, 6, 8, 9) and that this is
similar, in general, to that reported previously (25, 32, 53). In-
terestingly, we found that the absence of SHIP resulted in higher
expression of TLR1, 2, 3, 7, 8, and 9 in CTMCs. One caveat, of
course, with RT-PCR studies is that they may not reflect cell
surface protein levels, and this certainly appeared to be the case
for TLR9 because RT-PCR studies suggested no TLR9 expression
in BMMCs or MMCs, whereas we found that CpG significantly
increased IgE plus Ag-induced IL-6 and TNF-a production from
these MCs (Fig. 5). Because of this, we honed in on LPS-
stimulated events because RT-PCR studies suggested similar
TLR4 expression levels and subsequent flow cytometric analyses
confirmed equal cell surface expression of TLR4 and its co-
receptor, CD14, on SHIP+/+and SHIP2/2MCs (Supplemental Fig.
Because our subsequent cell signaling studies suggested that the
PI3K pathway was more activated in the absence of SHIP when
stimulated with either LPS or IgE plus Ag, we examined the effects
of PI3K inhibition and found that LY, W, and PI3Ka inhibitor-2
reduced IgE plus Ag-induced IL-6 production from SHIP+/+and
SHIP2/2BMMCs/MMCs, consistent with the PI3K pathway be-
ing a positive regulator of IgE plus Ag-induced cytokine pro-
duction. However, we found that even though SHIP was a positive
regulator of LPS-induced IL-6 production, PI3K inhibition by
three different PI3K inhibitors did not increase the levels of this
cytokine. The only exception to this finding was when very high
levels of W were employed and, even then, elevated IL-6 was only
seen with WT CTMCs. To avoid the off-target effects of PI3K
inhibitors, we also compared WTand PI3K p85a2/2BMMCs and
CTMCs and obtained results consistent with our small molecule
CTMCs also secrete substantially more IL-6 and
TNF-a than SHIP2/2cells in response to LPS.
SHIP+/+(black bars) and SHIP2/2(white bars)
BMMCs and MMCs were stimulated with 0.1 mg/
ml SPE-7 for 18 h at 37˚C, washed, and resus-
pended in MC starvation medium (N/S) 6 2 ng/ml
DNP-HSA. SHIP+/+(black bars) and SHIP2/2
(white bars) pCTMCs were stimulated 6 100 ng/
ml LPS for 24 h at 37˚C. Cell-free supernatants
were assessed for IL-6 (left panel) and TNF-a
(right panel) levels by ELISA. Values shown are
the mean 6 SD from one representative experi-
ment, measured in duplicate. Similar results were
obtained in three independent experiments. (B)
TLR expression of SHIP+/+and SHIP2/2CTMCs,
MMCs, and BMMCs. For CTMCs, 200 ng cDNA
was used for RT-PCR, whereas for MMCs and
BMMCs 160 ng cDNA was used. As a loading
control, 100 ng cDNA was used as template in
a PCR with primers specific for GAPDH. Geno-
mic DNA (10 ng) was used as a positive control.
(C) SHIP+/+(black bars) and SHIP2/2(white bars)
CTMCs were incubated for 24 h with buffer alone
(N/S) or with 10 mg/ml PGN, and cell-free
supernatants were assessed for IL-6, TNF-a, IL-
12, and IL-10 levels by ELISA. Values shown are
the mean 6 SD from one representative experi-
ment, measured in duplicate. Similar results were
obtained in three independent experiments.
(A) WT peritoneal cell-derived
The Journal of Immunology 3847
PI3K inhibitor results, that is, that SHIP may be acting as a posi-
tive regulator via its adaptor function(s). Our results are consistent
with very nice studies showing that W does not inhibit LPS-
induced IL-6 production from WT BMMCs (54) and that SHIP
promotes TLR2- and TLR4-induced cytokine production from
murine macrophages (55). However, in the latter study, Keck et al.
