SHIP1 Is a Repressor of Mast Cell Hyperplasia, Cytokine Production, and Allergic Inflammation In Vivo

D. James Haddon, Frann Antignano, Michael R. Hughes, Marie-Renée Blanchet, Lori Zbytnuik, Gerald Krystal and Kelly M. McNagny
J Immunol July 1, 2009, 183 (1) 228-236; DOI: https://doi.org/10.4049/jimmunol.0900427

Abstract

SHIP1 inhibits immune receptor signaling through hydrolysis of the PI3K product phosphatidylinositol 3,4,5-trisphosphate, forming phosphatidylinositol 3,4-bisphosphate. In mast cells, SHIP1 represses FcεRI- and cytokine-mediated activation in vitro, but little is known regarding the function of SHIP1 in mast cells in vivo or the susceptibility of Ship1−/− mice to mast cell-associated diseases. In this study, we found that Ship1−/− mice have systemic mast cell hyperplasia, increased serum levels of IL-6, TNF, and IL-5, and heightened anaphylactic response. Further, by reconstituting mast cell-deficient mice with Ship1+/+ or Ship1−/− mast cells, we found that the above defects were due to loss of SHIP1 in mast cells. Additionally, we found that mice reconstituted with Ship1−/− mast cells suffered worse allergic asthma pathology than those reconstituted with Ship1+/+ mast cells. In summary, our data show that SHIP1 represses allergic inflammation and mast cell hyperplasia in vivo and exerts these effects specifically in mast cells.

The Src homology 2-containing inositol 5′-phosphatase SHIP1 inhibits immune receptor signaling by hydrolysis of the PI3K product phosphatidylinositol 3,4,5-trisphosphate, forming phosphatidylinositol 3,4-bisphosphate SHIP1 represses the activation, survival, or proliferation of T cells, B cells, macrophages, NK cells, neutrophils, and mast cells (1, 2, 3, 4, 5, 6). Ship1−/− mice are viable and fertile but suffer from excessive granulocyte and macrophage numbers, profound splenomegaly, extramedullary hematopoiesis, massive myeloid infiltration of the lungs, osteoporosis, wasting, and a shortened lifespan (7, 8). Because SHIP1 is expressed in all hematopoietic cell lineages, it has been difficult to determine which cell types are responsible for given phenotypes.

Mast cells are immune effector cells known for releasing their cytoplasmic granules when IgE Abs, bound to FcεRI receptors, are crosslinked by multivalent exogenous Ag. During degranulation, mast cells release inflammatory and immunomodulatory molecules, including cytokines, chemokines, lipid-derived mediators, histamine, proteoglycans, and neutral proteases (reviewed in Ref. 9). Although SHIP1 is not required for mast cell differentiation, it functions as a repressor of mast cell degranulation and cytokine secretion in vitro. Specifically, Ship1−/− mast cells exhibit increased degranulation and cytokine secretion compared with Ship1+/+ mast cells in response to IgE crosslinking (6, 10). Further, stimulation with stem cell factor (SCF)4 or IgE without crosslinking Ag induces aberrant degranulation in Ship1−/− but not Ship1+/+ mast cells (6, 11).

Mast cells contribute to a wide variety of inflammatory diseases, including anaphylaxis and allergic asthma (reviewed in Refs. 12 and 13). Anaphylaxis is a type I hypersensitivity reaction primarily provoked by allergen-induced mast cell degranulation. Symptoms of anaphylaxis include vascular permeability, tachycardia, hypothermia, and mortality. Allergic asthma is a complex, chronic inflammatory disease of the airways and lungs. Naive Ship1−/− mice have symptoms of allergic asthma under steady-state conditions, including severe airway inflammation, mucus hyperproduction, and airway remodeling, but the contribution of Ship1−/− mast cells to these symptoms is unknown (14).

Mast cell physiology can be studied in vivo by using mast cell-deficient KitW-sh/W-sh mice, which lack mast cells due to a chromosomal inversion that disrupts the 5′ regulatory sequences of Kit (15, 16). Reconstituting KitW-sh/W-sh mice with Kit+/+ bone marrow derived mast cells (BMMCs) selectively repairs their mast cell deficiency (17). Furthermore, if KitW-sh/W-sh mice are reconstituted with BMMCs from knockout mice they can be used to study gene function in mast cells in vivo (18, 19, 20).

