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(American Journal of Pathology. 2005;167:835-848.)
© 2005 American Society for Investigative Pathology

Mast Cell-Deficient W-sash c-kit Mutant Kit^W-sh/W-sh Mice as a Model for Investigating Mast Cell Biology in Vivo

Michele A. Grimbaldeston^*, Ching-Cheng Chen^*, Adrian M. Piliponsky^*, Mindy Tsai^*, See-Ying Tam^* and Stephen J. Galli^*

From the Departments of Pathology^* and Microbiology and Immunology, Stanford University School of Medicine, Stanford, California

	Abstract

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Mice carrying certain mutations in the white spotting (W) locus (ie, c-kit) exhibit reduced c-kit tyrosine kinase-dependent signaling that results in mast cell deficiency and other phenotypic abnormalities. The c-kit mutations in Kit^W/W-v mice impair melanogenesis and result in anemia, sterility, and markedly reduced levels of tissue mast cells. In contrast, Kit^W-sh/W-sh mice, bearing the W-sash (W^sh) inversion mutation, have mast cell deficiency but lack anemia and sterility. We report that adult Kit^W-sh/W-sh mice had a profound deficiency in mast cells in all tissues examined but normal levels of major classes of other differentiated hematopoietic and lymphoid cells. Unlike Kit^W/W-v mice, Kit^W-sh/W-sh mice had normal numbers of TCR intraepithelial lymphocytes in the intestines and did not exhibit a high incidence of idiopathic dermatitis, ulcers, or squamous papillomas of the stomach, but like Kit^W/W-v mice, they lacked interstitial cells of Cajal in the gut and exhibited bile reflux into the stomach. Systemic or local reconstitution of mast cell populations was achieved in nonirradiated adult Kit^W-sh/W-sh mice by intravenous, intraperitoneal, or intradermal injection of wild-type bone marrow-derived cultured mast cells but not by transplantation of wild-type bone marrow cells. Thus, Kit^W-sh/W-sh mice represent a useful model for mast cell research, especially for analyzing mast cell function in vivo.

Several different W mutant rodents have been investigated as potential models for the analysis of mast cell function in vivo, including Kit^W/W-v and Kit^W-f/W-f mice and Kit^W-s/W-s rats.^1,6,7 In general, the utility of these animals as models in which to investigate mast cell function depends on both the extent of their mast cell deficiency and the nature and extent of their other c-kit-related phenotypic abnormalities. The WBB6F₁-Kit^W/W-v mouse is currently the most commonly used animal model for such investigations. Kit^W/W-v mice are profoundly mast cell-deficient; the adult mice contain no detectable mast cell populations in the peritoneal cavity, gastrointestinal tract, respiratory system, heart, brain, skeletal muscle, spleen, and multiple other anatomical sites, and they exhibit <1% of the wild-type levels of skin mast cells by the time they reach 6 to 8 weeks of age.¹ However, the reduced c-kit function in these mice results in many other phenotypic abnormalities, including a macrocytic anemia, sterility, impaired melanogenesis, a virtual lack of interstitial cells of Cajal (ICC), and a decline in the number of intestinal TCR intraepithelial lymphocytes (IELs) with age.^8-12 Kit^W/W-v mice also develop a high incidence of spontaneous dermatitis,¹³ squamous papillomas of the fore-stomach,¹⁴ gastric ulcers,¹⁵ and dilatation of the duodenum.¹⁶

The W-sash mutation, which arose spontaneously in a cross between two inbred strains of mice (C3H/HeH x 101/H), was first described 23 years ago.¹⁷ However, C57BL/6-Kit^W-sh/W-sh mice only recently have begun to be used as a model for studies of mast cell function in vivo.¹⁸ W-sash (W^sh) is an inversion mutation in the transcriptional regulatory elements upstream of the c-kit transcription start site on mouse chromosome 5.¹⁹ Earlier work indicated that the W^sh mutation results in fewer developmental abnormalities than do those c-kit/W mutations which are null mutations or alter the c-kit coding region and thereby cause varying degrees of impairment of intrinsic c-kit receptor function. For example, until very recently, the only reported phenotypic abnormalities in Kit^W-sh/W-sh mice were an impairment of skin pigmentation and a mast cell-deficiency in the skin and peritoneal cavity.^18,20-23 Subsequently, it was reported that adult (10 weeks old) Kit^W-sh/W-sh mice are mast cell-deficient in multiple anatomical sites.²⁴ Moreover, Kit^W-sh/W-sh mice, unlike Kit^W/W-v mice, are fertile (with appreciable numbers of germ cells in their gonads) and are not anemic.^17,20 Accordingly, Kit^W-sh/W-sh mice may have ad-vantages for some mast cell studies, compared to the Kit^W/W-v and Kit^W-f/W-f mutants. Although recent studies demonstrate that Kit^W-sh/W-sh mice can be transplanted successfully with bone marrow-derived mast cells,^18,24 a detailed comparison of the phenotypic aberrations that are observed in mice carrying the Kit^W/W-v or Kit^W-sh mutant alleles has not yet been reported.