(55) concluded, based on results obtained with high doses of W,
that PI3K inhibition increased LPS-induced cytokine production
in macrophages. Our results also contrast with those of Luyendyk
et al. (56), who carried out some very elegant genetic studies
showing that the PI3K/Akt pathway negatively regulates LPS-
induced TNF-a and IL-6 production in macrophages. However,
other studies in macrophages suggest that PI3K plays a positive
role during LPS-induced cytokine production. For example, Kuo
cytokine production from LPS-stimulated
MCs via an adaptor pathway. (A) SHIP+/+
and SHIP2/2BMMCs were stimulated with
IgE plus Ag or LPS for the indicated times,
and SDS-lysed total cell lysates were sub-
jected to Western analysis, as indicated.
Short and long refer to exposure times of
the x-ray film. (B) SHIP+/+and SHIP2/2
BMMCs were pretreated for 30 min with or
without the indicated concentration of LY,
W, or PI3Ka inhibitor-2, and then stimu-
lated with IgE plus Ag (left panel) or LPS
(right panel), and SDS-lysed total cell
lysates were subjected to Western analysis,
as indicated. The numbers under the IgE
plus Ag- and LPS-induced pAkt blots in (B)
refer to the levels of pAkt, relative to Grb2
(the loading control), determined via den-
sitometry. Similar results were obtained in
two other experiments. (C) SHIP+/+(black
bars) and SHIP2/2(white bars) BMMCs
were stimulated with IgE plus Ag (left
panel), or SHIP+/+and SHIP2/2pCTMCs
were stimulated with LPS (right panel) for
24 h with the indicated concentrations of
LY, W, or PI3Ka inhibitor-2, and cell-free
supernatants were assessed for IL-6 levels
by ELISA. Values shown are the means 6
SD from one representative experiment,
measured in duplicate. *p , 0.05 compared
with no inhibitor. Similar results were ob-
tained in three independent experiments for
(A and B) and five independent experiments
SHIP positively regulates
overnight in serum- and cytokine-free media and stimulated with IL-3 (10 ng/ml) or LPS (10 ng/ml) for 15 and 30 min. Equal amounts of protein lysates
were subjected to Western blot analysis using an anti–phospho-Akt or total Akt Ab. Similar results were obtained in two independent experiments. (B)
p85a+/+(black bars) and p85a2/2(white bars) CT-like BMMCs and CTMCs were stimulated with or without 100 ng/ml LPS for 24 h (left panel), or p85a+/+
(black bars) and p85a2/2(white bars) BMMCs were stimulated with or without 100 ng/ml LPS or 5 mg/ml IgE (as a positive control) for 24 h (right panel),
and cell-free supernatants were assessed for IL-6 levels by ELISA. Values shown are the means 6 SD from one representative experiment, measured in
duplicate. Similar results were obtained in two independent experiments with CT-like BMMCs, two with CTMCs and four with BMMCs.
PI3K p85a2/2BMMCs and CTMCs secrete less IL-6 than their WT counterparts. (A) BMMCs from WT or p85a2/2mice were starved
3848 SHIP’S ROLE IN CTMCs, MMCs, AND BMMCs
et al. (57) found that transfecting RAW264.7 cells with a class I
PI3K p110 dominant-negative mutant inhibited LPS-induced
TNF-a secretion. Our data with p85a2/2CTMCs suggest that,
at least in MCs, PI3K appears to be playing a positive role. We
cannot say at this time whether this is a difference between MCs
and macrophages or not. Of note, whereas our data are consistent
with SHIP acting as a positive regulator of TLR-induced cytokines
via its adaptor function(s), we cannot rule out that it may still be
acting as a positive regulator via its enzymatic activity by hydro-
lyzing another substrate (e.g., inositol tetraphosphate) that inhibits
TLR agonist-induced cytokine production.
The differences we have observed between CTMCs and MMCs/
BMMCs are in keeping with several previous studies showing
significant differences between these MC subsets, including dif-
ferences in TLR expression (49), in responsiveness to IL-18
(which induces BMMCs/MMCs, but not CTMCs to secrete the
chemokine, Ccll, to recruit Th2 cells) (58), in expression of
STAT4 (in CTMCs) and STAT6 (in MMCs/BMMCs) (59) and in
TLR-induced cytokines and chemokines (higher with CTMCs)
(25). Differences have also been reported in IgE plus Ag-induced
survival, with survival being more pronounced with CTMCs (60),
and this may explain why IgE is a more potent survival enhancer
of CTMCs than MMCs (Fig. 1C).