Although the role of SHIP1 in immune receptor signaling has been established in mast cells in vitro, little is known about its function in mast cells in vivo or about the susceptibility of Ship1−/− mice to mast cell-associated diseases. In this study, we found that Ship1−/− mice have systemic mast cell hyperplasia, increased serum levels of IL-6, TNF, and IL-5, and heightened susceptibility to anaphylaxis. Furthermore, by reconstituting mast cell-deficient KitW-sh/W-sh mice with Ship1+/+ or Ship1−/− BMMCs, we established that loss of SHIP1 in the mast cell lineage was responsible for these defects (with the exception of increased IL-5). In addition, we found that mice reconstituted with Ship1−/− mast cells suffered worse allergic asthma pathology than those reconstituted with Ship1+/+ mast cells. In summary, our data show that SHIP1 is a repressor of allergic inflammation and mast cell hyperplasia in vivo and that it exerts these effects specifically in mast cells.

Materials and Methods

Mice

All mice were housed in specific pathogen-free mouse facilities at The Biomedical Research Centre or the British Columbia Cancer Research Centre, with approval from the University of British Columbia Animal Care Committee and according to the Canadian Council on Animal Care guidelines. C57BL/6 (B6) and B6.Cg-KitW-sh/HNihrJaeBsmJ (KitW-sh/W-sh) mouse strains were acquired from The Jackson Laboratory, and each were maintained as homozygous colonies by sibling breeding. B6-congenic Ship1−/− mice were a kind gift from Dr. F. R. Jirik (University of Calgary, Calgary, Alberta, Canada) and were backcrossed at least 12 times. F2 Ship1−/− mice (F2-Ship1−/−) were maintained on a B6 × 129/Sv mixed background. All mice used were at least 4-wk old and controls were littermates or were age- and sex-matched from the same colony.

Cell culture

Bone marrow cells were flushed from the femurs of 6-wk-old Ship1+/+ and Ship1−/− mice and placed in IMDM supplemented with 15% FCS (StemCell Technologies), 150 μM monothioglycerol (Sigma-Aldrich), 100 U/ml penicillin, 100 μg/ml streptomycin (Sigma-Aldrich), and 30 ng/ml recombinant mouse IL-3 (R&D Systems). Cells were maintained at 37°C in a 5% CO2 humidified atmosphere, selected for nonadherence, and considered to be BMMCs after 6 wk in culture as verified by Wright-Giemsa staining and flow cytometry (data not shown).

Reconstitution of mast cell-deficient KitW-sh/W-sh mice

KitW-sh/W-sh mice were injected with 1 × 107 Ship1+/+ or Ship1−/− BMMCs in 200 μl of HBSS via the tail vein (designated Ship1+/+-BMMC and Ship1−/−-BMMC mice). After 12 wk the mice were bled for serum collection, subjected to passive systemic anaphylaxis, or sensitized for allergic asthma and then euthanized via CO2 inhalation. After euthanizing the mice, peritoneal washes were taken for subsequent analysis by flow cytometry. Samples of back skin, spleen, lung, mesentery, ileum, and colon were fixed in 10% neutral buffered formalin. Fixed samples were paraffin embedded, sectioned, and stained by Wax-it Histology Services. All tissues were stained with toluidine blue. Mast cells were enumerated by counting the number of toluidine blue-positive cells in each section at ×40 or ×100 objective on a Zeiss Axioplan 2 microscope. The area of the section was then determined using the polygon area tool with OpenLab 4.0.4 software (Improvision) at ×5 objective.

Cytokine assay

Serum (50 μl) was collected from the saphenous vein and assayed using BD mouse inflammation and mouse Th1/Th2 cytometric bead array kits according to manufacturer’s instructions (BD Biosciences).

Passive systemic anaphylaxis

Mice were sensitized by i.v. injection with 20 μg of IgE SPE-7 (Sigma-Aldrich) in 100 μl of HBSS plus 0.1% BSA. The following day, anaphylaxis was induced by i.v. injection with 1 mg of DNP-human serum albumin (HSA) in 100 μl of PBS. Rectal temperature was taken 5 min before and immediately preceding injection and then monitored every 5 min for 40 min and again at 75 and 90 min, at which time all mice were sacrificed. Temperature measurements were performed with a traceable expanded range metric digital thermometer (VWR International).

