Genetic Inversion in Mast Cell-Deficient Wsh Mice Interrupts Corin and Manifests as Hematopoietic and Cardiac Aberrancy
2008; Elsevier BV; Volume: 173; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2008.080407
ISSN1525-2191
AutoresPeter A. Nigrović, Daniel H.D. Gray, T. G. Jones, Jenny Hallgren, Frank C. Kuo, Blair Chaletzky, Michael F. Gurish, Diane Mathis, Christophe Benoist, David M. Lee,
Tópico(s)melanin and skin pigmentation
ResumoMast cells participate in pathophysiological processes that range from antimicrobial defense to anaphylaxis and inflammatory arthritis. Much of the groundwork for the understanding of mast cells was established in mice that lacked mast cells through defects in either stem cell factor or its receptor, Kit. Among available strains, C57BL/6-KitW-sh (Wsh) mice are experimentally advantageous because of their background strain and fertility. However, the genetic inversion responsible for the Wsh phenotype remains poorly defined, and its effects beyond the mast cell have been incompletely characterized. We report that Wsh animals exhibit splenomegaly with expanded myeloid and megakaryocyte populations. Hematopoietic abnormalities extend to the bone marrow and are reflected by neutrophilia and thrombocytosis. In contrast, mast cell-deficient WBB6F1-KitW/KitW-v (W/Wv) mice display mild neutropenia, but no changes in circulating platelet numbers. To help define the basis for the Wsh phenotype, a “DNA walking” strategy was used to identify the precise location of the 3′ breakpoint, which was found to reside 67.5 kb upstream of Kit. The 5′ breakpoint disrupts corin, a cardiac protease responsible for the activation of atrial natriuretic peptide. Consistent with this result, transcription of full-length corin is ablated and Wsh mice develop symptoms of cardiomegaly. Studies performed using mast cell-deficient strains must consider the capacity of associated abnormalities to either expose or compensate for the missing mast cell lineage. Mast cells participate in pathophysiological processes that range from antimicrobial defense to anaphylaxis and inflammatory arthritis. Much of the groundwork for the understanding of mast cells was established in mice that lacked mast cells through defects in either stem cell factor or its receptor, Kit. Among available strains, C57BL/6-KitW-sh (Wsh) mice are experimentally advantageous because of their background strain and fertility. However, the genetic inversion responsible for the Wsh phenotype remains poorly defined, and its effects beyond the mast cell have been incompletely characterized. We report that Wsh animals exhibit splenomegaly with expanded myeloid and megakaryocyte populations. Hematopoietic abnormalities extend to the bone marrow and are reflected by neutrophilia and thrombocytosis. In contrast, mast cell-deficient WBB6F1-KitW/KitW-v (W/Wv) mice display mild neutropenia, but no changes in circulating platelet numbers. To help define the basis for the Wsh phenotype, a “DNA walking” strategy was used to identify the precise location of the 3′ breakpoint, which was found to reside 67.5 kb upstream of Kit. The 5′ breakpoint disrupts corin, a cardiac protease responsible for the activation of atrial natriuretic peptide. Consistent with this result, transcription of full-length corin is ablated and Wsh mice develop symptoms of cardiomegaly. Studies performed using mast cell-deficient strains must consider the capacity of associated abnormalities to either expose or compensate for the missing mast cell lineage. Mast cells arise as precursors in the bone marrow and mature within the tissues, where they participate in both allergic and nonallergic immune processes. Phylogenetic studies confirm that this lineage predates the development of humoral immunity, implying that participation in IgE-mediated responses is a rather late specialization.1McNeil HP Adachi R Stevens RL Mast cell-restricted tryptases: structure and function in inflammation and pathogen defense.J Biol Chem. 2007; 282: 20785-20789Crossref PubMed Scopus (86) Google Scholar Indeed, mast cells have now been implicated in a broad range of pathophysiologic processes, where they most typically initiate or amplify immune responses via rapid release of preformed and newly synthesized mediators. One important role in this regard is the mobilization of defenses against bacteria and parasites.2Echtenacher B Mannel DN Hultner L Critical protective role of mast cells in a model of acute septic peritonitis.Nature. 