Cutaneous mast cells have important pathogenic roles in skin inflammation, but the signals regulating mast-cell numbers in healthy and inflamed skin are not fully understood. Mast-cell development depends on the receptor tyrosine kinase Kit as shown by a greater than 95% reduction of mast-cell numbers in hypomorphic (KitW/Wv) mutant mice that are widely used as a mast-cell deficiency model. Mast-cell numbers are normally very low in KitW/Wv mice, but numbers can strongly increase under inflammatory conditions. It remains elusive whether this inflammation-driven mast-cell accumulation is mediated by signals transmitted via the KitWv receptor or by other, Kit-independent stimuli. We show here, using viable Kit- null mice (KitW/W), that Kit is essential for mast-cell accumulation in phorbol-12-myristate-13-acetate (PMA)–treated, chronically inflamed skin. This increase in mast- cell numbers is strongly attenuated in KitW/Wv mice lacking mature lymphocytes (T, B, and natural killer [NK] cells). These data, together with reconstitution experiments, point at a role for lymphocytes in the regulation of mast-cell compartments under limiting Kit signaling. We conclude that inflammation-induced cutaneous mast-cell accumulation is dependent on Kit signaling strength, and, under limiting Kit signals, on cells of the adaptive immune system.

Connective tissue mast cells reside in a variety of tissues, notably skin and peritoneal cavity (reviewed in Galli et al1 ). The exact hematopoietic pathways that fill and maintain mast cell compartments are still poorly understood. Mast cell–committed progenitors have been identified in fetal blood,2  adult bone marrow (BM),3,4  and spleen,5  and their existence suggests that mast cell commitment can precede tissue immigration. A crucial growth factor receptor that drives mast cell development in vivo is the receptor tyrosine kinase Kit. The importance of Kit is documented by the drastic reduction of mast cell numbers in mice with inactivating mutations in Kit or Kit ligand (stem cell factor [Scf]) genes6  (reviewed in Besmer,7  Galli et al,8  and Broudy9 ). In the skin, Kit signals can regulate mast cell numbers as shown by local mast cell proliferation and activation after subcutaneous administration of exogenous Scf.10,11  In contrast to steady state mast cell development, less information exists on the requirement for Kit signaling in mast cells under inflammatory conditions.

KitW/Wv mice have been widely used as an in vivo model for mast cell deficiency. These mice have only approximately 1% of normal mast cell numbers in their skin, and they lack peritoneal mast cells.6 KitW/Wv mice express the KitW protein together with the KitWv protein. The KitW protein lacks the transmembrane domain and thus cannot be expressed on the cell surface. In KitW/Wv mice, Kit expression is reduced to about 5% of wild-type levels.12  In addition to this low cell-surface expression, the function of the KitWv receptor is impaired because of a point mutation that attenuates its kinase activity.13 

Skin mast cells are present constitutively (ie, in the absence of an immune response), but their numbers markedly increase under local inflammatory conditions. Despite impaired Kit expression and function, mast cell numbers can also increase strongly (10- to 100-fold) in the skin of KitW/Wv mice during the natural history of an idiopathic dermatitis14  or during phorbol-12-myristate-13-acetate (PMA)–induced chronic dermatitis.15 

Mice homozygous for KitW alleles die within a few days after birth,16  a fact that has precluded analyses of mast cells in adult KitW/W mice. KitW/W mice suffer from severe anemia16  that is the cause of their lethality.17  Stimulation of erythropoiesis by transgenic overexpression of a human erythropoietin (EPO) gene is sufficient to generate viable KitW/W mice.17  In these adult KitW/WTg(EPO) mice (referred to as KitW/W) the transgene has no salvage effects on hematopoietic cells outside the erythrocyte lineage, including stem/progenitor cells12,17  and mast cells (see Figure 2).

The fact that chronic inflammation induces mast cell accumulation even in KitW/Wv mice raises the possibility that Kit signaling is not essential under inflammatory conditions. Alternatively, the residual Kit expression/signaling in KitW/Wv mast cells is sufficient to increase mast cell numbers. To solve this question, we have challenged the skin of adult KitW/W mice by PMA to induce a chronic irritative dermatitis. In contrast to KitW/Wv mice that showed a massive mast cell accumulation, the skin of KitW/W mice remained free of mast cells. To address the role of lymphocytes during this inflammation-induced mast cell accumulation, we have also generated and analyzed new compound mutants. In these triple mutant mice, termed RγKitW/Wv, the mast cell deficiency of KitW/Wv mice was combined with lack of all lymphocytes owing to mutations in the recombination activating gene (Rag-2) and the common cytokine receptor gamma chain (γc) gene. Of note, inflammation-driven mast cell expansion was severely impaired in these compound RγKitW/Wv mutants. Adoptive transfer experiments suggest that this defect in mast cell reconstitution was caused by the absence of lymphocytes rather than lack of γc, a receptor subunit invoked in mast cell growth. Hence, in this report, we provide new insights into the cellular and molecular regulation of mast cell numbers in the skin.

Mice

WBKitW/+ (strain, WB), C57BL/6KitWv/+ (strain, C57BL/6) mice (Japan-SLC, Shizuoka, Japan) and WBB6F1KitW/Wv (strain, F1 of WB × C57BL/6) mice were continuously bred in our animal facility. Parental mouse strains for Rag-2−/−γcKitW/Wv (termed RγKitW/Wv) triple mutant mice were generated by crossing Rag-2−/−18 γc−19 double-mutant mice (strain, mixed C57BL/6 and C57BL/10 background; Taconic, Germantown, NY) to WBKitW/+ and C57BL/6KitWv/+ mice. White-spotted (W or Wv) F1 mice were intercrossed to obtain Rag-2−/−γcKitW/+ or Rag-2−/−γcKitWv/+ mice. Mice were typed for Rag-2 deficiency by lack of T and B cells in the peripheral blood by flow cytometry and for γc deficiency by polymerase chain reaction (PCR) on genomic DNA as described.19 RγKitW/Wv mice were obtained by crossing Rag-2−/−γcKitW/+ to Rag-2−/−γcKitWv/+ mice. KitW/W Tg(EPO) mice (strain, mixed WB and C57BL/6 background) were bred as described.17  The EPO transgene, its properties, and the promoter have been described previously.20 

Flow cytometry and cell sorting

Cells were stained, analyzed, or sorted using Calibur analysis or Aria cell sorter instruments (BD Biosciences [BD], Heidelberg, Germany) as described.21,22  Briefly, cells were blocked with 500 μg/mL mouse IgG (Dianova, Hamburg, Germany), prior to specific antibody staining. Peritoneal exudate cells were harvested by peritoneal lavage with 10 mL warm (37°C) PBS/5% FCS and stained with FITC-labeled anti-T1 and PE-labeled anti-FcϵRI antibodies. Peripheral-blood leukocytes were enriched as described23  and analyzed for the presence of lymphocytes using biotinylated anti-CD45, PE-Cy7–labeled anti-CD3 (all BD Pharmingen, San Diego, CA) antibodies. Second-step reagent was streptavidin-APC (Molecular Probes, Carlsbad, CA). Splenic mast cells were analyzed by staining of spleen cell suspensions with a FITC-labeled lineage (lin) cocktail (anti-CD4, anti-CD8, anti-CD19, anti-Ter119), APC-labeled anti-Kit (all BD Pharmingen), and PE-labeled anti-FcϵRI (eBioscience, San Diego, CA). Mast cells were identified by their linKit+FcϵRI+ phenotype. Numbers of lymphocytes in the blood and mast cells in the spleen were calculated by the addition of a defined number of flow cytometry beads (Calibrite; BD) to the stained cells. Lymphocytes were purified negatively by staining of spleen cells with a FITC-labeled myeloid-erythroid lineage cocktail (anti–Mac-1, anti–Gr-1, and anti-Ter119; all BD Pharmingen) followed by magnetic bead (Dynal Biotech, Oslo, Norway) depletion. Subsequently, cells were stained with APC-labeled anti-Kit, and Mac-1Gr-1Ter119Kitcells (ie, the nonmyeloid, nonerythroid, non–mast cell fraction) were cell-sorter purified. This fraction was composed of 53% B cells, 41% T cells, and the remaining cells being non-T and non-B cells (presumably natural killer [NK] cells). Kit+ cells were excluded from the sort to prevent transfer of contaminating wild-type mast cells together with the lymphocytes.

