Article Figures & Data
Figures
- FIGURE 1.
Mast cell infiltration in the glandular stomach of IL-9 transgenic mice. The mucosal region of the stomach located closed to the esophagus was analyzed by safranin (A and B) and chloroesterase-specific (C andD) staining for IL-9 transgenic (A and C) and normal FVB (B andD) mice. IL-9 transgenics showed safranin-positive (A, arrowhead) and chloroesterase-positive (C, arrowhead) intraepithelial mast cells that were not observed in the normal FVB mice (B and D). Meanwhile, a few positive cells for chloroesterase staining (D, arrow) located in the connective tissue surrounding stomach glands were observed in FVB as well as in IL-9-Tg (C and D, arrow).
- FIGURE 2.
Intestinal mast cell infiltration in IL-9 transgenic mice. The small intestine from IL-9 transgenic (A,C, D, and E) or control FVB (B and F) mice was analyzed after hematoxylin-eosin staining (A andB), Giemsa staining (C), or chloroesterase-specific staining (D–F). Arrows and boxes indicate intraepithelial mast cells showing a granular eosinophilic content after hematoxylin-eosin staining (A) and appearing as slightly pink blue with Giemsa staining (C) but negative after chloroesterase-specific staining (D). The large arrow in D points to a chloroesterase-positive submucosal mast cell. In the muscularis mucosae, chloroesterase staining reveals the presence of increased numbers of mast cells in IL-9 transgenic mice (E) compared with those in normal FVB mice (F), particularly in the vicinity of lymph nodes as illustrated here.
- FIGURE 3.
Electron microscopy of intestinal IL-9 transgenic mast cells. Electron microsopy of intraepithelial granulated cells (large arrow) in IL-9 transgenic mice shows features compatible with mast cells in the small gut (A) as well as in the stomach (B). The granule content in the small intestine seemed more crystalline (inset). In both pictures, comparison can be made with eosinophilic granules (small arrows).
- FIGURE 4.
Mast cell infiltration in IL-9 transgenic lungs and kidneys. In between epithelial cells of IL-9 transgenic tracheal cells, a large number of chloroesterase-positive cells (A, arrowheads) was observed, while they were not found in FVB normal mice (B). Quantitatively, six or seven mast cells were found per square millimeter of IL-9-transgenic trachea epithelium compared with <0.1 in wild-type mice. The same observation was made in the kidney of IL-9 transgenic mice stained with Giemsa (C) compared with that of normal FVB mice (D).
- FIGURE 5.
Expression of mast cell differentiation markers in the glandular stomach of IL-9 transgenic mice. Total RNA was extracted from the glandular stomach of 6- to 8-wk-old Tg5, Tg54, or normal FVB mice (three mice per group). RT-PCR amplification was performed as described in Materials and Methods, using the primers described in Table I. For mMCP-1, -2, and -4 (left panel), cDNAs were amplified by a single pair of primers corresponding to identical sequences in these genes, and the PCR product was hybridized with three specific probes, whose specificities are shown using cloned mMCP-1, -2, and -4 cDNAs. For all other genes, specific oligonucleotides were used for both PCR amplification and hybridization.
- FIGURE 6.
Derivation of mast cell cultures from bone marrow progenitors in the presence of IL-3, IL-9, and SCF. Bone marrow cells from 4-wk-old BALB/c mice were cultured for 15 days in enriched medium supplemented with the indicated combinations of IL-3 (1 ng/ml), IL-9 (200 U/ml), and SCF-transfected COS cell supernatant (5%). Supernatants from mock-transfected COS cells were used as a control and proved completely inactive. Every 4 days, the nonadherent cells in the cultures were transferred into new flasks and suspended in fresh culture medium supplemented with the appropriate combination of cytokines. After 15 days, the binding of biotinylated IgE was measured by FACS analysis (A). Mast cell numbers shown in B correspond to the numbers of cells positive for IgE binding.
- FIGURE 7.