Asthma disease course

Female mice were sensitized with 100 μl of 2 mg/ml OVA in alum (Al(OH)3; Sigma-Aldrich) via i.p. injection on days 1 and 8. Following sensitization, asthma was induced by introducing 50 μl of 20 mg/ml OVA in HBSS intranasally on days 22, 23, 24, 27, and 28. During intranasal sensitizations, mice were anesthetized via inhalation of 3% isoflurane in 0.8 liters of oxygen per minute. Mice were sacrificed by CO2 inhalation on day 29. Bronchoalveolar lavages (BAL) were performed by completing three separate additions and removals of 1 ml of PBS. Total CD45.2+ BAL cells were enumerated using flow cytometry, and differentials were performed on Wright-Giemsa stained cytospins.

A portion of the right lower lobe of the lung was taken from each mouse for histopathological grading and mast cell enumeration. Lung portions were fixed in 10% neutral buffered formalin, paraffin-embedded, sectioned, and H&E stained by Wax-it Histology Services. Samples were blinded and clinically scored from 0 (no infiltration) to 4 (profound infiltration) on each of the following parameters: perivascular infiltration, peribronchial infiltration, parenchymal infiltration, and epithelial damage (maximum score of 16).

Flow cytometry

Samples were blocked with anti-CD16/32 in 10% goat serum and then stained with anti-c-kit-PE (BD Pharmingen), anti-CD45.2-PE (eBioscience), or anti-FcεRI-biotin (eBioscience) followed by streptavidin-FITC (BD Pharmingen) or anti-CD45.2-PE (eBioscience). Latex beads (10 μm; Invitrogen) were added to HBSS with 0.1% BSA before peritoneal washes and to BAL to calculate total cell recovery via flow cytometry. All flow cytometry experiments were performed on a BD FACSCalibur with CellQuest software and postcollection analyses were performed with FlowJo (Tree Star).

Statistics

The unpaired t test (two-tailed) was used for analysis of tissue mast cell numbers, asthma BAL cellular infiltrate, BAL differentials, lung clinical scores, serum cytokine levels, and passive systemic anaphylaxis (PSA) assays. A Kruskal-Wallis test was used for the mesentery of BMMC-reconstituted mice (due to all 0 values in the Ship1+/+ condition). A log-rank (Mantel-Cox) test was used to compare the PSA survival rates. Separate allergic asthma experiments were normalized based on their OVA-treated B6 levels before analysis. All of the above tests were performed using GraphPad Prism 5 or Microsoft Excel X software. Missing samples from some tissues used in the enumeration of mast cells in Ship1−/− and BMMC-reconstituted mice were due to errors during sample processing.

Results

Ship1−/− mice have mast cell hyperplasia in multiple tissues

We suspected that the chronic inflammatory phenotype of Ship1−/− mice could be due in part to increased tissue mast cell numbers. With this in mind, we enumerated the tissue mast cells of Ship1+/+ and Ship1−/− mice and found significantly higher numbers in the spleen, lung, ileum, mesentery, and back skin of Ship1−/− mice (Fig. 1⇓A). We also found that Ship1−/− mice had higher numbers of colon mast cells, although the counts were more variable than those for other tissues assessed. Similarly, Ship1−/− mice had over two times more peritoneal mast cells than wild-type controls (Fig. 1⇓B). The frequency of peritoneal mast cells was similar between Ship1−/− and wild-type mice, reflecting a greater number of total peritoneal cells in Ship1−/− mice than in Ship1+/+ mice (6.8, SD 2.8 vs 2.0, SD 0.13 cells (×106); p < 0.0114). Thus, Ship1−/− mice have mast cell hyperplasia in the spleen, lungs, colon, ileum, mesentery, peritoneal cavity, and back skin.

FIGURE 1.
FIGURE 1.

Mast cell hyperplasia in multiple tissues of Ship1−/− mice. A, Toluidine blue histological staining revealed that Ship1−/− mice (n = 11) had significantly higher numbers of metachromatic tissue mast cells than Ship1+/+ controls (n = 10). B, Ship1−/− mice (n = 10) had a higher total number (graph) but similar frequency (gate statistics) of SSChigh/c-kithigh peritoneal wash mast cells compared to Ship1+/+ mice (n = 9), as determined by flow cytometry. Data represent pooled results from two independent experiments.