1996; 381: 75-77Crossref PubMed Scopus (818) Google Scholar, 3Malaviya R Ikeda T Ross E Abraham SN Mast cell modulation of neutrophil influx and bacterial clearance at sites of infection through TNF-alpha.Nature. 1996; 381: 77-80Crossref PubMed Scopus (996) Google Scholar, 4Gurish MF Bryce PJ Tao H Kisselgof AB Thornton EM Miller HR Friend DS Oettgen HC IgE enhances parasite clearance and regulates mast cell responses in mice infected with Trichinella spiralis.J Immunol. 2004; 172: 1139-1145PubMed Google Scholar, 5Thakurdas SM Melicoff E Sansores-Garcia L Moreira DC Petrova Y Stevens RL Adachi R The mast cell-restricted tryptase mMCP-6 has a critical immunoprotective role in bacterial infections.J Biol Chem. 2007; 282: 20809-20815Crossref PubMed Scopus (152) Google Scholar, 6Shin K Watts GF Oettgen HC Friend DS Pemberton AD Gurish MF Lee DM Mouse mast cell tryptase mMCP-6 is a critical link between adaptive and innate immunity in the chronic phase of trichinella spiralis infection.J Immunol. 2008; 180: 4885-4891PubMed Google Scholar Rapid mediator release also has pathogenic consequences, such as anaphylaxis or the initiation phase of inflammatory arthritis.7Martin TR Galli SJ Katona IM Drazen JM Role of mast cells in anaphylaxis. Evidence for the importance of mast cells in the cardiopulmonary alterations and death induced by anti-IgE in mice.J Clin Invest. 1989; 83: 1375-1383Crossref PubMed Scopus (92) Google Scholar, 8Nigrovic PA Binstadt BA Monach PA Johnsen A Gurish M Iwakura Y Benoist C Mathis D Lee DM Mast cells contribute to initiation of autoantibody-mediated arthritis via IL-1.Proc Natl Acad Sci USA. 2007; 104: 2325-2330Crossref PubMed Scopus (157) Google Scholar Beyond this “sentinel” role, mast cells participate in the neutralization of venoms and inflammatory mediators as well as other biological processes (reviewed in9Metz M Grimbaldeston MA Nakae S Piliponsky AM Tsai M Galli SJ Mast cells in the promotion and limitation of chronic inflammation.Immunol Rev. 2007; 217: 304-328Crossref PubMed Scopus (258) Google Scholar). These conclusions have been reached largely through experiments in mice with spontaneous genetic mutations that perturb mast cell differentiation. Mast cells at all stages of maturation express the receptor tyrosine kinase Kit (CD117) and require the Kit ligand, stem cell factor (SCF), for their survival. Most laboratory strains deficient in mast cells arise from mutations affecting the Kit/SCF axis. For example, the WCB6F1/J-KitlSl/KitlSl-d mouse lacks SCF on the surface of fibroblasts and other cells.10Zsebo KM Williams DA Geissler EN Broudy VC Martin FH Atkins HL Hsu RY Birkett NC Okino KH Murdock DC Frederick W Jacobsen Langley Keith E Smith Kent A Takeish Takashi Cattanach Bruce M Galli Stephen J Suggs Sidney V Stem cell factor is encoded at the Sl locus of the mouse and is the ligand for the c-kit tyrosine kinase receptor.Cell. 1990; 63: 213-224Abstract Full Text PDF PubMed Scopus (1221) Google Scholar The W/Wv mouse bears a compound mutation (one allele null, the other impaired) at the Kit locus W (white spotting), while the Wsh mouse carries an incompletely characterized inversion upstream of Kit that affects a key regulatory element.11Nocka K Tan JC Chiu E Chu TY Ray P Traktman P Besmer P 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; 9: 1805-1813Crossref PubMed Scopus (471) Google Scholar, 12Duttlinger R Manova K Chu TY Gyssler C Zelenetz AD Bachvarova RF Besmer P W-sash affects positive and negative elements controlling c-kit expression: ectopic c-kit expression at sites of kit-ligand expression affects melanogenesis.Development. 