In the present study, we assessed the effects of the W^sh mutation on the development of multiple different cell lineages, including mast cells, basophils, and other hematopoietic and lymphoid cells, and ICC, and on the incidence of certain types of pathology that have been reported in Kit^W/W-v mice. Our results show that adult Kit^W-sh/W-sh mice are profoundly mast cell-deficient in all tissues compared with age-matched wild-type littermates, and that the heterozygous (Kit^+/W-sh) mice exhibit a mild mast cell deficiency in certain anatomical sites. Moreover, we demonstrate that Kit^W-sh/W-sh mice can accept transplantation of genetically compatible bone marrow-derived cultured mast cells (BMCMCs) with normal c-kit expression, by adoptive transfer of these cells via intraperitoneal, intradermal, or intravenous injection, without the development of other donor-derived hematopoietic cells. And even though Kit^W-sh/W-sh mice lacked ICC and demonstrated significant bile reflux into the stomach, the levels of hematopoietic cells (other than mast cells) and lymphoid cells, including TCR IELs, were normal in adult Kit^W-sh/W-sh mice, and these animals did not exhibit a high incidence of spontaneous pathology affecting the skin, stomach, or duodenum.

	Materials and Methods

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Mice

Mast cell-deficient Kit^W-sh/W-sh, C57BL/Ka-Thy1.1-CD45.1 (Ly5.2), and green fluorescent protein (GFP)-expressing C57BL/Ka-Thy1.1-CD45.2 (Ly5.1) mice were bred and maintained at the Stanford University Research Animal Facility. Kit^W-sh/W-sh mice on the C57BL/6 background were generously provided by Dr. Peter Besmer (Molecular Biology Program, Memorial Sloan-Kettering Cancer Center and Cornell University Graduate School of Medical Sciences, New York, NY).²¹ ß-Actin/enhanced green fluorescent protein (eGFP) transgenic mice on the C57BL/Ka-Thy1.1-CD45.2 (Ly5.1) background were generated as previously described using the pCXEGFP vector,²⁵ and allowed to freely imbibe acidified water (pH 2.5). Female 6-week-old WBB6F₁-Kit^W/W-v mice and their wild-type littermates were obtained from Jackson Laboratories (Bar Harbor, ME). All experiments were performed in compliance with the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources, National Research Council, and published by the National Academy Press (revised 1996) and the Stanford University Committee on Animal Welfare.

Preparation and Adoptive Transfer of BMCMCs into Mast Cell-Deficient Mice

Bone marrow cells from female Kit^+/+ (C57BL/6-Ly5.1 wild-type littermates, or C57BLKa-Ly5.2, or ß-act-eGFP-C57BLKa-Ly5.1) mice were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma Chemical Co., St. Louis, MO) supplemented with 20% WEHI-3 conditioned medium (as a source of IL-3) for 4 to 6 weeks, at which time >95% of the cells were identified as BMCMCs by May Grunwald-Giemsa staining and by flow cytometric analysis (for details, see Supplementary Methods at http://ajp.amjpathol.org). For mast cell reconstitution studies, BMCMCs were transferred by intradermal (1 x 10⁶ cells in 40 µl of DMEM/ear, or 4 x 10⁶ cells in 8 x 50-µl aliquots in two rows down the length of shaved back skin), or by intraperitoneal (2.5 to 5 x 10⁶ cells in 200 µl of DMEM), or by tail-vein or retro-orbital intravenous (1 x 10⁷ cells in 200 µl of DMEM) injection into 4-week-old female Kit^W-sh/W-sh mice. Six to eight weeks after intradermal or intraperitoneal transfer, or 12 weeks after intravenous injection, mice were sacrificed and tissues were assessed for repair of mast cell deficiency.

Bone Marrow Transplantation

Bone marrow cells were collected from femurs of wild-type littermates (C57BL/6-Ly5.1) and C57BL/Ka-Thy1.1-CD45.1 (Ly5.2). Red blood cells were lysed with 1 ml of ACK lysis buffer (Cambrex Bioscience, Walkersville, MD) for 5 minutes, and the remaining whole bone marrow (WBM) cells centrifuged at 1200 rpm for 5 minutes, and then resuspended in 1x phosphate-buffered saline (PBS) for injection. Female 4- to 6-week-old Kit^W-sh/W-sh mice were injected intraperitoneally or intravenously (retro-orbital) with 1 x 10⁶ WBM cells (from femurs) in 200 µl of PBS, or 1 x 10⁷ WBM cells in 100 µl of PBS, respectively. Some recipient Kit^W-sh/W-sh mice were lethally irradiated with a split dose of 950 rad to facilitate the bone marrow reconstitution, as described.²⁶ For irradiated mice receiving 1 x 10⁷ Ly5.2-expressing BMCMCs intravenously, a radioprotective dose of 3 x 10⁵ WBM cells obtained from Kit^+/+-Ly5.1 colony littermates was transferred simultaneously.

Flow Cytometric Analysis of Hematopoietic Lineages

Cells were blocked with unconjugated anti-FcRII/III (2.4G2; BD Pharmingen, San Diego, CA) then stained with PE/Cy7-conjugated anti-CD45.1 (A20; eBioscience, San Diego, CA) and a combination of PE- or APC-labeled antibodies specific for B220 (6B2), CD3 (145-2C11), CD4 (GK1.5), CD8 (53-6.7), CD11c (N418), F4/80 (BM8), Gr-1 (8C5), FcRI (MAR-1), CD49b (DX5), and CD117. Cells were analyzed by using a triple laser (407-nm krypton laser, 488-nm argon laser, and 598-nm dye laser) FACS Vantage SE/DiVa (Becton Dickinson, Mountain View, CA).