Mast cell hyperplasia in Ship1−/− mice is mast cell autonomous

Because SHIP1 is expressed in many hematopoietic cell types, it was unclear whether the mast cell hyperplasia we observed in ubiquitous Ship1−/− mice was mast cell autonomous. To answer this question, we i.v. reconstituted KitW-sh/W-sh mast cell-deficient mice with Ship1+/+ or Ship1−/− BMMC (designated Ship1+/+-BMMC and Ship1−/−-BMMC mice) and, following 12 wk of engraftment, found mast cell hyperplasia in the spleen, lung, colon, ileum, mesentery, and back skin of Ship1−/−-BMMC mice (Fig. 2⇓A). Likewise, Ship1−/−-BMMC mice also had a greater number and frequency (0.92, SD 0.52% vs 0.10, SD 0.13%; p < 0.0015) of peritoneal mast cells than Ship1+/+-BMMC controls (Fig. 2⇓B). KitW-sh/W-sh mice were mast cell deficient in all tissues assessed, and the mast cell numbers in the tissues of B6 and KitW-sh/W-sh mice reconstituted with wild-type mast cells were similar to those of previous reports (17). Thus, the mast cell hyperplasia observed in Ship1−/− mice is a mast cell autonomous defect.

FIGURE 2.
FIGURE 2.

Mast cell hyperplasia in Ship1−/− mice is mast cell autonomous. KitW-sh/W-sh mice were injected with 1 × 107 Ship1+/+ or Ship1−/− BMMC via the tail vein and, after 12 wk of reconstitution, the mice were sacrificed and their tissue mast cells were enumerated. A, The number of metachromatic tissue mast cells was significantly higher in Ship1−/−-BMMC mice (n = 7) than in Ship1+/+-BMMC mice (n = 7) and, in most cases, B6 mice (n = 6), whereas KitW-sh/W-sh mice (n = 9) were mast cell deficient as determined by toluidine blue histological staining. B, Similar results were observed for the total number (graph) and frequency (gate statistics) of SSChigh/c-kithigh (where SSC is side scatter) peritoneal mast cells from B6 (n = 3), Ship1−/−-BMMC (n = 3), Ship1+/+-BMMC (n = 2), and KitW-sh/W-sh (n = 3) mice by flow cytometry. Data represent pooled results from two independent experiments.

Ship1−/− mice have mast cell-dependent systemic inflammation

Because Ship1−/− mast cells exhibit enhanced degranulation and cytokine secretion in response to FcεRI stimulation and react aberrantly to c-kit stimulation in vitro, we investigated the levels of inflammatory cytokines in the serum of adult Ship1−/− mice (6, 10, 11). Ship1−/− mouse serum contained significantly increased levels of IL-5, IL-6, and TNF (and a small increase in CCL2) compared with Ship1+/+ controls (Fig. 3⇓A). To determine whether Ship1−/− mast cells were responsible for the increased levels of inflammatory serum cytokines observed in Ship1−/− mice, we evaluated the serum of Ship1−/−-BMMC-reconstituted mice. We detected significantly higher levels of IL-6 and TNF in the serum of Ship1−/−-BMMC mice, whereas the levels of IL-5 and CCL2 were only marginally increased compared with those of controls (Fig. 3⇓B). We concluded that Ship1−/− mast cells induce increased serum levels of the inflammatory cytokines IL-6 and TNF in vivo.

FIGURE 3.
FIGURE 3.

Increased inflammatory cytokine levels in the serum of Ship1−/− mice and mice reconstituted with Ship1−/− mast cells. A, Cytometric bead arrays of mouse cytokines were used to assay the serum of both F2-Ship1−/− (n = 7) and F2-Ship1+/+ (n = 7) mice. Data represent pooled results from two independent experiments. B, Cytometric bead arrays were also used to assay the serum of B6 (n = 4), Ship1−/−-BMMC (n = 8), Ship1+/+-BMMC (n = 8), and KitW-sh/W-sh (n = 6) mice. Data represent pooled results from two independent experiments.