In terms of in vivo significance, our finding that TLR agonists
trigger far higher levels of inflammatory cytokines from WT
CTMCs than from WT MMCs makes sense because CTMCs are in
sterile sites of the body and should be more vigilant toward
microbes than MMCs, which are in “dirty” environments. As far as
MMCs are concerned, our work, coupled with recent studies by
Haddon et al. (61) showing that it is the absence of SHIP in MCs
that is responsible for both the increased IL-6 and TNF-a serum
levels and the chronic lung inflammation that is characteristic
of SHIP2/2mice, suggests that it is the high levels of IL-6 and
TNF-a specifically produced by IgE 6 Ag-induced SHIP2/2
MMCs that most likely drive this chronic lung inflammation (62–
64), by attracting and activating other immune cells (27, 65, 66).
As well, our finding that TLR stimulation augments IgE plus Ag-
induced TNF-a and IL-6 production from MMCs (Fig. 5) might
explain the exacerbation of IgE-mediated allergic episodes by
infectious agents (26) and suggests that activating SHIP specifi-
cally in MMCs might be useful for treating chronic inflammatory
diseases like asthma.
We thank Drs. Odile Malbec and Marc Daeron for SCF-secreting Chinese
hamster ovary (CHO-KL) cells and Christine Kelly for preparing the man-
to identifying small molecule activators and inhibitors of SHIP.
1. Welle, M. 1997. Development, significance, and heterogeneity of mast cells with
particular regard to the mast cell-specific proteases chymase and tryptase. J.
Leukoc. Biol. 61: 233–245.
2. Gurish, M. F., and J. A. Boyce. 2006. Mast cells: ontogeny, homing, and recruit-
ment of a unique innate effector cell. J. Allergy Clin. Immunol. 117: 1285–1291.
3. Beil, W. J., M. Schulz, A. R. McEuen, M. G. Buckley, and A. F. Walls. 1997.
Number, fixation properties, dye-binding and protease expression of duodenal
mast cells: comparisons between healthy subjects and patients with gastritis or
Crohn’s disease. Histochem. J. 29: 759–773.
4. Razin, E., R. L. Stevens, F. Akiyama, K. Schmid, and K. F. Austen. 1982.
Culture from mouse bone marrow of a subclass of mast cells possessing a dis-
tinct chondroitin sulfate proteoglycan with glycosaminoglycans rich in N-ace-
tylgalactosamine-4,6-disulfate. J. Biol. Chem. 257: 7229–7236.
5. Razin, E., J. N. Ihle, D. Seldin, J. M. Mencia-Huerta, H. R. Katz, P. A. LeBlanc,
A. Hein, J. P. Caulfield, K. F. Austen, and R. L. Stevens. 1984. Interleukin 3:
a differentiation and growth factor for the mouse mast cell that contains chon-
droitin sulfate E proteoglycan. J. Immunol. 132: 1479–1486.
6. Maltby, S., K. Khazaie, and K. M. McNagny. 2009. Mast cells in tumor growth:
angiogenesis, tissue remodelling and immune-modulation. Biochim. Biophys.
Acta 1796: 19–26.
7. Sonoda, S., T. Sonoda, T. Nakano, Y. Kanayama, Y. Kanakura, H. Asai,
T. Yonezawa, and Y. Kitamura. 1986. Development of mucosal mast cells after
injection of a single connective tissue-type mast cell in the stomach mucosa of
genetically mast cell-deficient W/Wv mice. J. Immunol. 137: 1319–1322.
8. Otsu, K., T. Nakano, Y. Kanakura, H. Asai, H. R. Katz, K. F. Austen,
R. L. Stevens, S. J. Galli, and Y. Kitamura. 1987. Phenotypic changes of bone
marrow-derived mast cells after intraperitoneal transfer into W/Wv mice that are
genetically deficient in mast cells. J. Exp. Med. 165: 615–627.
9. Metcalfe, D. D., R. D. Peavy, and A. M. Gilfillan. 2009. Mechanisms of mast
cell signaling in anaphylaxis. J. Allergy Clin. Immunol. 124: 639–646, quiz
10. Ryan, J. J., and J. F. Fernando. 2009. Mast cell modulation of the immune re-
sponse. Curr. Allergy Asthma Rep. 9: 353–359.