Adoptive cell transfers

Each mouse received 3 × 107 splenic lymphocytes by intravenous injections. Beginning on day 5 after lymphocyte transfer, mice were treated with PMA or acetone for 6 weeks. Numbers of lymphocytes were measured in the blood 13 days after transfer (not shown) and at the end of the experiment (6 weeks after transfer) in blood (Figure 6), spleen, and lymph nodes (not shown). Bone marrow mast cell cultures (BMMCs) were established as described.22  Because the relative contribution of local mast cell expansion versus increased colonization of mast cell progenitors from the circulation to the total mast cell increase in the inflamed skin is not fully understood, we choose intravenous cell transfer. Mice received a total of 3 intravenous BMMC injections (cell numbers were 1 × 107 on day 0, 2 × 106 on day 14, and 2 × 106 on day 22), and mast cell numbers in the skin (not shown) and the spleen (Figure 6) were determined on day 30.

Skin inflammation

One ear of each animal was treated 3 times per week on Monday, Wednesday, and Friday over a period of 6 weeks with 10 μg PMA (Sigma, Taufkirchen, Germany) dissolved in acetone (solvent). For controls, the contralateral ears were treated with solvent alone. Before each PMA treatment, ear thickness was determined using a caliper (Käfer Messuhrenfabrik, Villingen Schwenningen, Germany). After 6 weeks (Figures 45), mice were killed, and ear skin was processed for histology. The study protocol was approved by the Regierungsprasidium Tubingen (permit no. 761).

Histochemistry, immunofluorescence, and determination of mast-cell numbers

Histochemical analysis of tissue mast cells was done as described.22  Mast cells were also identified on acetone-fixed cryosections after blocking with 0.1 mg/mL mouse IgG in PBS/1% BSA using APC-labeled anti-Kit. The presence of granulocytes was determined by staining with FITC-labeled anti–Gr-1 (not shown). Tissue sections shown in Figures 2 and 4 were inspected with an Axioskop microscope using objectives with 10 × (0.30 aperture) and 20 × (0.50 aperture) magnification, respectively, at room temperature in air. Microscope and objectives were from Zeiss (Oberkochen, Germany). Fluorescence photomicrographs of berberine sulfate–stained cytospin preparations (Figure 2) were taken using the ORCA ER camera (Hamamatsu Photonics, Herrsching, Germany) and processed using Openlab software (Improvision, Coventry, England). Light microscopy photomicrographs of toluidin staining (Figure 4) were taken using the OM-11 color camera (Olympus, Hamburg, Germany).

RT-PCR

Ears and peritoneal exudate cells were homogenized in RNAzol (WAK-Chemie, Steinbach, Germany), and cDNA was prepared as described.12  Primers for reverse transcription (RT)–PCR were used as follows: Hprt sense, 5′-GCTGGTGAAAAGGACCTCT-3′; Hprt antisense, 5′-CACAGGACTAGAACACCTGC-3′; Mc-cpa sense, 5′-ACACAGGATCGAATGTGGAG-3′; Mc-cpa antisense, 5′-TAATGCAGGACTTCATGAGC-3′. Southern blot probe for the Mc-cpa PCR product was 5′-CCTCCTAACCACCAGGACCT-3′.

Single and compound Kit mouse mutants

To investigate the role of Kit signaling strength in mast cells and the role of lymphocytes in the regulation of mast cell numbers in chronically inflamed skin, we generated a series of informative single and compound mutants (Figure 1A). Skin inflammation was induced in the following mouse strains: (1) mice wild type for Kit, Rag-2, and γc in which normal Kit signaling was permissive, and all lymphocytes subsets were present; (2) KitW/Wv mice in which Kit expression and signaling were impaired but not completely abrogated, and all lymphocytes subsets were present; (3) Kit wild-type mice lacking T, B, and NK cells (Rag-2−/−γc [termed Rγ]); (4) KitW/Wv mice lacking T, B, and NK cells (Rag-2−/−γcKitW/Wv [termed RγKitW/Wv]); and (5) KitW/W (ie, Kit cell surface null having peripheral T, B, and NK cells).

Figure 1

Mouse mutants and experimental outline to assess mast cells in normal and inflamed skin. (A) Genotypes and their impact on lymphocytes (T cells, B cells, NK cells) and on mast cells are indicated. The generation of new compound mutants is explained in the text. (B) Experimental outline. Mice were treated topically on one ear with PMA in acetone and on the contralateral ear with acetone (solvent) alone 3 times per week. Ear thickness was measured in regular intervals (see Figure 3) during the course of the treatment. After 6 weeks, numbers of mast cells in the ears were determined histologically.

Figure 1

Mouse mutants and experimental outline to assess mast cells in normal and inflamed skin. (A) Genotypes and their impact on lymphocytes (T cells, B cells, NK cells) and on mast cells are indicated. The generation of new compound mutants is explained in the text. (B) Experimental outline. Mice were treated topically on one ear with PMA in acetone and on the contralateral ear with acetone (solvent) alone 3 times per week. Ear thickness was measured in regular intervals (see Figure 3) during the course of the treatment. After 6 weeks, numbers of mast cells in the ears were determined histologically.

Close modal

Mice of all genotypes where treated with PMA 3 times per week over a period of 6 weeks (Figure 1B). As a control, the contralateral ear of each mouse was treated with solvent alone. During the course of the treatment, ear thickness was determined as a measure of inflammation, and, after 4 to 6 weeks of treatment, mast cell numbers were counted by toluidine blue staining on skin sections of both ears.

Mast cell compartments in Kit and lymphocyte mutants

Mice bearing the viable KitW/Wv genotype have strongly reduced numbers (approximately 1% of wild-type mice) of mature dermal mast cells and lack peritoneal mast cells6,15  (Figure 2). In contrast to the widely used KitW/Wv mouse, up to now mast cells have not been analyzed in adult KitW/W mice. Cytospins of peritoneal exudate cells (PECs) were analyzed by toluidine blue reactivity which results in the mast cell–specific, metachromatic staining (Figure 2A-D) and by reactivity with the heparin-binding dye berberine sulfate24  (Figure 2E-H). To include cell numbers larger than those accessible on cytospins, PECs were also analyzed by flow cytometry using the mast cell marker T125  and the high-affinity IgE receptor (FcϵRI) (Figure 2I-L). Kit was excluded as a mast cell marker because it is only weakly or not at all expressed on hematopoietic cells in KitW/Wv and KitW/W mice, respectively.21 