Staining and Quantification of Mast Cells

Mice were euthanized and samples of back skin, ear pinna, tongue, lung, spleen, trachea, heart, stomach, jejunum, ileum, colon, kidney, bladder, tail, liver, brain, and lymph nodes (submaxillary, axillary, and inguinal) were fixed in 10% buffered formalin, embedded in paraffin ensuring a cross-sectional orientation of all tissues, and 4-µm sections were cut. Mesenteric windows were arranged onto slides and fixed for 1 hour in Carnoy’s solution (3:2:1 v/v/v of ethanol, chloroform, and acetic acid). All tissues, with the exceptions of ear pinna and brain, were stained with Csaba stain for mast cell detection.²⁷ Csaba stain contains both safranin (red, identifying mature mast cells) and alcian blue (blue, identifying less mature mast cells), which bind to mast cell granules. For ear and brain sections, mast cells were stained metachromatically with 0.1% toluidine blue, pH 1 (cytoplasmic granules appear purple). Mast cells were quantified according to area (mm²) or expressed in numbers per mm horizontal field length (back skin and ear pinna) using computer-generated image analysis (NIH Image J software, version 1.29x) (for details, see Supplementary Methods at http://ajp.amjpathol.org).

Immunohistochemical Detection of ICC

Samples of freshly dissected stomach, ileum, and colon were gently flushed in PBS to remove luminal contents, and prepared for cryopreservation as previously described.^28,29 Briefly, tissues were treated with a series of graded sucrose solutions (5%, 10%, 15%, and 20% sucrose in PBS) for 20 to 30 minutes each on ice, and embedded overnight at 4°C in a solution consisting of OCT (Tissue Tek, IL) and 20% sucrose in PBS (1 part/2 parts; v/v). Tissues were then embedded in OCT ensuring a cross-sectional orientation and rapidly frozen on dry ice. Frozen sections (10 µm) were cut and fixed in ice-cold acetone for 2 to 5 minutes. After incubation with 10% normal rabbit serum for 30 minutes at room temperature, sections were treated overnight at 4°C with 0.6 µg/ml goat polyclonal anti-c-kit IgG (sc-1494; Santa Cruz Biotechnology, Santa Cruz, CA). Immunoreactivity was detected with a 1:40 dilution of fluorescein isothiocyanate-conjugated rabbit anti-goat IgG (DAKO, Carpinteria, CA) incubated for 2 hours at room temperature. Control tissues were prepared in a similar manner but with the omission of the primary antibody. Images were captured using a confocal microscope (Eclipse TE300; Nikon, Melville, NY) with an excitation wavelength for fluorescein isothiocyanate fluorescence (488 nm), and LaserSharper2000, version 5.2, software. For detection of ICC in the stomach and ileum of 7-day-old, 14-day-old, and 4-week-old C57BL/6-Kit^+/+ and Kit^W-sh/W-sh mice (Supplementary Figure 1 at http://ajp.amjpathol.org), a Z-series of up to 11 images through a depth of 10 µm were collected and merged to form a confocal micrograph.

Measurement of Bile Acids in the Stomach

Mutant mice of the Kit^W-sh/W-sh and Kit^W/W-v genotypes, together with their respective wild-type littermates were assessed for stomach concentrations of total bile acids 7 days, 4 to 6 weeks, and 10 to 14 weeks after birth, as previously described.³⁰ Briefly, mice were starved for 6 hours, euthanized, and the stomachs ligated at the distal (anal) and proximal (oral) ends, and then removed. Distilled water (0.7 ml) was injected into each stomach with a syringe and needle, and the gastric contents were collected after gentle pipetting. The pH of the gastric contents was corrected to the range of pH 6.5 to 7.5 and all samples were adjusted to 1 ml with addition of distilled water. The concentration of total bile acids of each sample was measured using a bile acids kit (Trinity Biotech USA, MO). Bile acids are first oxidized to 3-oxo bile acids with the catalytic enzyme 3-hydroxysteroid dehydrogenase. During this reaction an equimolar quantity of nicotinamide adenine dinucleotide (NAD) is reduced to nicotinamide adenine dinucleotide (NADH). The NADH is subsequently oxidized to NAD with concomitant reduction of nitro blue tetrazolium salt to formazan by the catalytic action of diaphorase. The color of the resulting diformazan was measured at 530 nm with a spectrophotometer. The intensity of the color produced is directly proportional to the bile acid concentration in the sample. The concentration of total bile acids in the samples was calculated using a bile acids calibrator (Trinity Biotech USA), in which the difference in absorption between the test and blank reagents of each sample was divided by the difference in absorption between the test and blank reagents of the calibrator, and the resulting value multiplied by the concentration of the calibrator to yield gastric content bile acid concentration, expressed as µmol/L. Quality control of each assay and between assays was monitored with the inclusion of control sera with known bile acids concentration (bile acids control set, Trinity Biotech USA). Eight assays were performed and the mean ± SD together with the coefficient of variation (percent) of the calibrator, normal control serum, and abnormal control serum, were 0.405 ± 0.015 (3.7%), 6.61 ± 0.38 (5.7%), and 40.02 ± 1.8 (4.5%), respectively.