11. Gilfillan, A. M., and J. Rivera. 2009. The tyrosine kinase network regulating
mast cell activation. Immunol. Rev. 228: 149–169.
12. Echtenacher, B., D. N. Ma ¨nnel, and L. Hu ¨ltner. 1996. Critical protective role of
mast cells in a model of acute septic peritonitis. Nature 381: 75–77.
13. Metz, M., and M. Maurer. 2007. Mast cells: key effector cells in immune
responses. Trends Immunol. 28: 234–241.
14. Rauh, M. J., J. Kalesnikoff, M. Hughes, L. Sly, V. Lam, and G. Krystal. 2003.
Role of Src homology 2-containing-inositol 59-phosphatase (SHIP) in mast cells
and macrophages. Biochem. Soc. Trans. 31: 286–291.
15. Antignano, F., J. Ruschmann, M. Hamilton, V. Ho, V. Lam, E. Kuroda, L. M. Sly,
and G. Krystal. 2009. The Src homology 2 containing inositol 59 phosphatases.
In Handbook of Cell Signalling. R. A. Bradsaw, and E. A. Dennis, eds. Elsevier,
San Diego, CA, p. 1065–1084.
16. Sly, L. M., J. Kalesnikoff, V. Lam, D. Wong, C. Song, S. Omeis, K. Chan, C. W.
K. Lee, R. P. Siraganian, J. Rivera, and G. Krystal. 2008. IgE-induced mast cell
survival requires the prolonged generation of reactive oxygen species. J.
Immunol. 181: 3850–3860.
17. Malbec, O., K. Roget, C. Schiffer, B. Iannascoli, A. R. Dumas, M. Arock, and
M. Dae ¨ron. 2007. Peritoneal cell-derived mast cells: an in vitro model of mature
serosal-type mouse mast cells. J. Immunol. 178: 6465–6475.
18. Huber, M., C. D. Helgason, J. E. Damen, L. Liu, R. K. Humphries, and
G. Krystal. 1998. The src homology 2-containing inositol phosphatase (SHIP) is
the gatekeeper of mast cell degranulation. Proc. Natl. Acad. Sci. USA 95: 11330–
19. Nishizumi, H., and T. Yamamoto. 1997. Impaired tyrosine phosphorylation and
Ca2+mobilization, but not degranulation, in lyn-deficient bone marrow-derived
mast cells. J. Immunol. 158: 2350–2355.
20. Damen, J. E., H. Wakao, A. Miyajima, J. Krosl, R. K. Humphries, R. L. Cutler,
and G. Krystal. 1995. Tyrosine 343 in the erythropoietin receptor positively
regulates erythropoietin-induced cell proliferation and Stat5 activation. EMBO J.
21. Kataoka, T. R., N. Komazawa, E. Morii, K. Oboki, and T. Nakano. 2005. In-
volvement of connective tissue-type mast cells in Th1 immune responses via
Stat4 expression. Blood 105: 1016–1020.
22. Kalesnikoff, J., M. Huber, V. Lam, J. E. Damen, J. Zhang, R. P. Siraganian, and
G. Krystal. 2001. Monomeric IgE stimulates signaling pathways in mast cells
that lead to cytokine production and cell survival. Immunity 14: 801–811.
23. Asai, K., J. Kitaura, Y. Kawakami, N. Yamagata, M. Tsai, D. P. Carbone,
F. T. Liu, S. J. Galli, and T. Kawakami. 2001. Regulation of mast cell survival by
IgE. Immunity 14: 791–800.
24. Kitaura, J., J. Song, M. Tsai, K. Asai, M. Maeda-Yamamoto, A. Mocsai,
Y. Kawakami, F. T. Liu, C. A. Lowell, B. G. Barisas, et al. 2003. Evidence that
IgE molecules mediate a spectrum of effects on mast cell survival and activation
via aggregation of the FcepsilonRI. Proc. Natl. Acad. Sci. USA 100: 12911–
25. Matsushima, H., N. Yamada, H. Matsue, and S. Shimada. 2004. TLR3-, TLR7-,
and TLR9-mediated production of proinflammatory cytokines and chemokines
from murine connective tissue type skin-derived mast cells but not from bone
marrow-derived mast cells. J. Immunol. 173: 531–541.