Mast cell protease expression pattern of BMMC. RNA from bone marrow-derived cultures, obtained as described in Figure 6, was analyzed by RT-PCR as described in Materials and Methods. For mMCP-1, -2, and -4, cDNAs were amplified by a single pair of primers corresponding to identical sequences in these genes, and the PCR product was hybridized with three specific probes. For all other genes, specific oligonucleotides were used for both PCR amplification and hybridization.
- FIGURE 8.
SCF and IL-9 expression in the glandular stomach of IL-9 transgenic and control mice. Total RNA was extracted from the glandular stomach of 6- to 8-wk-old Tg5, Tg54, or normal FVB mice (three mice per group). Nonsaturating RT-PCR amplification and hybridization were performed as described in Materials and Methods, using the oligonucleotides described in Table I.
- FIGURE 9.
Inhibition of IL-9-transgenic mastocytosis by anti-c-Kit treatment. The rat anti-murine c-Kit mAb ACK-2 or control rat IgG was administered daily to 6- to 8-wk-old male Tg54 mice (n = 5) for 4 days. Animals received 0.5 mg on days 1 and 3, and 1 mg on days 2 and 4. On day 5, the animals were killed, and mast cell numbers were determined after toluidine blue staining. Results show the number of mast cells in 20 villus-crypt units for small intestine or for one high power field of view for stomach mucosa. In the stomach, p < 0.0002 for ACK-2 treatment vs no Ab or control (Ctl) Ab (by Student’s t test). In the small intestine, p < 0.0001 for ACK-2 treatment vs no Ab, and p = 0.0013 vs control Ab (Student’s t test). Differences between the no Ab and control Ab groups were not statistically significant. Control FVB mice usually had <10 mast cells/20 villus-crypt units (data not shown).
Tables
Target Primer Site Sequence β-actin Sense 5′-ATGGATGACGATATCGCTGC-3′ Antisense 5′-GCTGGAAGGTGGACAGTGAG-3′ Probe 5′-CGTACCACAGGCATTGTGATGG-3′ mMCP-1, -2, -4 Sense 5′-GCTGGAGCTGAGGAGATT-3′ Antisense 5′-GGTGAAGACTGCAGGGG-3′ mMCP-1 Probe 5′-AGTTAAAAACAGCATACATGG-3′ mMCP-2 Probe 5′-TAAGAACGGTTCGAAGGAGAGG-3′ mMCP-4 Probe 5′-TGAGAGAGGGTTCACAGCTAC-3′ mMCP-5 Sense 5′-GAACTACCTGTCGGCCTGCAG-3′ Antisense 5′-AGAACCTTCTGGAAGCTCAGG-3′ Probe 5′-GTCCACGACATCATGCTACTG-3′ mMCP-7 Sense 5′-CCTCACTGTGTCCAAATGCTA-3′ Antisense 5′-CCTCCTGCCTCAGAGACC-3′ Probe 5′-GGCAGGTGAGCCTGCGTGCCA-3′ Granzyme B Sense 5′-CTCCACGTGCTTTCACCAAA-3′ Antisense 5′-GGAAAATAGTACAGAGAGGCA-3′ Probe 5′-GCTCTGCTTCCCCTCAGTGCC-3′ FcεRIα Sense 5′-TTAGCACCTGAAGGTGCAGGG-3′ Antisense 5′-ACATGAGTGGCCTTTGACAGT-3′ Probe 5′-GGTACCGTCTCTGAGGTGAAC-3′ FcεRIβ Sense 5′-AAATGGACACAGAAAATAGGAGC-3′ Antisense 5′-GGCATCTGCAGAACAATCCAA-3′ Probe 5′-GTCTGCTCCGTACTCTATTGG-3′ SCF Sense 5′-TGGAGCTCTAGAACAGCTAAACGG-3′ Antisense 5′-GGCTGTCTAGAGTAGGCTGGAGTC-3′ Probe 5′-GCATCTGACACTAGTGACTGT-3′ IL-9 Sense 5′-GTGCTGGGCCAGAGATGCAGC Antisense 5′-GCATCTGTCTTCATGGTCGGC-3′ Probe 5′-GCCTGTTTTCCATCGGGTGAAA-3′
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