Ship1−/− mice are hyperresponsive to passive systemic anaphylaxis

Because Ship1−/− mice possess mast cell hyperplasia in multiple tissues and Ship1−/− mast cells exhibit increased degranulation in response to IgE crosslinking in vitro, we suspected that Ship1−/− mice would be more susceptible to anaphylaxis (6). To test this hypothesis, we subjected Ship1−/− mice and littermate controls to PSA by sensitizing them with anti-DNP IgE, challenging them with DNP-HSA the next day, and monitoring their rectal temperature for 90 min (Fig. 4⇓). Interestingly, the body temperature of Ship1−/− mice (before challenge) was on average 1°C lower than that of wild-type controls. This may reflect low-level degranulation induced by the IgE sensitization, as Ship1−/− mast cells aberrantly degranulate in response to IgE without crosslinking Ag in vitro (6). More importantly, we found that Ship1−/− mice suffered increased mortality, more severe hypothermia (at t =20 min; 5°C, SD 0.6°C vs 3°C, SD 0.7°C; p < 0.0001), and no body temperature recovery compared with wild-type controls (Figs. 4⇓, A and B).

FIGURE 4.
FIGURE 4.

Hyperresponsive anaphylaxis in Ship1−/− mice and in mice reconstituted with Ship1−/− mast cells. To induce passive systemic anaphylaxis, Ship1−/− (n = 10, red line) and Ship1+/+ (n = 9, blue line) mice were sensitized with anti-DNP IgE and challenged the following day with DNP-HSA. Increased mortality (A) and hypothermia (B), measured rectally, were observed in Ship1−/− mice. Anaphylaxis was induced and monitored in Ship1−/−-BMMC (n = 5, red line) and Ship1+/+-BMMC (n = 6, blue line) mice (C), as well as B6 (n = 5, black line) and KitW-sh/W-sh (n = 5, gray line) controls (D) as described above. Data for C and D were measured concurrently but separated for clarity. Data in each panel represent pooled results from two separate experiments. (*, p < 0.05; **, p < 0.01; ***, p < 0.001). Bars represent SE.

It was unclear whether the increased severity of anaphylaxis suffered by Ship1−/− mice was due to increased mast cell activation and mast cell numbers or to the reactivity of other cell types to the mediators released during PSA challenge (reviewed in Ref. 21). Therefore we subjected reconstituted Ship1−/−-BMMC mice to PSA and found that they also suffered a much larger temperature drop than Ship1+/+-BMMC mice (at t = 35; 7°C, SD 2.2°C vs 1°C, SD 2.5°C; p = 0.0037) (Fig. 4⇑C). Notably, unlike ubiquitous Ship1−/− mice, Ship1−/−-BMMC mice exhibited body temperature recovery 40 min postchallenge. Although KitW-sh/W-sh mice were resistant to PSA, B6 mice suffered a similar temperature drop as that of Ship1−/−-BMMC mice (Fig. 4⇑D). In summary, we find that the increased severity of anaphylaxis experienced by Ship1−/− mice is largely mast cell autonomous.

Allergic airway inflammation is greater in Ship1−/−-BMMC mice than in Ship1+/+-BMMC controls

Naive Ship1−/− mice have allergic asthma-like symptoms, including severe airway inflammation, mucus hyperproduction, and symptoms of airway remodeling (14). To determine the role of Ship1−/− mast cells in allergic asthma pathology, we induced an acute form of the disease in Ship1−/−-BMMC- and Ship1+/+-BMMC-reconstituted mice (Fig. 5⇓A). We found that asthmatic Ship1−/−-BMMC mice had over two times more infiltrating cells in their airways than Ship1+/+-BMMC mice, including increased eosinophils, which are the primary effector cells in asthma (Figs. 5⇓, B and C). Perivascular, peribronchial, and parenchymal infiltration, as well as epithelial damage, were most prominent in the lungs of asthmatic Ship1−/−-BMMC mice, and the clinical scores of asthmatic Ship1−/−-BMMC lung sections were significantly higher than those of Ship1+/+-BMMC mice (Fig. 5⇓, D and E). KitW-sh/W-sh mice suffered comparable symptoms as those of B6 mice, similar to the findings of a study published after we completed our asthma course that also used an OVA-induced (with alum adjuvant) model of allergic asthma (see Fig. E1 in Ref. 20). Ship1+/+-BMMC mice had slightly reduced number of infiltrating BAL cells compared with KitW-sh/W-sh mice, highlighting the importance of wild-type reconstituted controls. We concluded that Ship1−/−-BMMC mice suffered worse allergic asthma pathology than Ship1+/+-BMMC mice.