Statistics

A multiple comparison procedure using an analysis of variance and Fisher’s test was used to determine statistical significance between groups (n = 3 to 16 mice/group). Probabilities 0.05 were considered significant.

	Results

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Hematopoietic and Lymphoid Cell Populations

Adult Kit^W-sh/W-sh mice displayed normal levels of B cells in bone marrow and spleen (Figure 1A) ; T cells in thymus and spleen (Figure 1B) ; myeloid cells (granulocytes and macrophages) in bone marrow, spleen, and peritoneal cavity (Figure 1D) ; dendritic cells and natural killer cells in the spleen (Figure 1E) ; and basophils in bone marrow and spleen (Figure 1F) . In addition, these mice also exhibited normal levels of small intestinal TCR and TCRß IELs at 16 weeks of age (Figure 1C) . By contrast, in agreement with the findings of Puddington and colleagues,¹² we confirmed that age-matched WBB6F₁-Kit^W/W-v mice displayed a significant deficit in TCR IELs, with a concomitant increase in TCRß IELs (Figure 1C) .

View larger version (52K): [in this window] [in a new window]   Figure 1. Levels of hematopoietic cells in adult c-kit mutant C57BL/6-KitW-sh/W-sh mice and the congenic wild-type littermates. For analysis of each lineage, three to four mice per group were investigated, with very similar results obtained for all of the mice within each group. The plots presented in this figure are the results obtained from one mouse chosen as representative of the data from each group. A: B-cell populations in spleen and bone marrow. B: CD4+ and CD8+ T cells in thymus and spleen. C: KitW-sh/W-sh mice exhibit normal numbers of small intestinal TCR and TCRß IELs, whereas KitW/W-v mice exhibit depletion of TCR IELs and a corresponding increase in TCRß IELs. D: Myeloid cell populations (granulocytes and macrophages) in bone marrow, spleen, and peritoneal cavity. E: CD11c+ dendritic cell and CD49b+ natural killer cell populations in spleen (NKT cell populations were not specifically analyzed). F: c-Kitlo FcRI+ basophils in bone marrow and spleen.

In contrast to the normal levels of the aforementioned hematopoietic cell populations, morphological evidence indicates that adult (10 weeks of age) Kit^W-sh/W-sh mice, like Kit^W/W-v mice, lack the network of c-kit⁺ ICC that is associated with Auerbach’s nerve plexus and that provides interstitial pacemaker activity of the stomach, ileum, and colon (Figure 2 ; D to F and G to I). Further analysis of 7-day-old, 14-day-old, and 4-week-old Kit^W-sh/W-sh mice demonstrated that, unlike in the wild-type littermates, no ICC were detected in the longitudinal and circular muscle layers of the stomach and ileum at all ages investigated (Supplementary Figure 1 at http://ajp.amjpathol.org). This contrasts with mutants of the Kit^Wbd/Wbd genotype, which like the Kit^W-sh mutation, contain a 2.8-Mb inversion 5' of Kit.³¹ At postnatal day 5, Kit^Wbd/Wbd mice exhibited equivalent numbers of ICC compared to their wild-type littermates, and the numbers of ICC significantly diminished in the mutants by postnatal day 15, eventually decreasing to the extent that the adult Kit^Wbd/Wbd mice lacked a functional ICC network and intestinal pacemaker activity.³¹

View larger version (39K): [in this window] [in a new window]   Figure 2. Morphological identification of c-Kit expressing ICC in the stomach (A, D, G, J), ileum (B, E, H, K), and colon (C, F, I, L) in wild-type C57BL/6-Kit+/+ (A–C) and WBB6F1-Kit+/+ mice (G–I), but ICC were not detected in mutant C57BL/6-KitW-sh/W-sh (D–F) or WBB6F1-KitW/Wv mice (J–L). A and G: Cryostat cross-sections show detection of ICC within the longitudinal muscle (Lm) and in the circular muscle (Cm) layers of the gastric fundus, orientated parallel to longitudinal muscle cells (arrowheads) and observed in cross-section within the circular muscle bundles (arrows). B and H: Cross-sections through the ileum reveal ICC between the Lm and Cm at the level of the myenteric plexus (arrowheads) and at the level of the deep muscular plexus (arrows). C and I: ICC in the proximal colon were identified at the level of the myenteric plexus (arrowheads) within the Cm (small arrow) and along the submucosal surface of the circular muscle layer (arrows). Original magnifications, x400.