1993; 118: 705-717PubMed Google Scholar, 13Duttlinger R Manova K Berrozpe G Chu TY DeLeon V Timokhina I Chaganti RS Zelenetz AD Bachvarova RF Besmer P The Wsh and Ph mutations affect the c-kit expression profile: c-kit misexpression in embryogenesis impairs melanogenesis in Wsh and Ph mutant mice.Proc Natl Acad Sci USA. 1995; 92: 3754-3758Crossref PubMed Scopus (81) Google Scholar, 14Nagle DL Kozak CA Mano H Chapman VM Bucan M Physical mapping of the Tec and Gabrb1 loci reveals that the Wsh mutation on mouse chromosome 5 is associated with an inversion.Hum Mol Genet. 1995; 4: 2073-2079Crossref PubMed Scopus (48) Google Scholar, 15Berrozpe G Timokhina I Yukl S Tajima Y Ono M Zelenetz AD Besmer P The W(sh), W(57), and Ph Kit expression mutations define tissue-specific control elements located between -23 and -154 kb upstream of Kit.Blood. 1999; 94: 2658-2666PubMed Google Scholar, 16Berrozpe G Agosti V Tucker C Blanpain C Manova K Besmer P A distant upstream locus control region is critical for expression of the Kit receptor gene in mast cells.Mol Cell Biol. 2006; 26: 5850-5860Crossref PubMed Scopus (34) Google Scholar Mice with mutations affecting Kit (rather than SCF) are particularly useful because they can be engrafted with cultured mast cells.17Nakano T Sonoda T Hayashi C Yamatodani A Kanayama Y Yamamura T Asai H Yonezawa T Kitamura Y Galli SJ 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; 162: 1025-1043Crossref PubMed Scopus (400) Google Scholar, 18Grimbaldeston MA Chen CC Piliponsky AM Tsai M Tam SY Galli SJ Mast cell-deficient W-sash c-kit mutant Kit W-sh/W-sh mice as a model for investigating mast cell biology in vivo.Am J Pathol. 2005; 167: 835-848Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar An abnormal phenotype that can be corrected with such engraftment may presumptively be attributed to mast cell deficiency. Such complementation studies are important because mutations affecting Kit have effects beyond the mast cell lineage. For example, the W/Wv mouse is white, anemic, partially deaf, prone to dermatitis and gastritis, and lacks intestinal interstitial cells of Cajal, as well as intraepithelial γδ T lymphocytes (reviewed in18Grimbaldeston MA Chen CC Piliponsky AM Tsai M Tam SY Galli SJ Mast cell-deficient W-sash c-kit mutant Kit W-sh/W-sh mice as a model for investigating mast cell biology in vivo.Am J Pathol. 2005; 167: 835-848Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar). Bone marrow and circulating neutropenia have also been described.19Chervenick PA Boggs DR Decreased neutrophils and megakaryocytes in anemic mice of genotype W/W.J Cell Physiol. 1969; 73: 25-30Crossref PubMed Scopus (63) Google Scholar, 20Zhou JS Xing W Friend DS Austen KF Katz HR Mast cell deficiency in Kit(W-sh) mice does not impair antibody-mediated arthritis.J Exp Med. 2007; 204: 2797-2802Crossref PubMed Scopus (152) Google Scholar Further, W/Wv mice are sterile, so colony maintenance and breeding are cumbersome, and control mice are WBB6 heterozygotes. Wsh mice are also white (the heterozygote exhibiting a white abdominal sash, providing the strain name, the homozygote having residual ear pigment), but they are not anemic and are fully fertile. Other hematological lineages have been considered normal based on the comparison of limited numbers of Wsh and C57BL/6 animals.18Grimbaldeston MA Chen CC Piliponsky AM Tsai M Tam SY Galli SJ Mast cell-deficient W-sash c-kit mutant Kit W-sh/W-sh mice as a model for investigating mast cell biology in vivo.Am J Pathol. 