Figure 2

Peritoneal and skin mast cells in Kit mutant mice. Peritoneal exudate cells (PECs) (A-P) and skin (Q-T) from Kit+/+ (A,E,I,M,Q), KitW/Wv (B,F,J,N,R), RγKitW/Wv (C,G,K,O,S), and KitW/W (D,H,L,P,T) mice were analyzed for the presence of mast cells. PEC cytospins were stained with toluidine blue (A-D) and berberine sulfate (E-H). Absence of mast cells in all Kit mutants is evident by lack of staining with these dyes. Scale bars in A and E correspond to 60 μm. (I-L) Flow cytometric analysis of PECs for expression of the mast cell markers T1 and FcϵRI. T1+FcϵRI+ cells were absent in all Kit mutants. Cells on the diagonal, present in all plots, are myeloid cells that are unaffected by mutations in Rag-2, γc, or Kit. Cells on the bottom of the dot plots are lymphocytes that are absent in RγKitW/Wv owing to the mutations in Rag-2, and γc (M-T). Mice were 12 to 13 weeks of age. RT-PCR analysis for expression of mast cell carboxypeptidase A (Mc-cpa), a mast cell–specific gene, as a sensitive tool to detect mast cells in PECs (M-P) and ear skin (Q-T). The triangles indicate 5 cDNA dilutions in 1:10 steps. In line with the analysis shown in panels A through L, PECs lacked Mc-cpa expression in all Kit mutants (N-P). In contrast, Mc-cpa expression was detectable in KitW/Wv (R) and RγKitW/Wv (S) mice. This expression was 10- to 100-fold reduced compared with Kit+/+ skin (Q). Mc-cpa expression was undetectable in the skin of KitW/W mice (T). Mice were 24 weeks (Kit+/+ [C57BL/6]), 12 weeks (KitW/Wv), 31 weeks (RγKitW/Wv), and 17 weeks (KitW/W) old.

Figure 2

Peritoneal and skin mast cells in Kit mutant mice. Peritoneal exudate cells (PECs) (A-P) and skin (Q-T) from Kit+/+ (A,E,I,M,Q), KitW/Wv (B,F,J,N,R), RγKitW/Wv (C,G,K,O,S), and KitW/W (D,H,L,P,T) mice were analyzed for the presence of mast cells. PEC cytospins were stained with toluidine blue (A-D) and berberine sulfate (E-H). Absence of mast cells in all Kit mutants is evident by lack of staining with these dyes. Scale bars in A and E correspond to 60 μm. (I-L) Flow cytometric analysis of PECs for expression of the mast cell markers T1 and FcϵRI. T1+FcϵRI+ cells were absent in all Kit mutants. Cells on the diagonal, present in all plots, are myeloid cells that are unaffected by mutations in Rag-2, γc, or Kit. Cells on the bottom of the dot plots are lymphocytes that are absent in RγKitW/Wv owing to the mutations in Rag-2, and γc (M-T). Mice were 12 to 13 weeks of age. RT-PCR analysis for expression of mast cell carboxypeptidase A (Mc-cpa), a mast cell–specific gene, as a sensitive tool to detect mast cells in PECs (M-P) and ear skin (Q-T). The triangles indicate 5 cDNA dilutions in 1:10 steps. In line with the analysis shown in panels A through L, PECs lacked Mc-cpa expression in all Kit mutants (N-P). In contrast, Mc-cpa expression was detectable in KitW/Wv (R) and RγKitW/Wv (S) mice. This expression was 10- to 100-fold reduced compared with Kit+/+ skin (Q). Mc-cpa expression was undetectable in the skin of KitW/W mice (T). Mice were 24 weeks (Kit+/+ [C57BL/6]), 12 weeks (KitW/Wv), 31 weeks (RγKitW/Wv), and 17 weeks (KitW/W) old.

Close modal

On the basis of toluidine blue and berberine staining, and on flow cytometry, peritoneal mast cells were detectable in Kit+/+ (Figure 2A,E,I) and in Rγ mice (not shown). In contrast, peritoneal mast cells were undetectable in all Kit mutants, that is, KitW/Wv (Figure 2B,F,J), RγKitW/Wv (Figure 2C,G,K), and KitW/W (Figure 2D,H,L). Hence, the absence of lymphocytes did not affect the peritoneal mast cell compartment. In addition, the data confirmed the massive reduction of mast cells in KitW/Wv mice. At this level of analysis, there was no difference comparing hypomorphic KitW/Wv and null KitW/W mice with regard to mast cell development.

Gene expression analysis for mast cell–specific genes is a sensitive way to detect mast cells.26  The mast cell carboxypeptidase A (Mc-cpa) is strongly and specifically expressed in the mast cell lineage27  (references in Feyerabend et al22 ). Therefore, Mc-cpa expression was analyzed by RT-PCR in PECs (Figure 2M-P) or ears (Figure 2Q-T). In wild-type mice (Figure 2M,Q), Mc-cpa mRNA was strongly expressed in PECs and in skin. Consistent with the absence of mast cells in the peritoneal cavity of KitW/Wv mice,6 Mc-cpa mRNA was undetectable in KitW/Wv (Figure 2N) and RγKitW/Wv (Figure 2O) mice. Not surprisingly, KitW/W PECs also lacked Mc-cpa mRNA (Figure 2P), indicating a complete absence of peritoneal mast cells in KitW/W mice.

In the ears, Mc-cpa was again strongly expressed in wild-type mice (Figure 2Q). Confirming previous results,26  we detected Mc-cpa mRNA in the skin of both KitW/Wv mutants (Figure 2R,S). Titration of cDNA templates suggested that Mc-cpa expression was 10- to 100-fold lower in the ears of KitW/Wv mutants compared with wild-type mice (Figure 2R,S). Interestingly, in the ears of KitW/W mice Mc-cpa expression was undetectable by RT-PCR (Figure 2T). These data suggest that the residual Kit function in KitW/Wv mice is required for the low but significant mast cell production under steady state conditions in the KitW/Wv mutant.

Mast-cell dependency of initial versus long-term ear swelling after PMA treatment

PMA treatment elicits acute and chronic inflammatory responses, akin to irritative dermatitis, by nonspecific activation of a variety of PMA receptors (reviewed in Boutwell28  and Brose and Rosenmund29 ). The resulting immunologically nonspecific inflammation, measured by immediate (< 36 hours) tissue swelling, is amplified by mast cells after PMA treatment.30  To measure the impact of other immune cells on this inflammation process, we determined time course and extent of ear thickness in all mutants after PMA treatment (Figure 3).

Figure 3

Ear swelling during PMA treatment. Ear thickness of PMA-treated ears (■) and solvent-treated contralateral ears (▴) was monitored with a caliper over a period of 6 weeks. Means and SDs of 3 mice per genotype are shown. Data are representative for 1 of 2 complete and independent experiments. Mice used were 13 weeks (Kit+/+ [C57BL/6]), 8 weeks (KitW/Wv), 10 weeks (Rγ), 16 weeks (RγKitW/Wv), 2 × 9 weeks, and 1 × 16 weeks (KitW/W) old.

Figure 3

Ear swelling during PMA treatment. Ear thickness of PMA-treated ears (■) and solvent-treated contralateral ears (▴) was monitored with a caliper over a period of 6 weeks. Means and SDs of 3 mice per genotype are shown. Data are representative for 1 of 2 complete and independent experiments. Mice used were 13 weeks (Kit+/+ [C57BL/6]), 8 weeks (KitW/Wv), 10 weeks (Rγ), 16 weeks (RγKitW/Wv), 2 × 9 weeks, and 1 × 16 weeks (KitW/W) old.