In addition, Kit^W-sh/W-sh mice are profoundly mast cell-deficient (containing no detectable mast cells) in all tissues studied, with the exception of the skin (of back and ear pinna) where an age-dependent reduction in mast cell density occurred by 12 weeks of age (see Supplementary Table 1 at http://ajp.amjpathol.org). The numbers of dermal mast cells in back skin of 4-week-old Kit^W-sh/W-sh mice were 16.5% that of the age-matched wild-type littermates, whereas at 6 weeks of age the number dropped to 12.1% that of Kit^+/+ mice, and continued to decrease to 7.2% at 10 weeks and 1.2% at 12 weeks after birth; an observation consistent with the report of Yamazaki and colleagues.²² In the majority of tissues examined, with the exceptions of forestomach, trachea, and spleen, significantly fewer mast cells were observed in the heterozygous Kit^+/W-sh mice compared to the age-matched wild-type counterparts. Flow cytometric analysis of c-Kit levels expressed by peritoneal mast cells in adult Kit^+/W-sh mice demonstrated a lower mean fluorescent intensity of c-Kit and a reduced percentage of mast cells present in the peritoneal cavity, when compared with age-matched wild-type littermates [Supplementary Figure 3 at http://ajp.amjpathol.org; c-Kit MFI = 69 ± 7.8, FcRI MFI = 478 ± 15, percent peritoneal mast cells = 0.77 ± 0.23, and c-Kit MFI = 478 ± 15, FcRI MFI = 660 ± 213, percent peritoneal mast cells = 3.8 ± 0.1, P = 0.003, P = 0.1, and P = 0.0005 in Kit^+/W-sh and Kit^+/+ mice (three mice per group), respectively]. Reduced expression of functional c-Kit on the surface of peritoneal mast cells in the heterozygous mice may account for the lower mast cell percentages observed in the peritoneal lavage fluid from these mice; this mechanism also may contribute to the lower numbers of mast cells observed in other anatomical sites in the heterozygous mice (Supplementary Table 1 at http://ajp.amjpathol.org). The fact that there is c-Kit expression, albeit significantly reduced from wild-type levels, may explain why the diminished mast cell levels in the heterozygous mice were not as profoundly reduced as those observed in Kit^W-sh/W-sh mice.

Selective Repair of Mast Cell Deficiency in Kit^W-sh/W-sh Mice by Adoptive Transfer of Kit^+/+ BMCMCs

Cultured mast cells of C57BL/6-Kit^+/+ mouse origin were harvested 4 to 6 weeks after the initiation of culture, and 1 to 4 x 10⁶, 2.5 to 5 x 10⁶ or 10⁷ cells were injected intradermally, intraperitoneally, or intravenously, respectively, into individual Kit^W-sh/W-sh mice. Six to eight weeks after intradermal transfer of BMCMCs, mice displayed significant local repair of mast cell populations in back skin and ear pinna compared to untreated age- and gender-matched Kit^W-sh/W-sh mice (Figure 3, A and B , for dermis of back skin, P < 0.0001 for either GFP⁺ or WT BMCMCs; for ear pinna, P < 0.01 and P < 0.005, respectively; and Supplementary Table 2 at http://ajp.amjpathol.org). Although Kit^W-sh/W-sh mice successfully accepted intracutaneous transfer of congenic BMCMCs, only those mice that received wild-type-derived mast cells from littermates had dermal mast cell densities in back skin that were comparable to the levels observed in the littermate wild-type Kit^+/+ mice (Figure 3A ; Supplementary Table 2 at http://ajp.amjpathol.org). Eight weeks after intradermal reconstitution of Kit^W-sh/W-sh mice, the anatomical distributions of back skin mast cells in the upper dermis and the dermal fat were similar to those observed in wild-type mice (Figure 4; A to C) .

View larger version (41K): [in this window] [in a new window]   Figure 3. Mast cell numbers (per mm horizontal length of skin or per mm2) in dorsal (back) skin (A); ear pinna (B); peritoneal cavity (percent mast cells in lavage) (C); mesenteric window (D); jejunum, ileum, and colon (E); stomach (F); lung parenchyma (G); and spleen (H) of C57BL/6-Kit+/+ (black diamond), KitW-sh/W-sh (white diamond) and Kit+/+ (gray triangle), or GFP+ (gray diamond), or Ly5.2+ (gray circle) BMCMC-engrafted KitW-sh/W-sh mice. Samples of tissues were obtained 6 to 8 weeks after intraperitoneal or intradermal or 12 weeks after intravenous adoptive transfer of BMCMCs. The mean for each group is indicated on the graphs. *, **, and ***: P < 0.05, 0.01, and 0.001,respectively, versus corresponding values for mast cell-deficient KitW-sh/W-sh mice. Supplementary Table 2 at http://ajp.amjpathol.org shows the mean ± SD data for mast cell numbers in all tissues/sites examined in each group, as well as the number of mice per group and additional statistical analysis of the results.

View larger version (121K): [in this window] [in a new window]   Figure 4. Histological sections showing the dermis and dermal fat of back skin (A–C), mesenteric windows (D–F), and the submucosa and muscularis propria of the glandular stomach (E-I) in C57BL/6-Kit+/+ (A, D, G), KitW-sh/W-sh (B, E, H), and mast cell knockin KitW-sh/W-sh mice that had been injected with congenic GFP+ BMCMCs intradermally (C), intraperitoneally (F), or intravenously (I). Mast cells were not detected in the majority of tissue sections from KitW-sh/W-sh mice, but a few mast cells were sometimes detected in back skin. Arrows: mast cells. Original magnifications: x200; x1000 (insets).

Development of Mast Cell Populations in Kit^W-sh/W-sh Mice by Transfer of Congenic Kit^+/+ WBM Cells

To determine whether Kit^W-sh/W-sh mice can be repaired of their mast cell deficiency by the transfer of Kit^+/+ WBM cells, we injected into recipient Kit^W-sh/W-sh mice 1 x 10⁶ cells intraperitoneally or 1 x 10⁷ cells by retro-orbital intravenous injection, using bone marrow cells derived from C57BL/Ka-Thy1.1-CD45.1 (Ly5.2) donor mice. Six weeks after intraperitoneal transfer, mast cell populations were observed in the mesenteric windows and submucosa of the stomach (Table 2) . With the exception of the glandular stomach, mast cell densities in Kit^W-sh/W-sh mice injected with bone marrow cells were significantly lower than those observed in Kit^+/+ littermates or in Kit^W-sh/W-sh mice that had been reconstituted with Ly5.2⁺ BMCMC. At 12 weeks after transfer, mice that received WBM cells intravenously did not display any cell lineages of Ly5.2⁺ donor origin, including mast cells, in all of the tissues studied (Table 2) .