26. Qiao, H., M. V. Andrade, F. A. Lisboa, K. Morgan, and M. A. Beaven. 2006.
FcepsilonR1 and Toll-like receptors mediate synergistic signals to markedly
augment production of inflammatory cytokines in murine mast cells. Blood 107:
27. Supajatura, V., H. Ushio, A. Nakao, S. Akira, K. Okumura, C. Ra, and H. Ogawa.
2002. Differential responses of mast cell Toll-like receptors 2 and 4 in allergy
and innate immunity. J. Clin. Invest. 109: 1351–1359.
28. Fehrenbach, K., F. Port, G. Grochowy, C. Kalis, W. Bessler, C. Galanos,
G. Krystal, M. Freudenberg, and M. Huber. 2007. Stimulation of mast cells via
FcεR1 and TLR2: the type of ligand determines the outcome. Mol. Immunol. 44:
29. Nigo, Y. I., M. Yamashita, K. Hirahara, R. Shinnakasu, M. Inami, M. Kimura,
A. Hasegawa, Y. Kohno, and T. Nakayama. 2006. Regulation of allergic airway
inflammation through Toll-like receptor 4-mediated modification of mast cell
function. Proc. Natl. Acad. Sci. USA 103: 2286–2291.
30. Huber, M., C. D. Helgason, M. P. Scheid, V. Duronio, R. K. Humphries, and
G. Krystal. 1998. Targeted disruption of SHIP leads to Steel factor-induced
degranulation of mast cells. EMBO J. 17: 7311–7319.
The Journal of Immunology3849
31. Metz, M., M. A. Grimbaldeston, S. Nakae, A. M. Piliponsky, M. Tsai, and
S. J. Galli. 2007. Mast cells in the promotion and limitation of chronic inflam-
mation. Immunol. Rev. 217: 304–328.
32. Stelekati, E., Z. Orinska, and S. Bulfone-Paus. 2007. Mast cells in allergy: innate
instructors of adaptive responses. Immunobiology 212: 505–519.
33. Nakano, N., C. Nishiyama, S. Kanada, Y. Niwa, N. Shimokawa, H. Ushio,
M. Nishiyama, K. Okumura, and H. Ogawa. 2007. Involvement of mast cells in
IL-12/23 p40 production is essential for survival from polymicrobial infections.
Blood 109: 4846–4855.
34. Carty, M., and A. G. Bowie. 2010. Recent insights into the role of Toll-like
receptors in viral infection. Clin. Exp. Immunol. 161: 397–406.
35. Kalesnikoff, J., N. Baur, M. Leitges, M. R. Hughes, J. E. Damen, M. Huber, and
G. Krystal. 2002. SHIP negatively regulates IgE + antigen-induced IL-6 pro-
duction in mast cells by inhibiting NF-kB activity. J. Immunol. 168: 4737–4746.
36. Poderycki, M., Y. Tomimori, T. Ando, W. Xiao, M. Maeda-Yamamoto, K. Sauer,
Y. Kawakami, and T. Kawakami. 2010. A minor catalytic activity of Src family
kinases is sufficient for maximal activation of mast cells via the high-affinity IgE
receptor. J. Immunol. 184: 84–93.
37. Zorn, C. N., S. Keck, R. W. Hendriks, M. Leitges, M. A. Freudenberg, and
M. Huber. 2009. Bruton’s tyrosine kinase is dispensable for the Toll-like
receptor-mediated activation of mast cells. Cell. Signal. 21: 79–86.
38. Yang, Y. J., W. Chen, S. O. Carrigan, W. M. Chen, K. Roth, T. Akiyama, J. Inoue,
J. S. Marshall, J. N. Berman, and T. J. Lin. 2008. TRAF6 specifically contributes
to FcepsilonRI-mediated cytokine production but not mast cell degranulation. J.
Biol. Chem. 283: 32110–32118.
39. Hamilton, M. J., V. W. Ho, E. Kuroda, J. Ruschmann, F. Antignano, V. Lam, and
G. Krystal. 2011. Role of SHIP in cancer. Exp. Hematol. 39: 2–13.
40. Koncz, G., G. K. To ´th, G. Bo ¨ko ¨nyi, G. Ke ´ri, I. Pecht, D. Medgyesi, J. Gergely,
and G. Sa ´rmay. 2001. Co-clustering of Fcgamma and B cell receptors induces
dephosphorylation of the Grb2-associated binder 1 docking protein. Eur. J.