2005; 167: 835-848Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar, 20Zhou JS Xing W Friend DS Austen KF Katz HR Mast cell deficiency in Kit(W-sh) mice does not impair antibody-mediated arthritis.J Exp Med. 2007; 204: 2797-2802Crossref PubMed Scopus (152) Google Scholar The breeding advantage and C57BL/6 background strain (from backcrossing, after the mutation arose spontaneously during a cross between C3H/HeH and 101/H)21Lyon MF Glenister PH A new allele sash (Wsh) at the W-locus and a spontaneous recessive lethal in mice.Genet Res. 1982; 39: 315-322Crossref PubMed Scopus (73) Google Scholar have made Wsh mice recent favorites for work in the mast cell field. In initial analyses of Wsh mice, it was noted that some animals had strikingly enlarged and histologically abnormal spleens. Given the increasing importance of the Wsh strain in mast cell research, we wished to better understand “off-target” hematological effects of the large gene inversion in these animals, since associated abnormalities could alter experimental interpretation. The results presented herein provide an expanded phenotypic and genotypic characterization of this important experimental strain, and suggest that experimental results obtained in both Wsh and W/Wv must be interpreted in light of the potential role of accompanying abnormalities on the phenotype of interest. WBB6 F1-Kitw/KitW-v (W/Wv), WBB6 littermate controls and C57BL/6J (B6) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). C57BL/6-KitW-sh/W-sh mice22Wolters PJ Mallen-St Clair J Lewis CC Villalta SA Baluk P Erle DJ Caughey GH Tissue-selective mast cell reconstitution and differential lung gene expression in mast cell-deficient Kit(W-sh)/Kit(W-sh) sash mice.Clin Exp Allergy. 2005; 35: 82-88Crossref PubMed Scopus (133) Google Scholar were maintained at The Jackson Laboratory. Animals were housed for at least 2 weeks in the specific-pathogen-free animal facility of the Dana-Farber Cancer Institute before sacrifice for phenotyping experiments. All procedures were approved by the animal care and use committees of the Dana-Farber Cancer Institute or Harvard Medical School. Age-matched male mice (W/Wv and WBB6: 12 to 20 weeks, Wsh and B6: 10 to 15 weeks, except as noted) were anesthetized with isofluorane for bleeding by cardiac puncture. Whole blood was collected in the presence of EDTA to prevent clotting. A complete blood count and automated leukocyte differential was obtained using an Advia 120 Hematology System (Siemens, Tarrytown, NY) and appropriate species-specific standards and software. The accuracy of the automated neutrophil assessment was confirmed by parallel flow cytometric examination of selected blood samples stained for CD45 and Gr-1 (data not shown). Spleens were removed, cleaned of attached tissues, and weighed. Remaining splenic tissue was disaggregated and filtered through 100 μm mesh for cytofluorometric analysis. Hearts were removed, cleaned of excess soft tissue, and compressed to expel luminal blood before weighing. Femoral bone marrow was obtained by flush and passed through 100-μm mesh to separate adherent cells and exclude bone fragments. Tissue taken for histology was fixed in 4% paraformaldehyde in PBS. Samples were washed in PBS with 10% fetal bovine serum and stained with appropriate antibodies and isotype controls. Cytofluorometric analysis was performed using a FACSDiva cytometer (Becton-Dickinson, Franklin Lakes, NJ). All samples were co-stained with 7-amino-actinomycin D (7-AAD) and anti-CD45.2 (BD Biosciences, Franklin Lakes, NJ), and gated to include only viable (7-AAD negative) CD45+ cells. Other antibodies used were as follows: CD3-flurorescein isothiocyanate, CD41-phycoerythrin (BD Biosciences), CD11b-flurorescein isothiocyanate, CD117-Alexa647, F4/80-phycoerythrin, Gr-1-flurorescein isothiocyanate, Gr-1-Alexa647 (Invitrogen, Carlsbad, CA), CD11c-allophycocyanin, CD19-phycoerythrin (eBioscience, San Diego, CA). Mast cell progenitors were enumerated by limiting dilution analysis, as