Irradiation (950 rad, split dose) of recipient Kit^W-sh/W-sh mice before bone marrow transplantation rendered the mice able to develop mast cell populations, with mast cell densities in the stomach, jejunum, ileum, colon, lung, spleen, and lymph nodes that were significantly higher than those found in wild-type mice (Table 2) . However, such mice also developed other hematopoietic lineages of donor origin, with donor-derived cells accounting for 90% of cells in the femoral bone marrow, 92% of cells in the spleen, as well as 95% of cells in the peritoneal cavity. Irradiation also permitted mast cell development in mice injected intravenously with Ly5.2⁺ BMCMCs (last column of Table 2 ), as mast cell levels were significantly higher in the stomach (in which mast cells were detected not only in the muscularis, but also the submucosa), lung, and skin compared to those in nonirradiated Kit^W-sh/W-sh mice that received BMCMCs intravenously (Figure 3; A, C, and G) .

	Discussion

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Our data demonstrate that although there are some similarities in the phenotypes of Kit^W-sh/W-sh and Kit^W/W-v mice, there are also significant differences. As is discussed in more detail below, each mutant exhibits a profound mast cell deficiency in all tissues examined. Moreover, we have shown that, like Kit^W/W-v mice, adult Kit^W-sh/W-sh mice essentially lack the network of ICC in the gastrointestinal tract (Figure 2) . However, as summarized in Table 3 , Kit^W-sh/W-sh mice exhibit a more restricted set of other phenotypic abnormalities than do Kit^W/W-v mice.

Our data demonstrate that Kit^W-sh/W-sh mice, like Kit^W/W-v mice, can be selectively repaired of their mast cell deficiency by systemic intravenous or local intraperitoneal or intradermal injection of genetically compatible BMCMC populations. BMCMCs expressing either CD45.1 (Ly5.2) or GFP were used for injection into Kit^W-sh/W-sh mice, thereby providing either of two markers (ie, Ly5.2 or GFP) by which donor-derived cells could be identified in the recipient animals. This approach not only proved that the mast cells that were identified later in these Kit^W-sh/W-sh mice were of donor origin, but also demonstrated that no other cell types of donor origin could be detected in these mice. That is, Kit^W-sh/W-sh mice, like Kit^W/W-v mice, can be used to make mast cell knockin mice (ie, c-kit mutant genetically mast cell-deficient mice in which mast cell populations have been selectively transferred).

Moreover, like Kit^W/W-v mice^2,32,33 Kit^W-sh/W-sh mice that have been locally repaired of their mast cell deficiency by injection of BMCMCs into the skin or peritoneal cavity exhibit anatomical localization and tissue prevalence of mast cells that are similar to those of the corresponding mast cell populations in wild-type mice (Figures 3 and 4 ; Supplementary Table 2 at http://ajp.amjpathol.org). However, after intravenous administration of BMCMCs into irradiated Kit^W-sh/W-sh mice, we repeatedly observed that these recipient mice had significantly higher densities of mast cells in the lung and spleen than those found in the same tissues of the Kit^+/+ littermates (Figure 3 ; Supplementary Table 2 at http://ajp.amjpathol.org). This phenomenon has also been observed in the lung tissue of Kit^W/W-v mice that received BMCMCs intravenously,^34,35 perhaps because the lung is the first organ that BMCMCs encounter after tail vein injection.

Although Kit^W-sh/W-sh mice readily accepted transplantation of in vitro-derived mast cells, as has also been reported by others,^18,24 establishment of mast cell populations in these mice by intravenous injection of congenic Kit^+/+ bone marrow cells was less successful (Table 2) . In nonirradiated Kit^W-sh/W-sh mice, intravenous transfer of 10 million WBM cells failed to establish any detectable mast cells in the recipients, as assessed 12 weeks after injection (Table 2) . By contrast, 6 weeks after injection of such bone marrow cells into the peritoneal cavity, mast cell populations were detected in some mesenteric windows and in the submucosa of the stomach of recipient Kit^W-sh/W-sh mice, indicating that these WBM cell populations indeed contained cells capable of giving rise to mature mast cells in Kit^W-sh/W-sh mice in vivo. Irradiation of the recipient Kit^W-sh/W-sh mice before intravenous transfer of 10 million WBM cells enabled mast cell reconstitution (Table 2) , but also resulted in the establishment of other hematopoietic cell lineages in these mice, as indicated by detection of Ly5.2 expression in these donor-derived cells. Thus, this approach cannot be used to achieve selective repair of the mast cell deficiency of Kit^W-sh/W-sh mice.

