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

2005; Elsevier BV; Volume: 167; Issue: 3 Linguagem: Inglês

10.1016/s0002-9440(10)62055-x

1525-2191

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

Polyamine Metabolism and Applications

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 KitW/W-v mice impair melanogenesis and result in anemia, sterility, and markedly reduced levels of tissue mast cells. In contrast, KitW-sh/W-sh mice, bearing the W-sash (Wsh) inversion mutation, have mast cell deficiency but lack anemia and sterility. We report that adult KitW-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 KitW/W-v mice, KitW-sh/W-sh mice had normal numbers of TCRγδ intraepithelial lymphocytes in the intestines and did not exhibit a high incidence of idiopathic dermatitis, ulcers, or squamous papillomas of the stomach, but like KitW/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 KitW-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, KitW-sh/W-sh mice represent a useful model for mast cell research, especially for analyzing mast cell function in vivo. 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 KitW/W-v mice impair melanogenesis and result in anemia, sterility, and markedly reduced levels of tissue mast cells. In contrast, KitW-sh/W-sh mice, bearing the W-sash (Wsh) inversion mutation, have mast cell deficiency but lack anemia and sterility. We report that adult KitW-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 KitW/W-v mice, KitW-sh/W-sh mice had normal numbers of TCRγδ intraepithelial lymphocytes in the intestines and did not exhibit a high incidence of idiopathic dermatitis, ulcers, or squamous papillomas of the stomach, but like KitW/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 KitW-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, KitW-sh/W-sh mice represent a useful model for mast cell research, especially for analyzing mast cell function in vivo. Genetically mast cell-deficient c-kit mutant mice have become a powerful tool for identifying and quantifying the contributions of mast cells in many biological responses in vivo. Mice carrying spontaneous loss-of-function mutations at both alleles of the dominant white spotting (W) locus (ie, c-kit), exhibit a marked reduction in c-kit tyrosine kinase-dependent signaling, resulting in disrupted normal mast cell development and survival,1Kitamura Y Go S Hatanaka K Decrease of mast cells in W/Wv mice and their increase by bone marrow transplantation.Blood. 1978; 52: 447-452Crossref PubMed Google Scholar, 2Kitamura Y Heterogeneity of mast cells and phenotypic change between subpopulations.Annu Rev Immunol. 1989; 7: 59-76Crossref PubMed Google Scholar and therefore mast cell function, as well as many other phenotypic abnormalities that are unrelated to the mast cell deficiency.3Russell ES Hereditary anemias of the mouse: a review for geneticists.Adv Genet. 1979; 20: 357-459Crossref PubMed Scopus (844) Google Scholar, 4Galli SJ Kitamura Y Genetically mast-cell-deficient W/Wv and Sl/Sld mice: their value for the analysis of the roles of mast cells in biologic responses in vivo.Am J Pathol. 1987; 127: 191-198PubMed Google Scholar, 5Galli SJ Kalesnikoff J Grimbaldeston MA Piliponsky AM Williams CMM Tsai M Mast cells as “tunable” effector and immunoregulatory cells: recent advances.Annu Rev Immunol. 2005; 23: 749-786Crossref PubMed Scopus (1075) Google Scholar Several different W mutant rodents have been investigated as potential models for the analysis of mast cell function in vivo, including KitW/W-v and KitW-f/W-f mice and KitW-s/W-s rats.1Kitamura Y Go S Hatanaka K Decrease of mast cells in W/Wv mice and their increase by bone marrow transplantation.Blood. 1978; 52: 447-452Crossref PubMed Google Scholar, 6Geissler EN McFarland EC Russell ES Analysis of pleiotropism at the dominant white-spotting (W) locus of the house mouse: a description of ten new W alleles.Genetics. 1981; 97: 337-361PubMed Google Scholar, 7Niwa Y Kasugai T Ohno K Morimoto M Yamazaki M Dohmae K Nishimune Y Kondo K Kitamura Y Anemia and mast cell depletion in mutant rats that are homozygous at “white spotting (Ws)” locus.Blood. 1991; 78: 1936-1941PubMed Google Scholar 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-KitW/W-v mouse is currently the most commonly used animal model for such investigations. KitW/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 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 × 106 cells in 40 μl of DMEM/ear, or 4 × 106 cells in 8 × 50-μl aliquots in two rows down the length of shaved back skin), or by intraperitoneal (2.5 to 5 × 106 cells in 200 μl of DMEM), or by tail-vein or retro-orbital intravenous (1 × 107 cells in 200 μl of DMEM) injection into 4-week-old female KitW-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 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 1× phosphate-buffered saline (PBS) for injection. Female 4- to 6-week-old KitW-sh/W-sh mice were injected intraperitoneally or intravenously (retro-orbital) with 1 × 106 WBM cells (from femurs) in 200 μl of PBS, or 1 × 107 WBM cells in 100 μl of PBS, respectively. Some recipient KitW-sh/W-sh mice were lethally irradiated with a split dose of 950 rad to facilitate the bone marrow reconstitution, as described.26Morrison SJ Weissman IL The long-term repopulating subset of hematopoietic stem cells is deterministic and isolatable by phenotype.Immunity. 