FIGURE 5.
FIGURE 5.

Increased allergic asthma pathology in mice reconstituted with Ship1−/− mast cells. A, Asthma was induced in B6 (n = 10), Ship1−/−-BMMC (n = 13), Ship1+/+-BMMC (n = 10), and KitW-sh/W-sh (n = 11) mice via two i.p. sensitizations (with alum adjuvant; days 1 and 7) and five intranasal (i.n.) challenges (days 21, 22, 23, 26, and 27) of OVA before sacrificing (Sac.) the mice (day 28). B, Inflammation was assessed by enumerating the cellular infiltrate in the BAL using flow cytometry. C, Differentials were performed with a modified Wright-Giemsa stain; B6 (n = 7), Ship1−/−-BMMC (n = 9), Ship1+/+-BMMC (n = 6), and KitW-sh/W-sh (n = 7). BAL data represent pooled results from three independent experiments, and differentials from two independent experiments. D, Selected representative micrographs of H&E-stained lungs from PBS- or OVA-treated Ship1−/−-BMMC-reconstituted mice and controls (bars = 100 μm). E, Clinical scores were assigned to each lung based on the following parameters: perivascular infiltration, peribronchial infiltration, parenchymal infiltration, and epithelial damage (maximum score of 16). Data represent pooled results from three independent experiments. Each circle represents a mouse.

Discussion

Our results show the following: 1) Ship1−/− mice have systemic mast cell hyperplasia, increased serum levels of the inflammatory cytokines IL-6, TNF, and IL-5, and increased susceptibility to anaphylaxis; 2) the above phenotypes are mast cell autonomous (with the exception of increased serum IL-5); and 3) mice reconstituted with Ship1−/− mast cells suffered worse allergic asthma pathology than Ship1+/+ controls. Previous studies have focused on the function of SHIP1 in mast cells in vitro. In this study we demonstrate that SHIP1 is also a repressor of mast cells in vivo and that the loss of SHIP1 increases the symptoms of two mast cell-associated diseases. Furthermore, we demonstrate a cell-autonomous role for SHIP1 in mast cell homeostasis.

Mast cell hyperplasia is observed in chronic inflammatory processes, fibrotic disorders, wound healing, and neoplastic tissue transformation, but little is known about normal mast cell homeostasis (reviewed in Ref. 22). Our results demonstrate that SHIP1 is a cell-autonomous repressor of mast cell hyperplasia and a key player in mast cell homeostasis. Ship1−/− mice have increased numbers of granulocyte-macrophage progenitors in their bone marrow that exhibit enhanced sensitivity to multiple cytokines (7). In our studies, because the reconstituted BMMCs were composed exclusively of mast cells and their committed precursors and the numbers of mast cells observed in the tissues of Ship1−/−-BMMC mice were comparable to or greater than those in ubiquitous Ship1−/− mice, we concluded that the mast cell hyperplasia observed in Ship1−/− mice is not due to the increased number or sensitivity of granulocyte-macrophage progenitors. Furthermore, we found that the loss of SHIP1 specifically in mast cells was not sufficient to induce the excessive granulocyte and macrophage numbers previously observed in the blood, spleen, and lungs of Ship1−/− mice (data not shown) (7).

Interestingly, we found that Ship1−/− mice have elevated serum levels of the inflammatory cytokines IL-5, IL-6, and TNF compared with those of Ship1+/+ controls. This is in agreement with a previous report that Ship1−/− mice have increased levels of serum IL-6 (8). We demonstrated that the increased levels of IL-6 and TNF were due to loss of SHIP1 specifically in mast cells. Because Ship1−/− mast cells produce more IL-6 and TNF than wild-type mast cells upon FcεRI-mediated stimulation in vitro, we suspect that the increased serum levels of these cytokines arose directly from Ship1−/− mast cells via stimulation with endogenous IgE (10). Although the high level of IL-6 in the serum of Ship1−/− mice was previously attributed to macrophages, in this study we demonstrate that Ship1−/− mast cells also contribute to the increased IL-6 levels in vivo (8). Furthermore, the increased IL-6 was previously proposed to inhibit B cells and enhance myeloid cell development, but we found the frequency of B cells and myeloid cells in the bone marrow, peripheral blood, and spleen of Ship1−/−-BMMC-reconstituted mice were similar to that in controls (data not shown) (23). Thus, our data suggest that increased serum IL-6 is not sufficient to cause the hematopoietic lineage skewing observed in Ship1−/− mice, and a local increase of IL-6 in the bone marrow may be required.