These results differ from previously published findings of successful, albeit nonselective, mast cell reconstitution of nonirradiated Kit^W/W-v mice by intravenous injection of WBM cells.^1,34 Our study is similar in experimental design to the one performed by Kitamura and colleagues¹, in that donors and recipients of the cells were female and mice were sacrificed 12 weeks after transplantation. Why these two lines of c-kit mutant mast cell-deficient mice differ in their receptivity to rescue by systemic bone marrow transplantation is not clear, but may be due to availability of suitable niches for the donor cells in the hematopoietic stem cell compartment; a site that is dependent on functional c-Kit signaling for stem cell survival.³⁶ In Kit^W/W-v mice, hematopoietic stem cells do not express any c-Kit protein³⁷ whereas there is expression of c-Kit, albeit at levels slightly lower than those observed in the wild-type mice, in the hematopoietic stem cells of Kit^W-sh/W-sh mice.³⁸

An interesting phenotypic feature of the W^sh mutation that has not previously been reported is the dilution effect on mast cell prevalence observed in the heterozygous Kit^+/W-sh mice. Studies of the parental heterozygous mice of the WBB6F₁-Kit^W/W-v hybrid show that back skin mast cell density is slightly (36%) decreased at 8 weeks of age in C57BL/6-Kit^+/W-v mice but not in WBRej-Kit^+/W mice.³⁹ By contrast, mast cell levels in the back skin of 8-week-old Kit^+/W-sh mice are 59% reduced compared to those in the age-matched wild-type mice, and decrease further to a reduction of 65% by 12 weeks of age. Kit^+/W-sh mice (12 weeks old) also exhibited significant reductions in mast cell populations of 24%, 67%, 59%, 88%, and 43% (compared to those in wild-type mice) in the skin of the ear pinna and the mesentery, glandular stomach, heart, and tongue, respectively (Supplementary Table 1 at http://ajp.amjpathol.org).

The differential effect on mast cell numbers of the various mutant alleles of c-kit may be explained by their molecular nature. The W mutant allele produces truncated c-Kit proteins without the transmembrane domain, which, in turn, results in no expression of these proteins on the cell surface.⁴⁰ By contrast, W^v is a point mutation at the tyrosine kinase-encoding domain of c-kit; although this mutation reduces tyrosine kinase activity, the c-Kit protein encoded by W^v is normally expressed on the cell surface.^40,41 Cell surface expression is important to permit dimerization of c-Kit receptors on ligand binding, and thereby to initiate signaling.⁴⁰ Heterozygous Kit^+/W mice produce c-Kit receptors that are composed only of normal c-Kit proteins, whereas receptors of Kit^+/W-v mice consist of both wild-type and abnormal c-Kit proteins, with consequences for c-kit receptor signaling and mast cell development.⁴¹ However, because the W-sash mutation is not in the c-kit coding sequences, the production of a functionally impaired c-Kit protein cannot be the explanation for the reduction in the numbers of certain mast cell populations in Kit^+/W-sh mice. Indeed, in more general terms, because the W-sash mutation occurs in the transcriptional regulatory region of the c-kit gene, this mutation can affect the temporal and/or anatomical expression of c-kit in mast cells²² and other cell lineages²³ in the developing and adult mutant animals. Such effects, in turn, may account for some of the W-sash phenotypes observed,^22,23 including the decline in skin mast cell c-kit expression in embryonic versus newborn Kit^W-sh/W-sh mice.²²

The phenotypic features of Kit^W-sh/W-sh mice are of interest both because of their relevance to decisions about whether such mice would be suitable for certain studies of mast cell function and also because of insights they might provide into the mechanisms that underlie some of the phenotypic abnormalities that have been reported in other c-kit mutant mice. For example, the absence of the c-Kit-dependent ICC population in Kit^W/W-v mice causes defective intestinal pacemaker activity and absence of slow wave peristalsis on gastric emptying,^11,42 which in turn is thought to contribute to bile reflux from the duodenum into the stomach.¹⁶ In aggregate, these defects are thought to contribute to the development of forestomach papillomas and antral ulcers, lesions that some studies report occur in 40% of Kit^W/W-v mice by the age of 1 to 6 months.^14,43

By contrast, although we have established that Kit^W-sh/W-sh mutants lack detectable gastrointestinal tract ICC networks as early as postnatal day 7, and they exhibit greater concentrations of bile acids in the stomach compared with those in the wild-type littermates (Table 1) , we have not yet detected the development of spontaneous stomach lesions (either antral ulcers or forestomach papillomas) that are grossly detectable at autopsy in any of the Kit^W-sh/W-sh mutants raised in our laboratory that so far have been examined. This represents more than 550 mice, which were analyzed at ages ranging from birth to older than 1 year, including >60 that were older than 6 months in age. Furthermore, we have not yet observed significant dilatation of the duodenum, or gross abnormalities of the gastroduodenal junction, in Kit^W-sh/W-sh mice. Thus, the width of the uncut duodenum, measured 2 mm from the pylorus, was 2.8 ± 0.4 mm and 2.9 ± 0.3 mm for female, 10- to 12-week-old Kit^W-sh/W-sh (n = 11) and wild-type (n = 12) mice, respectively, whereas the circumference of the duodenum, after cutting it open longitudinally, flattening it out, and measuring it 2 mm from the pylorus in the same mice was 5.8 ± 0.9 mm and 5.7 ± 1.0 mm for Kit^W-sh/W-sh mice and wild-type mice, respectively. These observations suggest that factors in addition to a lack of ICC may contribute to the gastric pathology observed in Kit^W/W-v mice. However, it is possible that the higher concentrations of bile acids in the stomachs of young Kit^W/W-v as opposed to Kit^W-sh/W-sh mutants may contribute, at least in part, to the apparently increased susceptibility of Kit^W/W-v as opposed to Kit^W-sh/W-sh mutants to develop stomach lesions.