Close modal

Ear swelling of fully immune-competent mice peaked at day 10 and declined thereafter to approximately double the normal thickness compared with nontreated contralateral ears (Figure 3A). Such strong initial reactions were not observed in any of the immune-compromised mice (Figure 3B-E), suggesting that mast cells and lymphocytes can both contribute to the initial swelling in the first 10 days. Beginning on day 12, and for the remainder of the observation time, ear thickness of wild-type (Figure 3A), KitW/Wv (Figure 3B), Rγ (Figure 3C), and KitW/W (Figure 3E) mice had at least doubled compared with the starting point (swelling ≥ 0.5 mm). In contrast, PMA-treated ears of RγKitW/Wv mice showed reduced swelling (∼ 0.35 mm) in the long term (Figure 3D). Collectively, initial ear swelling was augmented by the presence of immune cells (either T, B, NK, and/or mast cells) (compare Figure 3A with B-E). Long-term ear swelling occurred in all mutants, but in the absence of all adaptive (T and B cells) and parts of innate (NK and mast cells) immune cells long-term ear swelling was reduced.

To obtain information about other cell types that are recruited during this inflammatory response, we examined infiltration of Gr-1+ leukocytes to the sites of chronic dermatitis. Immunofluorescence analysis of skin sections revealed an accumulation of polymorphonuclear Gr-1+ granulocytes in all treated ears, indicating that granulocyte recruitment was mast cell and lymphocyte independent (not shown).

Kit-mediated signals are pivotal for mast-cell accumulation during chronic dermatitis

To assess the contribution of Kit-mediated signals to the mast cell accumulation during chronic dermatitis, ears of PMA-treated mice of all genotypes were analyzed histochemically for the presence of mast cells (Figures 45). Skin thickness of PMA-treated ears (Figure 4B,D,F,H) was clearly increased compared with solvent-treated control ears (Figure 4A,C,E,G) visualizing the findings obtained by micrometer measurements (Figure 3). Mast cells were identified by their morphology and by their metachromatic staining with toluidine blue. Mast cells in control ears were only present in wild-type (Figure 4A) and Rγ (not shown) mice. Following control solvent treatment, we could not detect skin mast cells in KitW/Wv (Figure 4C), RγKitW/Wv (Figure 4E), or KitW/W (Figure 4G) mice, demonstrating the paucity or absence of mast cells in the healthy skin of these mutants. As expected, PMA treatment resulted in increased mast cell numbers in wild-type mice (Figure 4B). Mast cell numbers increased not only in wild-type mice but also in KitW/Wv (Figure 4D) and RγKitW/Wv (Figure 4F) mice. Interestingly, mast cells were undetectable in skin sections of KitW/W mice (Figure 4H).

Figure 4

Mast-cell accumulation and skin morphology after local PMA treatment. Six weeks after treatment of ears with solvent (acetone) (A,C,E,G) or with PMA (B,D,F,H) ear sections were stained with toluidine blue to assess the overall skin morphology and to obtain mast cell counts. Strongly increased skin thickness is apparent in all PMA-treated ears (B-H). Arrows indicate metachromatically stained mast cells in PMA-treated ears. Mast cells accumulated exclusively in the ears of Kit+/+ (B) and KitW/Wv (D) and RγKitW/Wv (F) mice. Mice used were 8 weeks (Kit+/+ [WB]), 11 weeks (KitW/Wv), 25 weeks (RγKitW/Wv), and 13 weeks (KitW/W) of age. The skin of KitW/W mice that lack all Kit cell-surface expression was completely devoid of mast cells. This was true before (G) and after (H) PMA-induced inflammation. The scale bar (A) applies to all panels and corresponds to 40 μm.

Figure 4

Mast-cell accumulation and skin morphology after local PMA treatment. Six weeks after treatment of ears with solvent (acetone) (A,C,E,G) or with PMA (B,D,F,H) ear sections were stained with toluidine blue to assess the overall skin morphology and to obtain mast cell counts. Strongly increased skin thickness is apparent in all PMA-treated ears (B-H). Arrows indicate metachromatically stained mast cells in PMA-treated ears. Mast cells accumulated exclusively in the ears of Kit+/+ (B) and KitW/Wv (D) and RγKitW/Wv (F) mice. Mice used were 8 weeks (Kit+/+ [WB]), 11 weeks (KitW/Wv), 25 weeks (RγKitW/Wv), and 13 weeks (KitW/W) of age. The skin of KitW/W mice that lack all Kit cell-surface expression was completely devoid of mast cells. This was true before (G) and after (H) PMA-induced inflammation. The scale bar (A) applies to all panels and corresponds to 40 μm.

Close modal
Figure 5

Steady state and inflammation-induced mast cell numbers in the skin of Kit+/+, KitW/Wv, Rγ, RγKitW/Wv, and KitW/W mice. After 6 weeks of PMA-induced chronic dermatitis, numbers of metachromatic mast cells were determined on toluidine blue–stained sections of ear skin. Closed symbols represent control (acetone)–treated ears, and open symbols represent PMA-treated ears. The figure summarizes data from 2 independent experiments with 3 mice per genotype and experiment (total number of mice = 6). Horizontal lines indicate the mean cell number for each group of mice (A-E). Numbers in healthy skin and the inflammation-induced increase were comparable in Kit+/+ (A) and Rγ (C) mice. Numbers in healthy skin were very low in untreated KitW/Wv (B) and RγKitW/Wv (D) skin. In PMA-treated skin, the increase in mast cell numbers was approximately 10-fold larger in KitW/Wv compared with RγKitW/Wv mice. In the skin from KitW/W mice, mast cells were undetectable and remained absent after PMA-induced chronic dermatitis (E). The differences in mast cell counts comparing untreated (closed symbols) and PMA-treated (open symbols) skin samples had significant one-sided P values (< .05) (Mann-Whitney test) in panels A through C. The P value (.09) in panel D was not significant. Data from mice of 2 independent experiments were pooled in this figure. Mice used in these experiments were 13 to 21 weeks (Kit+/+ [3 × C57BL/6; 3 × WB]), 8 to 19 weeks (KitW/Wv), 10 to 19 weeks (Rγ), 9 to 16 weeks (RγKitW/Wv), or 9 to 36 weeks (KitW/W) of age.

Figure 5

Steady state and inflammation-induced mast cell numbers in the skin of Kit+/+, KitW/Wv, Rγ, RγKitW/Wv, and KitW/W mice. After 6 weeks of PMA-induced chronic dermatitis, numbers of metachromatic mast cells were determined on toluidine blue–stained sections of ear skin. Closed symbols represent control (acetone)–treated ears, and open symbols represent PMA-treated ears. The figure summarizes data from 2 independent experiments with 3 mice per genotype and experiment (total number of mice = 6). Horizontal lines indicate the mean cell number for each group of mice (A-E). Numbers in healthy skin and the inflammation-induced increase were comparable in Kit+/+ (A) and Rγ (C) mice. Numbers in healthy skin were very low in untreated KitW/Wv (B) and RγKitW/Wv (D) skin. In PMA-treated skin, the increase in mast cell numbers was approximately 10-fold larger in KitW/Wv compared with RγKitW/Wv mice. In the skin from KitW/W mice, mast cells were undetectable and remained absent after PMA-induced chronic dermatitis (E). The differences in mast cell counts comparing untreated (closed symbols) and PMA-treated (open symbols) skin samples had significant one-sided P values (< .05) (Mann-Whitney test) in panels A through C. The P value (.09) in panel D was not significant. Data from mice of 2 independent experiments were pooled in this figure. Mice used in these experiments were 13 to 21 weeks (Kit+/+ [3 × C57BL/6; 3 × WB]), 8 to 19 weeks (KitW/Wv), 10 to 19 weeks (Rγ), 9 to 16 weeks (RγKitW/Wv), or 9 to 36 weeks (KitW/W) of age.