Biochem. 268: 3898–3906.
41. Song, M., M. J. Kim, S. Ha, J. B. Park, S. H. Ryu, and P. G. Suh. 2005. Inositol
59-phosphatase, SHIP1 interacts with phospholipase C-g1 and modulates EGF-
induced PLC activity. Exp. Mol. Med. 37: 161–168.
42. Ott, V. L., I. Tamir, M. Niki, P. P. Pandolfi, and J. C. Cambier. 2002. Downstream
of kinase, p62(dok), is a mediator of FcgIIB inhibition of FcεRI signaling. J.
Immunol. 168: 4430–4439.
43. Isnardi, I., R. Lesourne, P. Bruhns, W. H. Fridman, J. C. Cambier, and
M. Dae ¨ron. 2004. Two distinct tyrosine-based motifs enable the inhibitory re-
ceptor FcgammaRIIB to cooperatively recruit the inositol phosphatases SHIP1/2
and the adapters Grb2/Grap. J. Biol. Chem. 279: 51931–51938.
44. Knight, Z. A., B. Gonzalez, M. E. Feldman, E. R. Zunder, D. D. Goldenberg,
O. Williams, R. Loewith, D. Stokoe, A. Balla, B. Toth, et al. 2006. A pharma-
cological map of the PI3-K family defines a role for p110a in insulin signaling.
Cell 125: 733–747.
45. Hazeki, K., K. Nigorikawa, and O. Hazeki. 2007. Role of phosphoinositide 3-
kinase in innate immunity. Biol. Pharm. Bull. 30: 1617–1623.
46. Hayakawa, M., K. Kawaguchi, H. Kaizawa, T. Koizumi, T. Ohishi, M. Yamano,
M. Okada, M. Ohta, S. Tsukamoto, F. I. Raynaud, et al. 2007. Synthesis and
biological evaluation of sulfonylhydrazone-substituted imidazo[1,2-a]pyridines
as novel PI3 kinase p110a inhibitors. Bioorg. Med. Chem. 15: 5837–5844.
47. Ma, P., R. S. Mali, V. Munugalavadla, S. Krishnan, B. Ramdas, E. Sims,
H. Martin, J. Ghosh, S. Li, R. J. Chan, et al. 2011. The PI3K pathway drives the
maturation of mast cells via microphthalmia transcription factor. Blood 118:
48. Nakano, T., T. Sonoda, C. Hayashi, A. Yamatodani, Y. Kanayama, T. Yamamura,
H. Asai, T. Yonezawa, Y. Kitamura, and S. J. Galli. 1985. Fate of bone marrow-
derived cultured mast cells after intracutaneous, intraperitoneal, and intravenous
transfer into genetically mast cell-deficient W/Wv mice: evidence that cultured
mast cells can give rise to both connective tissue type and mucosal mast cells. J.
Exp. Med. 162: 1025–1043.
49. Mrabet-Dahbi, S., M. Metz, A. Dudeck, T. Zuberbier, and M. Maurer. 2009.
Murine mast cells secrete a unique profile of cytokines and prostaglandins in
response to distinct TLR2 ligands. Exp. Dermatol. 18: 437–444.
50. Takenaka, H., H. Ushio, F. Niyonsaba, S. T. Jayawardana, S. Hajime, S. Ikeda,
H. Ogawa, and K. Okumura. 2010. Synergistic augmentation of inflammatory
cytokine productions from murine mast cells by monomeric IgE and Toll-like
receptor ligands. Biochem. Biophys. Res. Commun. 391: 471–476.
51. Inami, M., K. Inokuchi, H. Yamaguchi, K. Nakayama, A. Watanabe, N. Uchida,
S. Tanosaki, and K. Dan. 2006. Oral administration of imatinib to P230 BCR/
ABL-expressing transgenic mice changes clones with high BCR/ABL comple-
mentary DNA expression into those with low expression. Int. J. Hematol. 84:
52. Kasakura, K., K. Takahashi, T. Aizawa, A. Hosono, and S. Kaminogawa. 2009.
A TLR2 ligand suppresses allergic inflammatory reactions by acting directly on
mast cells. Int. Arch. Allergy Immunol. 150: 359–369.