Our studies also suggest that the systemic mast cell hyperplasia observed in Ship1−/− mice is due to the high levels of IL-6 and TNF produced by Ship1−/− mast cells. TNF is required for mast cell development and stimulates mast cell colony formation in vitro (24, 25). In addition, IL-6 promotes mast cell development from mixed cultures, likely via a secondary mediator such as PGE (24). We suspect that the systemic mast cell hyperplasia observed in Ship1−/− mice is also due to the hypersensitivity of Ship1−/− mast cells to stimulation with SCF or IgE (without crosslinking Ag) (6, 11). SCF and IgE induce mast cell proliferation and survival, respectively, and endogenous levels of these factors may be capable of sustaining higher levels of hypersensitive Ship1−/− mast cells in vivo (26, 27).

We also found that Ship1−/− mice suffer considerably more severe anaphylaxis than controls. Although mast cells are the primary effector cells in the PSA model of anaphylaxis, other cell types also play a role in its pathology. For example, IL-4 or IL-13 treatment increases the severity of anaphylaxis by enhancing the responsiveness of target cells to the vasoactive mediators released during anaphylaxis (reviewed in Ref. 21). We found that Ship1−/−-BMMC mice also suffered more severe anaphylaxis than controls, suggesting that this effect is largely mast cell autonomous. We suspect that the increased severity of anaphylaxis observed in Ship1−/− (and Ship1−/−-BMMC) mice is due to their systemic mast cell hyperplasia and the enhanced degranulation of the Ship1−/− mast cells, but we have not ruled out the possibility that the increased levels of IL-6 and TNF in their serum could have also contributed to this effect (6).

In our studies, we also found that Ship1−/−-BMMC mice suffered worse allergic asthma pathology than Ship1+/+-BMMC mice in an acute, OVA-induced (with alum adjuvant) model of allergic asthma (28). Because KitW-sh/W-sh mice also developed symptoms of asthma, it made it difficult to determine to what extent Ship1−/− mast cells were proinflammatory or whether they instead lacked the anti-inflammatory activity present in Ship1+/+ mast cells. Indeed, asthmatic Ship1+/+-BMMC mice had lower numbers of infiltrating cells and clinical scores than KitW-sh/W-sh controls, suggesting an anti-inflammatory role for mast cells in this model. With this in mind, we believe that Ship1−/− mast cells also contributed to inflammation for the following reasons. First, mast cells amplify allergic asthma symptoms in a similar model of asthma (without alum adjuvant) (29). Second, mast cells are hyperplasic in the lungs of asthmatic mice, and the pre-existing mast cell hyperplasia of the lungs in Ship1−/−-BMMC mice would likely predispose them to heightened allergic asthma pathology (30). Third, IL-6 and TNF, inflammatory cytokines associated with increased asthma pathology, are elevated in the serum of Ship1−/−-BMMC mice (20, 31). Finally, Ship1−/− mast cells intensified the severity of anaphylaxis, another mast cell-dependent inflammatory disease model. The fact that Ship1−/−-BMMC mice exhibit greater allergic asthma pathology than Ship1+/+-BMMC mice strongly suggests a mast cell-autonomous role for SHIP1 in allergic asthma response.