described.23Gurish MF Tao H Abonia JP Arya A Friend DS Parker CM Austen KF Intestinal mast cell progenitors require CD49dbeta7 (alpha4beta7 integrin) for tissue-specific homing.J Exp Med. 2001; 194: 1243-1252Crossref PubMed Scopus (202) Google Scholar Briefly, splenic mononuclear cells were isolated from disaggregated splenocytes via Percoll gradient, enumerated, and cultured by limiting dilution in the presence of irradiated syngenic feeder splenocytes and baculovirus-generated SCF and interleukin-3 (both at 20 ng/ml final). Culture media was RPMI 1640 supplemented with 10% fetal calf serum, glutamine (2 mmol/L), penicillin (100 U/ml), streptomycin (100 μg/ml), gentamicin (50 μg/ml), pyruvate (1 mmol/L), nonessential amino acids, Hepes (10 mmol/L), and 2-mercaptoethanol (50 μmol/L). The mast cell colonies with their distinct morphology were counted at 10 to 14 days. While committed mast cell progenitors have typically been enumerated in peripheral tissues with this technique,24Hallgren J Jones TG Abonia JP Xing W Humbles A Austen KF Gurish MF Pulmonary CXCR2 regulates VCAM-1 and antigen-induced recruitment of mast cell progenitors.Proc Natl Acad Sci USA. 2007; 104: 20478-20483Crossref PubMed Scopus (78) Google Scholar myeloid progenitors upstream of committed mast cell progenitors that are SCF- and/or interleukin-3-responsive would also be expected to form colonies in this assay.25Arinobu Y Iwasaki H Gurish MF Mizuno S Shigematsu H Ozawa H Tenen DG Austen KF Akashi K Developmental checkpoints of the basophil/mast cell lineages in adult murine hematopoiesis.Proc Natl Acad Sci USA. 2005; 102: 18105-18110Crossref PubMed Scopus (280) Google Scholar Thus this assay enumerates the cells capable of giving rise to committed mast cells within this tissue. Histological sections were stained with H&E, and examined at ×100 and ×400 using a Leica DM LB2 microscope and Leica digital camera. The 5′-breakpoint of the Wsh inversion mutation was located by PCR with primers that yield a 600-bp product from wild-type (C57BL/6J), but not Wsh template (WT-for, 5′-TTTGCACGTGCTA GTTACAC-3′; WT-rev, 5′-TTAAGATGGCACCCTGCTG-3′). A series of three nested PCR primers was designed for sequencing 5′ to the distal breakpoint (TSP1, 5′-C CTCAGCCTGTCACACTTATG-3′; TSP2, 5′-GACAACGAAATGATACAGAG GATTC-3′; TSP3, 5′-GAGGATTCATAGTTGTTCAATGTCC-3′), and were used with the DNA Walking Speedup Kit (Seegene, Rockville, MD) to amplify a single 500-bp product from Wsh, but not WT template. Confirmatory PCR primers flanking the predicted breakpoint were designed (Wsh-for, 5′-AGGCTTGCAGCGCATTAT-3′; Wsh-rev, 5′-GAGGATTCATAGTTGTTCAATGTCC-3′). The “WT” and “Wsh” primers can be used for genotyping purposes with the PCR program: 94°C for 5 minutes, followed by 35 cycles of 94°C for 20 seconds; 57°C for 30 seconds; 72°C for 1 minute, followed by 72°C for 10 minutes. Regions of corin intron 5 and exon 6 were amplified using the following primers: Int 5A-for, 5′-GGGGGATTGTTTCCAATTCT-3′; Int 5A-rev, 5′-TGGA CCTAGAGGGCATCATC-3′, Int 5B-for, 5′-TCAGCCATTCGGTATTCCTC-3′; Int 5B-rev, 5′-AAAAGGCCACCAACAGATTG-3′; Exo 6-for, 5′-GGGGTTCT GGAAGGAAATCT-3′; Exo 6-rev, 5′-TCGCTCCAGTCAT CACAGTC-3′. Total RNA was prepared from atria dissected from wild-type or Wsh mice and cDNA synthesized using random primers. PCR was performed with SYBR Green Mastermix (Applied Biosystems, UK) using primers designed to corin exons 3 to 4 (forward-5′-TCCT TCTCCAGAGGACCAGA-3′; reverse-5′-AGGGCAGAATTTGACACTGG-3′), 5 to 6 (forward-5′-GCAGGAACATGGAAAGCAAT-3′; reverse-5′-TGGTACACAGGAA GCTCTCG-3′), or 10 to 11 (forward-5′-GACAGCAGCCTGAGTAACTGC-3′; reverse-5′-TGTAGGG CAAATTCATGCAG-3′) on a Mx3000p PCR machine (Stratagene, La Jolla, CA). Data were analyzed using the 2−ΔΔCt method normalized to a single wild-type data set.26Livak KJ Schmittgen TD Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.Methods. 