Close modal

Next, we obtained mast cell counts that were normalized on cartilage length (Figure 5). In wild-type mice (Figure 5A) and in Rγ mice (Figure 5C), 6 of 6 mice analyzed for each genotype showed a 3- to 4-fold increase in mast cells after PMA treatment (Kit+/+ mice control ears [mean ± SD, 540 ± 274 mast cells/cm; Kit+/+ mice PMA-treated ears, 1733 ± 454 mast cells/cm; Rγ mice control ears, 372 ± 96 mast cells/cm; Rγ mice PMA-treated ears, 999 ± 373 mast cells/cm). Mast cells also accumulated in 6 of 6 PMA-treated ears from KitW/Wv mice (Figure 5B) (KitW/Wv mice control ears, 1.3 ± 0.8 mast cells/cm; KitW/Wv mice PMA-treated ears, 179 ± 136 mast cells/cm) as reported previously.15  Although absolute mast cell numbers remained lower in the skin of KitW/Wv mice compared with wild-type or Rγ mice, the relative increase was larger in KitW/Wv compared with wild-type or Rγ mice. Interestingly, PMA treatment did not lead to the appearance of mast cells in the skin of KitW/W mice (Figure 5E) (KitW/W mice control ears, 0 mast cell/cm; KitW/W mice PMA-treated ears, 0 mast cell/cm), demonstrating an essential role for Kit-mediated signals in mast cell development in healthy and inflamed skin.

Role of lymphocytes and the common γ chain for mast cells in KitW/Wv mice

Analysis of Rγ mice (Figure 5C) indicated that the absence of lymphocytes and lack of γc expression did not impair the number of constitutive skin mast cells or their increase following skin inflammation in mice expressing wild-type levels of Kit. Thus, in contrast to peritoneal mast cells,31  γc expression is not required for normal numbers of skin mast cells. Because Kit signaling can act in marked synergy with γc signaling in other cell types such as thymocytes,32  we next examined the inflammation-driven mast cell increase in combined Rag-2 and γc mutants on a KitW/Wv background (RγKitW/Wv mice). In these mutants, the overall increase in mast cell numbers was strongly blunted (Figure 5D) (control ears, 2 ± 2 mast cells/cm, PMA, 12 ± 19 mast cells/cm) compared with KitW/Wv mice (Figure 5B). Moreover, 2 of 6 RγKitW/Wv mice did not respond at all. Thus, on a KitW/Wv background, the presence of lymphocytes or γc expression is important for the regulation of mast cell numbers in inflamed skin.

To distinguish between effects mediated by lack of Rag-2 (ie, loss of T and B cells) or γc expression (ie, loss of signaling via γc-dependent cytokines), we performed 2 sets of further experiments. In the first set, cell-sorter purified, wild-type (R+γ+Kit+/+) lymphocytes (see “Materials and methods”) were injected intravenously into RγKitW/Wv mice. This adoptive transfer lead to reconstitution of circulating T cells in RγKitW/Wv mice reaching about 50% (2157 ± 819 T cells/μL blood) (Figure 6D) of the numbers found in Kit+/+ (3960 ± 1017 T cells/μL blood) (Figure 6A) or KitW/Wv (4250 ± 780 T cells/μL blood) (Figure 6B) mice. No lymphocytes were detectable in RγKitW/Wv mice (Figure 6C). Ears in these groups of mice were subjected to PMA treatment over a period of 6 weeks. In agreement with the experiments shown in Figure 5, Kit+/+ showed a robust increase in skin mast cell numbers (control ears, 318 ± 64 mast cells/cm; PMA-treated ears, 1526 ± 139 mast cells/cm) (Figure 6E). Likewise, KitW/Wv demonstrated a marked response (control ears, 1.7 ± 2.8 mast cells/cm; PMA-treated ears, 216 ± 80 mast cells/cm) (Figure 6F), whereas RγKitW/Wv mice showed only a very modest mast cell response (control ears, 1 ± 1.7 mast cells/cm; PMA-treated ears, 22 ± 6 mast cells/cm) (Figure 6G). Lymphocyte-reconstituted RγKitW/Wv mice harbored 4 ± 4.5 mast cells/cm in control ears and 95 ± 71 mast cells/cm in PMA-treated ears (Figure 6H). Although the comparison of the PMA-induced cell numbers in non–lymphocyte-reconstituted RγKitW/Wv (open symbols in Figure 6G) versus in lymphocyte-reconstituted RγKitW/Wv mice (open symbols in Figure 6H) has a P value of only .2 (Mann Whitney rank test) and hence does not reach statistical significance, it should be noted that 2 of 3 mice showed mast cell numbers above 100 cells/cm (106 and 160). We did not observe similarly high mast cell numbers in any of the nonreconstituted, PMA-treated RγKitW/Wv ears (Figures 56), suggesting that the addition of lymphocytes to RγKitW/Wv mice might promote mast cell accumulation in the inflamed skin.

Figure 6

Role of lymphocytes and the common γ chain for mast cells in KitW/Wv mice. Adoptive transfer of wild-type (R+γ+Kit+/+) splenic lymphocytes into RγKitW/Wv mice (D) lead to approximately 50% reconstitution of circulating T cells compared with Kit+/+ (A) or KitW/Wv (B) mice. Nonreconstituted RγKitW/Wv mice are shown as controls (C). Ears in these groups of mice were subjected to PMA treatment over a period of 6 weeks, and mast cell numbers in treated and untreated ears from all mice were determined by histology (E-H). The differences in mast cell counts comparing untreated (closed symbols) and PMA-treated (open symbols) skin samples had significant one-sided P values (P = .05, Mann-Whitney test) in panels E through H. The P value (.2) comparing the PMA-treated skins in panel G versus panel H was not significant (see text). Kit+/+ were 13-week-old C57BL/6 mice (A,E). KitW/Wv (B,F), noninjected RγKitW/Wv (C,G), and lymphocyte-recipient RγKitW/Wv (D,H) mice were 17 to 20 weeks old. Donors (D,H) were 25-week-old C57BL/6 mice. To determine the possible role of γc for the defective mast cell reconstitution in RγKitW/Wv mice, wild-type (R+γ+Kit+/+) BMMCs were injected repeatedly (see “Material and methods” for cell numbers, frequency of injections, and duration of the experiment) into RγKitW/Wv (J) and KitW/Wv (K) mice. Numbers of mast cells in the spleen, a site that is more effectively colonized than skin following intravenous transfer of BMMCs,33  were determined by flow cytometry in noninjected controls (solid symbols in I-K) and in injected RγKitW/Wv (J) and KitW/Wv (K). Age of the mice was 17 weeks (C57BL/6) (I), 9 to 11 weeks (noninjected RγKitW/Wv) (J), 13 to 30 weeks (BMMC-injected RγKitW/Wv) (J), 21 weeks (noninjected KitW/Wv) (K), and 39 weeks (BMMC-injected KitW/Wv) (K). Mast cells appeared in the spleens of both types of recipients, but their numbers were at least one order of magnitude higher in KitW/Wv compared with RγKitW/Wv. The one-sided P value (.004) of the comparison of the BMMC-injected animals in panel J versus panel K by Mann-Whitney test was significant. Horizontal lines indicate the mean cell number for each group of mice (A-K). Hence, γc-competent mast cells did not rescue the defective mast cell reconstitution in RγKitW/Wv mice.