The PI3K pathway is involved in cytokine production and the amplification of FcεRI-mediated degranulation in mast cells in vitro and enhances anaphylaxis in vivo (reviewed in Ref. 32). In mast cells, FcεRI and c-kit receptors activate heterodimeric class IA PI3Ks, whereas G protein-coupled receptors, including the adenosine A3 receptor, activate class IB PI3Ks. SHIP1 negatively regulates PI3K activation via its phosphatase activity and can also act as a signaling adaptor (33). Other investigators have reported that the loss of the p85α subunit of class IA PI3K causes selective loss of gastrointestinal mast cells but does not affect mast cell degranulation in vitro or PSA in vivo (34). Loss of the p110δ subunit of PI3K (class IA) results in reductions in numbers of some dermal mast cells, degranulation, cutaneous anaphylaxis, and TNF and IL-6 production (35). PI3Kγ−/− (class IB)-cultured mast cells, in contrast, have reduced degranulation, and PI3Kγ−/− mice experience less severe PSA (36). The PI3K inhibitor LY294002 has also been reported to reduce asthma symptoms in vivo (37). In combination with our results, the studies outlined above indicate that SHIP1 may repress p85α subunit-dependent PI3K activity (class IA) during the formation or maintenance of gastrointestinal mast cells, p110δ-dependent PI3K activity (class IA) in the formation or maintenance of some dermal mast cells and TNF and IL-6 production, and both PI3Kγ−/− (class IB) and p110δ-dependent (class IA) PI3K activity in degranulation and anaphylaxis. This suggests that SHIP1 phosphatase activity (rather than adaptor function) is central to negative regulation of these processes. It is interesting to note the breadth of SHIP1 activity compared with the functional specificity of the PI3K family potentially making SHIP1 a more extensive target for therapy.

We propose that Ship1−/−-BMMC-reconstituted mice can serve as an in vivo model of hyperactive and hyperplasic mast cells for the investigation of mast cell-associated diseases and immune reactions. In this study we investigated the harmful roles that Ship1−/− mast cells play in inflammatory diseases. It would be interesting to test whether Ship1−/− mast cells also have an enhanced function in the beneficial roles of mast cells, such as clearing peritoneal and helminth infections (38, 39).

SHIP1 repressor activity in mast cells is conserved in humans, and agonists and antagonists of SHIP1 function have recently been identified and tested in vivo (40, 41). Our study suggests that SHIP1 agonists could reduce the severity of anaphylaxis, allergic asthma, and other mast cell-associated diseases in humans. Given that mast cells also have a role in such diverse diseases as rheumatoid arthritis, inflammatory bowel disease, and tumor progression and conversely aid in clearing peritoneal and helminth infections, modulators of SHIP1 function in mast cells may offer a variety of therapeutic strategies against key human diseases (25, 38, 39, 42, 43).

Acknowledgments

We thank Drs. Jami Bennett and Colby Zaph for thoughtful reviews of the manuscript.

Disclosures

G.K. is a founder and Chief Science Officer of Aquinox Pharmaceuticals Inc., a company dedicated to developing small molecule activators and inhibitors of SHIP1. Aquinox did not support this work in any way. All other authors have no conflicting financial interests.

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • ↵1 This study was supported by operating grants from the Canadian Institutes for Health Research (CIHR) and AllerGen Network Centre of Excellence. D.J.H. was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) and the University of British Columbia (UBC) Faculty of Medicine. F.A. was supported by NSERC, the UBC Faculty of Medicine, and the Michael Smith Foundation for Health Research (MSFHR). M.R.H. was supported by the UBC Centre for Blood Research CIHR/Heart and Stroke Foundation of Canada Strategic Training Program in Transfusion Science. M.-R.B. was supported through the CIHR/Canadian Asthma, Allergy and Immunology Foundation Training Program. K.M.M. is a MSFHR Senior Scholar.

  • D.J.H. designed and performed research, analyzed and interpreted data, and wrote the manuscript. F.A. and M.R.H. designed and performed research, analyzed and interpreted data, and edited the manuscript. M.-R.B. and L.Z. performed research. G.K. and K.M.M. analyzed and interpreted data and edited the manuscript.

  • ↵2 F.A. and M.R.H. contributed equally to this study.

  • ↵3 Address correspondence and reprint requests to Dr. Kelly M. McNagny, The Biomedical Research Centre, 2222 Health Sciences Mall, Vancouver, British Columbia, V6T 1Z3 Canada. E-mail address: kelly{at}brc.ubc.ca

  • ↵4 Abbreviations used in this paper: SCF, stem cell factor; BAL, bronchoalveolar lavage; BMMC, bone marrow cultured mast cells; HSA, human serum albumin; PSA, passive systemic anaphylaxis.

  • Received February 9, 2009.
  • Accepted April 23, 2009.

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