2001; 25: 402-408Crossref PubMed Scopus (130715) Google Scholar Experimental strains were compared with background-control animals using the Student's t-test without correction for multiple comparisons. Data are expressed as mean ± SEM. P values ≤0.05 were considered significant. Initial work with the Wsh mice was notable for several splenic abnormalities. Even among littermates, a striking divergence of spleen size was readily observed (Figure 1A–B). These changes were not explained by body mass, which was equivalent between Wsh and B6 animals (body weight mean of male 12-week old mice n = 15 to 18/group: Wsh 25.5 ± 0.5g, B6 26.1 ± 0.4g, P = 0.32). Histologically, splenic architecture ranged from normal to strikingly abnormal, with disrupted white pulp and abundant large multinucleated cells (Figure 1C). At higher power, these large cells were seen to be normal-appearing megakaryocytes, while abundant neutrophils were also evident (Figure 1D). Erythroid precursors were increased as well, indicating exaggerated trilineage hematopoiesis. In contrast, W/Wv spleens were slightly smaller than WBB6 controls and appeared histologically normal (Figure 1B and data not shown). To evaluate these abnormalities, splenocyte populations were quantified by cytofluorimetry and histomorphometry. Consistent with an expansion of the myeloid compartment in many of the Wsh mice, the proportion of cells expressing CD11b was elevated 2- to 3-fold, accompanied by a fivefold increase in cells expressing the myeloid marker Gr-1 and an almost twofold increase in cells expressing F4/80 (Figure 2, A–C). Reciprocal decreases were found in the proportion of splenic B and T cells (data not shown). Cells expressing the dendritic cell marker CD11c were present in normal proportions (data not shown). Histomorphometric enumeration of megakaryocytes demonstrated a greater than 20-fold increase in the number of cells visible in a splenic cross section, corresponding to a more than hundred-fold expansion extrapolated across three dimensions (Figure 2D). Since expression of CD11b together with Gr-1 is thought to mark a regulatory myeloid phenotype, co-expression of these antigens was examined.27Delano MJ Scumpia PO Weinstein JS Coco D Nagaraj S Kelly-Scumpia KM O'Malley KA Wynn JL Antonenko S Al-Quran SZ Swan R Chung CS Atkinson MA Ramphal R Gabrilovich DI Reeves WH Ayala A Phillips J Laface D Heyworth PG Clare-Salzler M Moldawer LL MyD88-dependent expansion of an immature GR-1(+)CD11b(+) population induces T cell suppression and Th2 polarization in sepsis.J Exp Med. 2007; 204: 1463-1474Crossref PubMed Scopus (547) Google Scholar Both Gr-1hiCD11bhi and Gr-1intCD11bint populations were expanded in Wsh animals (Figure 2, E–F). Unlike Wsh animals, W/Wv mice showed a reduction in splenic CD11b+ cells, Gr-1+ cells, and Gr-1hi CD11bhi splenocytes compared with their control strain (Figure 2, A, B, F). Although the Wsh mutation impairs Kit expression in mast cells, its effect on Kit expression is variable among tissues, and enhanced expression may be observed in a regionally and developmentally regulated manner.12Duttlinger R Manova K Chu TY Gyssler C Zelenetz AD Bachvarova RF Besmer P W-sash affects positive and negative elements controlling c-kit expression: ectopic c-kit expression at sites of kit-ligand expression affects melanogenesis.Development. 1993; 118: 705-717PubMed Google Scholar Further, signaling via Kit has been implicated in the recruitment of myeloid precursors to the spleen.28Broudy VC Lin NL Priestley GV Nocka K Wolf NS Interaction of stem cell factor and its receptor c-kit mediates lodgment and acute expansion of hematopoietic cells in the murine spleen.Blood. 