1994; 1: 661-673Abstract Full Text PDF PubMed Scopus (854) Google Scholar For irradiated mice receiving 1 × 107 Ly5.2-expressing BMCMCs intravenously, a radioprotective dose of 3 × 105 WBM cells obtained from Kit+/+-Ly5.1 colony littermates was transferred simultaneously. Cells were blocked with unconjugated anti-FcγRII/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), FcεRIα (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). 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.27Culling CFA Dunn WL Handbook of Histopathological and Histochemical Techniques (Including Museum Techniques). ed 3. Butterworths and Co., London1974: 419Google Scholar 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 1.29×) (for details, see Supplementary Methods at http://ajp.amjpathol.org). Samples of freshly dissected stomach, ileum, and colon were gently flushed in PBS to remove luminal contents, and prepared for cryopreservation as previously described.28Burns AJ Herbert TM Ward SM Sanders KM Interstitial cells of Cajal in the guinea-pig gastrointestinal tract as revealed by c-Kit immunohistochemistry.Cell Tissue Res. 1997; 290: 11-20Crossref PubMed Scopus (199) Google Scholar, 29Malysz J Thuneberg L Mikkelsen HB Huizinga JD Action potential generation in the small intestine of W mutant mice that lack interstitial cells of Cajal.Am J Physiol. 1996; 271: G387-G399PubMed Google Scholar 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 KitW-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. Mutant mice of the KitW-sh/W-sh and KitW/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.30Isozaki K Hirota S Nakama A Miyagawa J-I Shinomura Y Xu Z Nomura S Kitamura Y Disturbed intestinal movement, bile reflux to the stomach, and deficiency of c-kit-expressing cells in Ws/Ws mutant rats.Gastroenterology. 1995; 109: 456-464Abstract Full Text PDF PubMed Scopus (167) Google Scholar 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 3α-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. 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. Adult KitW-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 TCRγδ and TCRαβ IELs at 16 weeks of age (Figure 1C). By contrast, in agreement with the findings of Puddington and colleagues,12Puddington L Olson S Lefrancois L Interactions between stem cell factor and c-Kit are required for intestinal immune system homeostasis.Immunity. 1994; 1: 733-739Abstract Full Text PDF PubMed Scopus (117) Google Scholar we confirmed that age-matched WBB6F1-KitW/W-v mice displayed a significant deficit in TCRγδ IELs, with a concomitant increase in TCRαβ IELs (Figure 1C). In contrast to the normal levels of the aforementioned hematopoietic cell populations, morphological evidence indicates that adult (10 weeks of age) KitW-sh/W-sh mice, like KitW/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 KitW-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 KitWbd/Wbd genotype, which like the KitW-sh mutation, contain a 2.8-Mb inversion 5′ of Kit.31Klüppel M Huizinga JD Malysz J Bernstein A Developmental origin and Kit-dependent development of the interstitial cells of Cajal in the mammalian small intestine.Dev Dyn. 1998; 211: 60-71Crossref PubMed Scopus (217) Google Scholar At postnatal day 5, KitWbd/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 KitWbd/Wbd mice lacked a functional ICC network and intestinal pacemaker activity.31Klüppel M Huizinga JD Malysz J Bernstein A Developmental origin and Kit-dependent development of the interstitial cells of Cajal in the mammalian small intestine.Dev Dyn. 1998; 211: 60-71Crossref PubMed Scopus (217) Google Scholar Examination of stomachs from suckling postnatal 7-day-old KitW-sh/W-sh pups and their wild-type littermates revealed that the contents were not yellow in appearance (Supplementary Figure 2 at http://ajp.amjpathol.org); a feature associated with substantial bile reflux and previously noted in suckling KitW/W-v mice14Kitamura Y Yokoyama M Matsuda Shimada M Coincidental development of forestomach papilloma and prepyloric ulcer in nontreated mutant mice of W/Wv and Sl/Sld genotypes.Cancer Res. 1980; 40: 3392-3397PubMed Google Scholar, 16Yokoyama M Tatsuta M Baba M Kitamura Y Bile reflux: a possible cause of stomach ulcer in nontreated mutant mice of W/Wv genotype.Gastroenterology. 1982; 82: 857-863Abstract Full Text PDF PubMed Scopus (31) Google Scholar and KitW-s/W-s rats.30Isozaki K Hirota S Nakama A Miyagawa J-I Shinomura Y Xu Z Nomura S Kitamura Y Disturbed intestinal movement, bile reflux to the stomach, and deficiency of c-kit-expressing cells in Ws/Ws mutant rats.Gastroenterology. 1995; 109: 456-464Abstract Full Text PDF PubMed Scopus (167) Google Scholar However, we found that concentrations of total bile acids in the stomachs of 7-day-old KitW-sh/W-sh pups were significantly higher than those in littermates of the same age (P < 0.05, Table 1). Concentrations of bile acids remained significantly greater than the levels in the corresponding Kit+/+ mice in 4- to 6-week-old or 10-to 14-week-old KitW-sh/W-sh mutants as well (P < 0.0001, Table 1). We also evaluated 4- to 6-week-old and 10- to 14-week-old KitW/W-v mutants and their WBB6F1 wild-type littermates and, like the KitW-sh/W-sh mice, the KitW/W-v mutants exhibited greater concentrations of bile acids compared with wild-type littermates of the corresponding age (P < 0.0001 and P < 0.0008, respectively). Interestingly, the concentrations of bile acids in the stomachs of 4- to 6-week-old KitW/W-v mice were almost double those of the KitW-sh/W-sh mutants (P < 0.0001), whereas, in the 10- to 14-week-old mice, levels were higher in the KitW-sh/W-sh mutants than