Figure 6

Role of lymphocytes and the common γ chain for mast cells in KitW/Wv mice. Adoptive transfer of wild-type (R+γ+Kit+/+) splenic lymphocytes into RγKitW/Wv mice (D) lead to approximately 50% reconstitution of circulating T cells compared with Kit+/+ (A) or KitW/Wv (B) mice. Nonreconstituted RγKitW/Wv mice are shown as controls (C). Ears in these groups of mice were subjected to PMA treatment over a period of 6 weeks, and mast cell numbers in treated and untreated ears from all mice were determined by histology (E-H). The differences in mast cell counts comparing untreated (closed symbols) and PMA-treated (open symbols) skin samples had significant one-sided P values (P = .05, Mann-Whitney test) in panels E through H. The P value (.2) comparing the PMA-treated skins in panel G versus panel H was not significant (see text). Kit+/+ were 13-week-old C57BL/6 mice (A,E). KitW/Wv (B,F), noninjected RγKitW/Wv (C,G), and lymphocyte-recipient RγKitW/Wv (D,H) mice were 17 to 20 weeks old. Donors (D,H) were 25-week-old C57BL/6 mice. To determine the possible role of γc for the defective mast cell reconstitution in RγKitW/Wv mice, wild-type (R+γ+Kit+/+) BMMCs were injected repeatedly (see “Material and methods” for cell numbers, frequency of injections, and duration of the experiment) into RγKitW/Wv (J) and KitW/Wv (K) mice. Numbers of mast cells in the spleen, a site that is more effectively colonized than skin following intravenous transfer of BMMCs,33  were determined by flow cytometry in noninjected controls (solid symbols in I-K) and in injected RγKitW/Wv (J) and KitW/Wv (K). Age of the mice was 17 weeks (C57BL/6) (I), 9 to 11 weeks (noninjected RγKitW/Wv) (J), 13 to 30 weeks (BMMC-injected RγKitW/Wv) (J), 21 weeks (noninjected KitW/Wv) (K), and 39 weeks (BMMC-injected KitW/Wv) (K). Mast cells appeared in the spleens of both types of recipients, but their numbers were at least one order of magnitude higher in KitW/Wv compared with RγKitW/Wv. The one-sided P value (.004) of the comparison of the BMMC-injected animals in panel J versus panel K by Mann-Whitney test was significant. Horizontal lines indicate the mean cell number for each group of mice (A-K). Hence, γc-competent mast cells did not rescue the defective mast cell reconstitution in RγKitW/Wv mice.

Close modal

In the second set of experiments, we addressed the possibility that the poor mast cell increase in RγKitW/Wv mice was due to lack of γc expression in mast cells31  in these mutants. To this end, we transplanted γc-competent (R+γ+Kit+/+) BMMCs into RγKitW/Wv and KitW/Wv hosts. Despite repeated intravenous injections of BMMCs (see “Material and methods” for reasoning for route of administration, cell numbers, and frequency of injections), we failed to detect a significant increase in mast cell numbers in the ears of both types of recipients (RγKitW/Wv without BMMCs [n = 3], 1.3 ± 1.5 mast cells/cm; RγKitW/Wv with BMMCs [n = 5], 3.2 ± 5.5 mast cells/cm; KitW/Wv without BMMCs [n = 3], 7.7 ± 6.5 mast cells/cm; KitW/Wv with BMMCs [n = 5], 5.4 ± 3.4 mast cells/cm). Moreover, RγKitW/Wv mice transplanted with wild-type (R+γ+Kit+/+) BMMCs and challenged with PMA (as described for Figure 6E-H) also failed to generated skin mast cells (not shown).

These findings are in agreement with previous experiments that revealed very inefficient mast cell reconstitution in the skin following intravenous transfer.33  However, the spleen has been reported to be a more effective site of mast cell reconstitution following intravenous transfer of BMMCs.33  We therefore compared by flow cytometry mast cell numbers in the spleens of the same RγKitW/Wv and KitW/Wv recipient mice that had received γc-competent (R+γ+Kit+/+) BMMCs. Mast cells were present in the spleens of both types of recipients, but, interestingly, numbers of mast cells were at least one order of magnitude higher in KitW/Wv (1648 ± 780 mast cells/cm) compared with RγKitW/Wv (87 ± 112 mast cells/cm) recipients (Figure 6J,K) (P = .004). Because γc-competent BMMCs reconstituted KitW/Wv mice well, but RγKitW/Wv only poorly, these experiments support the notion that lymphocytes contribute to the regulation of mast cell compartments upon reconstitution of mast cell–deficient mice. Under these conditions, γc-competent mast cells did not rescue the defective mast cell reconstitution in RγKitW/Wv mice.

Despite the important contributions of mast cells to the pathophysiology of skin inflammation, little is known about the cellular and molecular signals that regulate mast cell numbers in inflamed skin. The crucial role of Kit and its ligand, Scf, for normal mast cell development is evident in mice bearing hypomorphic mutations in the genes encoding Kit6  or Scf.34  The mast cell paucity is, however, not absolute in the KitW/Wv mutant as shown by a significant enrichment of mast cells at sites of idiopathic14  or chemically15  induced chronic dermatitis. These findings raised the question whether residual Kit signaling, mediated by the KitW/Wv receptor, was facilitating this response or whether mechanisms unrelated to Kit signaling were able to drive mast cell accumulation under these conditions. Whatever the nature of these signals, they are insufficient to promote significant numbers of skin mast cells in KitW/Wv mice under nonpathologic conditions.

Here, we have taken advantage of the recently developed viable KitW/W mutants17  to address the role of Kit in the regulation of mast cell numbers during chronic dermatitis. Adult KitW/W mice have previously given insight into the crucial role of Kit in adult lymphopoiesis.12,35  Although in vitro–derived mast cell lines were used to characterize the loss of Kit expression in viable KitW/W mutants,12  mast cell compartments have not been analyzed in these mice. Because the hypomorphic KitW/Wv mutant has been widely used as a “mast cell–deficiency model,” we have now compared mast cells in KitW/W and KitW/Wv mice. The results show that KitW/W mice differ in several aspects from KitW/Wv mice. On the basis of mast cell protease expression (Figure 2) and histologic analysis (Figure 4), we confirm previous reports (reviewed in Tsai et al26 ) that KitW/Wv mice have very low but measurable numbers of skin but not peritoneal mast cells. In contrast, using the same criteria, we now find no evidence for skin mast cells in KitW/W mice. The absence of mast cells in the peritoneal cavity in both strains, but the presence of mast cells in the skin of KitW/Wv mice, suggests that skin mast cells are slightly less dependent on Kit signaling compared with peritoneal mast cells. However, the most dramatic difference between mast cells in KitW/Wv and KitW/W mice became only apparent following experimentally induced chronic dermatitis. Although mast cell numbers increased strongly in KitW/Wv mice, mast cells remained undetectable in KitW/W mice. These experiments demonstrate an active role of the KitWv protein in the regulation of mast cell numbers in response to skin inflammation. Collectively, Kit-mediated signals are essential for skin mast cell formation under steady state conditions and under inflammatory conditions.

Short- and long-term reactions during PMA-induced chronic dermatitis include ear swelling. PMA nonspecifically activates a wide variety of cell types28  by directly interacting with the signaling component protein kinase C and other PMA receptors.29  To determine whether the residual Kit function in KitW/Wv mast cells was in itself sufficient for the mast cell response to inflammation, we eliminated T, B, and NK cells from KitW/Wv mice by generating RγKitW/Wv mice. In short-term reactions (≤ 30 hours) mast cells play a role in ear swelling.30  After 3 days of PMA treatment we observed a slower onset of ear swelling in all analyzed mutants. Thus, not only mast cells, but also T, B, or NK cells, are required for initial ear swelling. In contrast, after 15 days, only mice lacking T, B, and NK cells showed reduced swelling. Ear thickness was indistinguishable after 6 weeks of PMA treatment comparing Kit+/+ and KitW/Wv mice15  (Figure 3). The fact that Rγ mice showed a similar swelling demonstrates that lymphocytes are not required for this skin reaction. Moreover, RγKitW/Wv that had initially very few mast cells and constantly lacked lymphocytes showed the same degree of swelling.