The presence of normal levels of TCR and TCRß IELs in 16-week-old Kit^W-sh/W-sh mice is another potentially significant difference in phenotype compared to that of Kit^W/W-v mice (Figure 1C) . Puddington and colleagues¹² first demonstrated the depletion of CD8⁺ TCR IELs and a concomitant increase in CD4⁺CD8⁺ TCRß IELs beginning at 6 weeks of age in Kit^W/W-v mutants, and we confirmed their finding in this study. Although the role of this population of T cells in immunity is yet to be fully understood, evidence from studies of parasite infections with Eimeria vermiformis suggests that TCR IELs may have an important function in attenuating the inflammatory response induced by TCRß T cells.⁴⁴ Accordingly, alterations in the levels of IEL populations must be kept in mind when interpreting data regarding immunoregulation and/or clearance of parasites or other enteric pathogens in studies using Kit^W/W-v versus wild-type mice. By contrast, our data indicate that this issue is less of a concern with Kit^W-sh/W-sh mice, which have normal levels of TCR IELs in the small intestines. In addition to having no apparent deficit in small intestinal TCR IELs, adult Kit^W-sh/W-sh mice, like adult Kit^W/W-v mice, also exhibit normal levels of populations of B cells, T cells, myeloid cells (including basophils), dendritic cells, and NK cells (NKT cell populations were not analyzed).

Another important advantage of Kit^W-sh/W-sh mice is that these mutants do not exhibit a high incidence of spontaneous idiopathic dermatitis with advancing age, at least in comparison with WBB6F₁-Kit^W/W-v mice. In fact, no Kit^W-sh/W-sh mice with grossly evident spontaneous dermatitis have been detected in our colony, including >40 that ranged in age from 6 months to 1 year. By contrast, we found that a high incidence of spontaneous idiopathic dermatitis occurs in Kit^W/W-v mice (50% of mice developed lesions affecting the ear pinna and/or other sites by 8 months of age) and that this condition is associated with the development of significant populations of mast cells at sites of severe dermatitis in the affected mice.¹³ WBB6F₁-Kit^W/W-v mice with severe idiopathic dermatitis of the ear can develop mast cell numbers as high as 33 ± 5 mast cells/mm², a level that is one third that of age-matched WBB6F₁-Kit^+/+ mice and 50-fold higher than that in unaffected Kit^W/W-v mice.¹³ Moreover, these lesions can occur even in WBB6F₁-Kit^W/W-v mice as young as 3 months of age (our unpublished data). The development of such skin lesions in Kit^W/W-v mice can be very problematic when the mice are being used for long-term experiments, such as those that require transplantation of BMCMCs and/or those that investigate chronic disease models, particularly if these are to be analyzed in the skin. Accordingly, the apparently much lower incidence of such lesions in C57BL/6-Kit^W-sh/W-sh mice represents a significant advantage of this mutant for studies that require the use of relatively old mice.

As summarized in Table 3 , C57BL/6-Kit^W-sh/W-sh mice offer some appealing advantages over WBB6F₁-Kit^W/W-v mice as a model for analyzing certain mast cell functions in vivo. The fertility of the Kit^W-sh/W-sh mice, as well as the fact that they are on the C57BL/6 background (as are many other mutant mice with interesting phenotypes), are advantageous when one wishes to produce mast cell-deficient mice that also have other defined genetic abnormalities. By comparison, more elaborate breeding strategies using the parental heterozygous mice must be used for this purpose in the case of the sterile WBB6F₁-Kit^W/W-v mice.^45,46 The Kit^W-sh/W-sh mice are also not anemic and express normal levels of TCR IELs in the small intestine, and, based on our experience to date, appear less likely than Kit^W/W-v mice to develop either pathology affecting the stomach or chronic idiopathic dermatitis. However, the deficiency of ICC in adult Kit^W-sh/W-sh mice appears to be as significant as that in the Kit^W/W-v mice.

In conclusion, our data provide further phenotypic characterization of Kit^W-sh/W-sh mice, and indicate that mast cell-deficient mice of this genotype, whose mutation is outside of the c-kit coding region and which are not anemic, are fertile and have a more limited panel of other phenotypic abnormalities than Kit^W/W-v mice, may represent a useful model for mast cell research, and, especially, for analyzing certain mast cell functions in vivo.

	Acknowledgements

 
We thank Z.-S. Wang, Alian Xu, and David Guthrie for their help with histological processing and analysis of tissues; and Peter Besmer for providing Kit^W-sh/W-sh mice.

	Footnotes

 
Address reprint requests to Stephen J. Galli, Department of Pathology, L-235, Stanford University School of Medicine, 300 Pasteur Dr., Stanford, CA 94305-5324. E-mail: sgalli{at}stanford.edu

Supported by the Australian National Health and Medical Research Council (C.J. Martin fellowship to M.A.G.) and the United States Public Health Service (grants AI23990, CA72074, and HL67674, project 1 to S.J.G.).

The authors have no conflicting financial interests.

Accepted for publication May 17, 2005.

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