Mast cell-deficient W-sash c-kit mutant Kit W-sh/W-sh mice as a model for investigating mast cell biology in vivo

Abstract

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 gammadelta 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.

Figure 1

Levels of hematopoietic cells in adult c-kit mutant C57BL/6-Kit^W-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: Kit^W-sh/W-sh mice exhibit normal numbers of small intestinal TCRγδ and TCRαβ IELs, whereas Kit^W/W-v mice exhibit depletion of TCRγδ IELs and a corresponding increase in TCRαβ 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-Kit^lo FcεRIα⁺ basophils in bone marrow and spleen.

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-Kit^W-sh/W-sh (D–F) or WBB6F₁-Kit^W/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, ×400.

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), Kit^W-sh/W-sh (white diamond) and Kit^+/+ (gray triangle), or GFP⁺ (gray diamond), or Ly5.2⁺ (gray circle) BMCMC-engrafted Kit^W-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 Kit^W-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.

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), Kit^W-sh/W-sh (B, E, H), and mast cell knockin Kit^W-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 Kit^W-sh/W-sh mice, but a few mast cells were sometimes detected in back skin. Arrows: mast cells. Original magnifications: ×200; ×1000 (insets).