The unexpected mast cell reconstitution defect in RγKitW/Wv, when compared with KitW/Wv mice, might be due to lack of γc expression on mast cells or due to the absence of lymphocytes. Mice deficient for γc or a downstream signaling molecule Jak-3 were reported to have 2-fold reduced numbers of peritoneal mast cells.31  We did not notice a reduction in skin mast cells in Rγ mice, thus ruling out an important function for γc in this mast cell population. However, γc signaling in mast cells might be important if the Kit signal is limiting. Although we cannot definitively exclude a role for γc under such conditions, the relative inability of adoptively transferred γc+ BMMCs to reconstitute splenic mast cells in RγKitW/Wv mice (Figure 6) argues against a major role of γc in mast cell reconstitution. The inability of robust reconstitution of skin mast cells following intravenous transfer of BMMCs prevented a more direct test whether the same rules apply to the skin as shown here for the spleen.

Lack of lymphocytes played no role for the accumulation of mast cells in Kit+/+ mice but T, B, or NK cells were required on the KitW/Wv background. These data suggest that mast cells can expand in the skin by 2 mechanisms. The first possibility is a strong signal via Kit alone that can be transmitted by the wild-type (Kit+) but not by the kinase-weak (KitWv) receptor. The second route would require 2 signals, a weak Kit signal that could be transmitted via KitWv in KitW/Wv mice and a second signal that originates directly or indirectly from lymphocytes. It is conceivable that a weak Kit signal could also be generated in normal mice (Kit+/+) under conditions of limiting availability of Kit ligand. The strongly blunted mast cell response in RγKitW/Wv compared with KitW/Wv mice agrees with a supportive role of lymphocytes in mast cell recruitment, generation, or local proliferation. In addition to T and B cells, NK cells that are absent in RγKitW/Wv mice could also play a role. The lymphocyte support for mast cell expansion could occur via direct cellular interactions or lymphocyte-derived factors may drive mast cell expansion. In vitro, IL-3 allows outgrowth of mast cells from bone marrow or peripheral blood of KitW/Wv36 and KitW/W12 mice. Furthermore, perfusion of KitW/Wv mice with IL-3 normalizes skin mast cell numbers,37  and Kit ligand and IL-3 cooperate in the mast cell–driven worm expulsion.38  In KitW/Wv mice, T-cell–derived IL-3 might, therefore, be a crucial factor for mast cell accumulation during the course of chronic dermatitis.

Presently, it is difficult to distinguish whether the defect in KitW/W mice is due to reduced availability of circulating mast cell progenitors or due to impaired skin colonization of mast cell progenitors or due to impaired survival or abrogated proliferation of mast cells within the skin. After 6 weeks of PMA treatment of normal mice, histologic analysis revealed that skin mast cells lacked expression of a proliferation marker, phospho-histone H3 (Ser10) (data not shown), indicating that, at least at this time point, mast cells did not proliferate locally. It is, however, possible that mast cells proliferated locally at earlier time points during the course of the inflammation. Progenitors with in vitro mast cell potential are present in peripheral blood of adult KitW/W mice,12  but these cells have not been accessible for direct analysis, partly due to lack of Kit expression that is an important marker of stem/progenitor cells. Hence, the precise stage at which mast cell development is absolutely blocked in adult KitW/W mice has to await the identification or development of specific mast cell progenitor markers. These could also be useful to study mast cell progenitor mobilization and their recruitment to healthy and inflamed tissues.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

We thank Dr Michael Huber, Freiburg, for generous supply of BMMC, Thorsten Feyerabend for proficient cell transfers, Dr Hans Jörg Fehling for critical reading of the manuscript, and members of the animal facility for careful mouse keeping.

This work was supported by the Landesstiftung (P-LS-AL/11) (C.W. and H.-R.R.) and the Landesgraduierten-Förderung (S.M.S.) Baden-Württemberg and by the Deutsche Forschungsgemeinschaft (DFG-RO754/2-2) (H.-R.R.).

Contribution: C.W. and H.-R.R. designed research, analyzed data, and wrote the paper; C.W., S.B., S.M.S., and C.C. performed research.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Hans-Reimer Rodewald, Institute for Immunology, University of Ulm, D-89081 Ulm, Germany; e-mail: hans-reimer.rodewald@uni-ulm.de.

1
Galli
 
SJ
Kalesnikoff
 
J
Grimbaldeston
 
MA
Piliponsky
 
AM
Williams
 
CM
Tsai
 
M
Mast cells as “tunable” effector and immunoregulatory cells: recent advances.
Annu Rev Immunol
2005
, vol. 
23
 (pg. 
749
-
786
)
2
Rodewald
 
H-R
Dessing
 
M
Dvorak
 
AM
Galli
 
SJ
Identification of a committed precursor for the mast cell lineage.
Science
1996
, vol. 
271
 (pg. 
818
-
822
)
3
Jamur
 
MC
Grodzki
 
AC
Berenstein
 
EH
Hamawy
 
MM
Siraganian
 
RP
Oliver
 
C
Identification and characterization of undifferentiated mast cells in mouse bone marrow.
Blood
2005
, vol. 
105
 (pg. 
4282
-
4289
)
4
Chen
 
CC
Grimbaldeston
 
MA
Tsai
 
M
Weissman
 
IL
Galli
 
SJ
Identification of mast cell progenitors in adult mice.
Proc Natl Acad Sci U S A
2005
, vol. 
102
 (pg. 
11408
-
11413
)
5
Arinobu
 
Y
Iwasaki
 
H
Gurish
 
MF
, et al. 
Developmental checkpoints of the basophil/mast cell lineages in adult murine hematopoiesis.
Proc Natl Acad Sci U S A
2005
, vol. 
102
 (pg. 
18105
-
18110
)
6
Kitamura
 
Y
Go
 
S
Hatanaka
 
K
Decrease in mast cells in W/Wv mice and their increase by bone marrow transplantation.
Blood
1978
, vol. 
52
 (pg. 
447
-
452
)
7
Besmer
 
P
The kit ligand encoded at the murine Steel locus: a pleiotropic growth and differentiation factor.
Curr Opin Cell Biol
1991
, vol. 
3
 (pg. 
939
-
946
)
8
Galli
 
SJ
Zsebo
 
KM
Geissler
 
EN
The kit ligand, stem cell factor.
Adv Immunol
1994
, vol. 
55
 (pg. 
1
-
96
)
9
Broudy
 
VC
Stem cell factor and hematopoiesis.
Blood
1997
, vol. 
90
 (pg. 
1345
-
1364
)
10
Tsai
 
M
Takeishi
 
T
Thompson
 
H
, et al. 
Induction of mast cell proliferation, maturation, and heparin synthesis by the rat c-kit ligand, stem cell factor.
Proc Natl Acad Sci U S A
1991
, vol. 
88
 (pg. 
6382
-
6386
)
11
Wershil
 
BK
Tsai
 
M
Geissler
 
EN
Zsebo
 
KM
Galli
 
SJ
The rat c-kit, stem cell, factor, induces c-kit receptor-dependent mouse mast cell activation in vivo. Evidence that signaling through the c-kit receptor can induce expression of cellular function.
J Exp Med
1992
, vol. 
175
 (pg. 
245
-
255
)
12
Waskow
 