53. Supajatura, V., H. Ushio, A. Nakao, K. Okumura, C. Ra, and H. Ogawa. 2001.
Protective roles of mast cells against enterobacterial infection are mediated by
Toll-like receptor 4. J. Immunol. 167: 2250–2256.
54. Chiba, N., A. Masuda, Y. Yoshikai, and T. Matsuguchi. 2007. Ceramide inhibits
LPS-induced production of IL-5, IL-10, and IL-13 from mast cells. J. Cell.
Physiol. 213: 126–136.
55. Keck, S., M. Freudenberg, and M. Huber. 2010. Activation of murine macro-
phages via TLR2 and TLR4 is negatively regulated by a Lyn/PI3K module and
promoted by SHIP1. J. Immunol. 184: 5809–5818.
56. Luyendyk, J. P., G. A. Schabbauer, M. Tencati, T. Holscher, R. Pawlinski, and
N. Mackman. 2008. Genetic analysis of the role of the PI3K-Akt pathway in
lipopolysaccharide-induced cytokine and tissue factor gene expression in
monocytes/macrophages. J. Immunol. 180: 4218–4226.
57. Kuo, C. C., W. T. Lin, C. M. Liang, and S. M. Liang. 2006. Class I and III
phosphatidylinositol 39-kinase play distinct roles in TLR signaling pathway. J.
Immunol. 176: 5943–5949.
58. Wiener, Z., P. Pocza, M. Racz, G. Nagy, G. Tolgyesi, V. Molnar, J. Jaeger,
E. Buzas, E. Gorbe, Z. Papp, et al. 2008. IL-18 induces a marked gene ex-
pression profile change and increased Ccl1 (I-309) production in mouse mucosal
mast cell homologs. Int. Immunol. 20: 1565–1573.
59. Kataoka, T. R., and Y. Nishizawa. 2008. Stat4 suppresses the proliferation of
connective tissue-type mast cells. Lab. Invest. 88: 856–864.
60. Ekoff, M., A. Strasser, and G. Nilsson. 2007. FcepsilonRI aggregation promotes
survival of connective tissue-like mast cells but not mucosal-like mast cells. J.
Immunol. 178: 4177–4183.
61. Haddon, D. J., F. Antignano, M. R. Hughes, M. R. Blanchet, L. Zbytnuik,
G. Krystal, and K. M. McNagny. 2009. SHIP1 is a repressor of mast cell hy-
perplasia, cytokine production, and allergic inflammation in vivo. J. Immunol.
62. Helgason, C. D., J. E. Damen, P. Rosten, R. Grewal, P. Sorensen, S. M. Chappel,
A. Borowski, F. Jirik, G. Krystal, and R. K. Humphries. 1998. Targeted dis-
ruption of SHIP leads to hemopoietic perturbations, lung pathology, and
a shortened life span. Genes Dev. 12: 1610–1620.
63. Rauh, M. J., V. Ho, C. Pereira, A. Sham, L. M. Sly, V. Lam, L. Huxham,
A. I. Minchinton, A. Mui, and G. Krystal. 2005. SHIP represses the generation of
alternatively activated macrophages. Immunity 23: 361–374.
64. Oh, S. Y., T. Zheng, M. L. Bailey, D. L. Barber, J. T. Schroeder, Y. K. Kim, and
Z. Zhu. 2007. Src homology 2 domain-containing inositol 5-phosphatase 1 de-
ficiency leads to a spontaneous allergic inflammation in the murine lung. J.
Allergy Clin. Immunol. 119: 123–131.
65. Malaviya, R., T. Ikeda, E. Ross, and S. N. Abraham. 1996. Mast cell modulation
of neutrophil influx and bacterial clearance at sites of infection through TNF-a.
Nature 381: 77–80.
66. Sutherland, R. E., J. S. Olsen, A. McKinstry, S. A. Villalta, and P. J. Wolters.
2008. Mast cell IL-6 improves survival from Klebsiella pneumonia and sepsis by
enhancing neutrophil killing. J. Immunol. 181: 5598–5605.
3850 SHIP’S ROLE IN CTMCs, MMCs, AND BMMCs