C
Paul
 
S
Haller
 
C
Gassmann
 
M
Rodewald
 
HR
Viable c-Kit(W/W) mutants reveal pivotal role for c-kit in the maintenance of lymphopoiesis.
Immunity
2002
, vol. 
17
 (pg. 
277
-
288
)
13
Nocka
 
K
Tan
 
JC
Chiu
 
E
, et al. 
Molecular bases of dominant negative and loss of function mutations at the murine c-kit/white spotting locus: W37, Wv, W41 and W.
EMBO J
1990
, vol. 
9
 (pg. 
1805
-
1813
)
14
Galli
 
SJ
Arizono
 
N
Murakami
 
T
Dvorak
 
AM
Fox
 
JG
Development of large numbers of mast cells at sites of idiopathic chronic dermatitis in genetically mast cell-deficient WBB6F1-W/Wv mice.
Blood
1987
, vol. 
69
 (pg. 
1661
-
1666
)
15
Gordon
 
JR
Galli
 
SJ
Phorbol 12-myristate 13-acetate-induced development of functionally active mast cells in W/Wv but not Sl/Sld genetically mast cell-deficient mice.
Blood
1990
, vol. 
75
 (pg. 
1637
-
1645
)
16
Russell
 
E
Hereditary anemias of the mouse: a review for geneticists.
Adv Genetics
1979
, vol. 
20
 (pg. 
357
-
459
)
17
Waskow
 
C
Terszowski
 
G
Costa
 
C
Gassmann
 
M
Rodewald
 
HR
Rescue of lethal c-KitW/W mice by erythropoietin.
Blood
2004
, vol. 
104
 (pg. 
1688
-
1695
)
18
Shinkai
 
Y
Rathbun
 
G
Lam
 
KP
, et al. 
RAG-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement.
Cell
1992
, vol. 
68
 (pg. 
855
-
867
)
19
Cao
 
X
Shores
 
EW
Hu-Li
 
J
, et al. 
Defective lymphoid development in mice lacking expression of the common cytokine receptor gamma chain.
Immunity
1995
, vol. 
2
 (pg. 
223
-
238
)
20
Ruschitzka
 
FT
Wenger
 
RH
Stallmach
 
T
, et al. 
Nitric oxide prevents cardiovascular disease and determines survival in polyglobulic mice overexpressing erythropoietin.
Proc Natl Acad Sci U S A
2000
, vol. 
97
 (pg. 
11609
-
116013
)
21
Waskow
 
C
Rodewald
 
HR
Lymphocyte development in neonatal and adult c-Kit-deficient (c-KitW/W) mice.
Adv Exp Med Biol
2002
, vol. 
512
 (pg. 
1
-
10
)
22
Feyerabend
 
TB
Hausser
 
H
Tietz
 
A
, et al. 
Loss of histochemical identity in mast cells lacking carboxypeptidase A.
Mol Cell Biol
2005
, vol. 
25
 (pg. 
6199
-
6210
)
23
Rodewald
 
HR
Sato
 
TN
Tie1, a receptor tyrosine kinase essential for vascular endothelial cell integrity, is not critical for the development of hematopoietic cells.
Oncogene
1996
, vol. 
12
 (pg. 
397
-
404
)
24
Enerbäck
 
L
Berberine sulphate binding to mast cell polyanions: a cytofluometric method for the quantification of heparin.
Histochemistry
1974
, vol. 
42
 (pg. 
301
-
313
)
25
Moritz
 
DR
Rodewald
 
HR
Gheyselinck
 
J
Klemenz
 
R
The IL-1 receptor-related T1 antigen is expressed on immature and mature mast cells and on fetal blood mast cell progenitors.
J Immunol
1998
, vol. 
161
 (pg. 
4866
-
4874
)
26
Tsai
 
M
Miyamoto
 
M
Tam
 
SY
Wang
 
ZS
Galli
 
SJ
Detection of mouse mast cell-associated protease mRNA. Heparinase treatment greatly improves RT-PCR of tissues containing mast cell heparin.
Am J Pathol
1995
, vol. 
146
 (pg. 
335
-
343
)
27
Reynolds
 
DS
Stevens
 
RL
Lane
 
WS
Carr
 
MH
Austen
 
KF
Serafin
 
WE
Different mouse mast cell populations express various combinations of at least six distinct mast cell serine proteases.
Proc Natl Acad Sci U S A
1990
, vol. 
87
 (pg. 
3230
-
3234
)
28
Boutwell
 
RK
The function and mechanism of promoters of carcinogenesis.
CRC Crit Rev Toxicol
1974
, vol. 
2
 (pg. 
419
-
443
)
29
Brose
 
N
Rosenmund
 
C
Move over protein kinase C, you've got company: alternative cellular effectors of diacylglycerol and phorbol esters.
J Cell Sci
2002
, vol. 
115
 (pg. 
4399
-
4411
)
30
Wershil
 
BK
Murakami
 
T
Galli
 
SJ
Mast cell-dependent amplification of an immunologically nonspecific inflammatory response. Mast cells are required for the full expression of cutaneous acute inflammation induced by phorbol 12-myristate 13-acetate.
J Immunol
1988
, vol. 
140
 (pg. 
2356
-
2360
)
31
Suzuki
 
K
Nakajima
 
H
Watanabe
 
N
, et al. 
Role of common cytokine receptor gamma chain (gamma(c))- and Jak3-dependent signaling in the proliferation and survival of murine mast cells.
Blood
2000
, vol. 
96
 (pg. 
2172
-
2180
)
32
Rodewald
 
HR
Waskow
 
C
Haller
 
C
Essential requirement for c-kit and common gamma chain in thymocyte development cannot be overruled by enforced expression of Bcl-2.
J Exp Med
2001
, vol. 
193
 (pg. 
1431
-
1437
)
33
Nakano
 
T
Sonoda
 
T
Hayashi
 
C
, et al. 
Fate of bone marrow-derived cultured mast cells after intracutaneous, intraperitoneal, and intravenous transfer into genetically mast cell-deficient W/Wv mice. Evidence that cultured mast cells can give rise to both connective tissue type and mucosal mast cells.
J Exp Med
1985
, vol. 
162
 (pg. 
1025
-
1043
)
34
Kitamura
 
Y
Go
 
S
Decreased production of mast cells in S1/S1d anemic mice.
Blood
1979
, vol. 
53
 (pg. 
492
-
497
)
35
Rodewald
 
HR
Making a Notch in the lymphocyte kit.
Eur J Immunol
2006
, vol. 
36
 (pg. 
508
-
511
)
36
Ihle
 
JN
Keller
 
J
Oroszlan
 
S
, et al. 
Biologic properties of homogeneous interleukin 3, I: demonstration of WEHI-3 growth factor activity, mast cell growth factor activity, p cell-stimulating factor activity, colony-stimulating factor activity, and histamine-producing cell-stimulating factor activity.
J Immunol
1983
, vol. 
131
 (pg. 
282
-
287
)
37
Ody
 
C
Kindler
 
V
Vassalli
 
P
Interleukin 3 perfusion in W/Wv mice allows the development of macroscopic hematopoietic spleen colonies and restores cutaneous mast cell number.
J Exp Med
1990
, vol. 
172
 (pg. 
403
-
406
)
38
Lantz
 
CS
Boesiger
 
J
Song
 
CH
, et al. 
Role for interleukin-3 in mast-cell and basophil development and in immunity to parasites.
Nature
1998
, vol. 
392
 (pg. 
90
-
93
)

Sign in via your Institution