1996; 88: 75-81Crossref PubMed Google Scholar Accordingly, splenocyte expression of Kit was studied. Unlike splenocytes from W/Wv mice, where surface Kit was rare, Kit+ cells were present in elevated proportion in Wsh mice compared to control (Figure 3A). This finding was supported by analysis of the mast cell potential of splenocytes by stimulation with SCF and interleukin-3. These conditions are optimized to grow mast cell progenitors and thus the numbers represent the number of cells potentially capable of becoming committed mast cell progenitors, which includes the early myeloid progenitors as well as those committed to the mast cell lineage.23Gurish MF Tao H Abonia JP Arya A Friend DS Parker CM Austen KF Intestinal mast cell progenitors require CD49dbeta7 (alpha4beta7 integrin) for tissue-specific homing.J Exp Med. 2001; 194: 1243-1252Crossref PubMed Scopus (202) Google Scholar, 25Arinobu Y Iwasaki H Gurish MF Mizuno S Shigematsu H Ozawa H Tenen DG Austen KF Akashi K Developmental checkpoints of the basophil/mast cell lineages in adult murine hematopoiesis.Proc Natl Acad Sci USA. 2005; 102: 18105-18110Crossref PubMed Scopus (280) Google Scholar Indeed, the density of mast cell colonies emerging from Wsh spleen was fourfold greater than that from B6 and 20-fold greater than that from W/Wv mice (Figure 3B). Whether these hematological abnormalities extended to the bone marrow was then investigated. Consistent with earlier results, cells expressing CD11b and Gr-1 were expanded in Wsh marrow and diminished in W/Wv marrow, though F4/80 expression was inversely affected (Figure 4A–C). Both Gr-1hiCD11bhi and Gr-1intCD11bint populations were expanded in Wsh mice (Figure 4, D and E). Kit expression in Wsh was similar or slightly decreased, while it was essentially undetectable in W/Wv individuals (Figure 4F). Again, B and T cells proportions were reciprocally reduced while CD11c expression was equivalent (data not shown). Finally, circulating blood parameters in Wsh and W/Wv mice were quantified. As has been described, W/Wv mice were anemic, while Wsh mice displayed mean hematocrit values similar to those of B6 animals, though with a wider scatter among individuals (data not shown). Consistent with observations in the spleen and marrow, Wsh mice exhibited an almost 70% increase in absolute circulating neutrophils compared with B6 controls (0.92 ± 0.10 vs. 0.55 ± 0.09 × 103 cells/μL, P = 0.008), while W/Wv mice manifested a 40% decrease relative to WBB6 (0.32 ± 0.05 vs. 0.53 ± 0.07 × 103 cells/μL, P = 0.03) (Figure 5A). Finally, in keeping with the megakaryocytosis observed in spleen, and quite distinct from the other strains tested, Wsh mice exhibited a previously undescribed thrombocytosis (1824 ± 114 vs. 1155 ± 37 × 103 cells/μL, P < 0.0001) (Figure 5B). Since these results differed in certain respects from those of other authors who have compared Wsh and B6 mice aged 7 to 9 weeks,20Zhou JS Xing W Friend DS Austen KF Katz HR Mast cell deficiency in Kit(W-sh) mice does not impair antibody-mediated arthritis.J Exp Med. 2007; 204: 2797-2802Crossref PubMed Scopus (152) Google Scholar a potential age-dependence of the hematopoietic phenotype was examined using 7 to 8 week-old mice (9 male animals pooled from 2 independent experiments). Indeed, splenomegaly was less pronounced in the younger animals, though still evident (Wsh 0.086 ± 0.004g versus B6 0.069 ± 0.002g, P = 0.0016). However, the relative neutrophilia in the blood and bone marrow occurred at a magnitude similar to older animals (bone marrow %Gr-1+ Wsh 51.9 ± 2.5 vs. B6 39.4 ± 1.0, P = 0.0003; blood absolute circulating neutrophil count Wsh 0.34 ± 0.04 × 103 cells/μL versus B6 0.14 ± 0.01 × 103 cells/μL, P < 0.0001). Similarly, splenic neutrophilia and circulating thrombocytosis were also observed at this younger age (data not shown). Accordingly, these findings suggest that younger Wsh animals are not free of hematopoietic aberrancy. To understand further the genetic basis for the hematological defects observed in Wsh mice, the causative inversion on chromosome 5 was defined. The 5′-breakpoint of the inversion had been shown to reside 2.8 to 3.3 Mb upstream of the 3′-breakpoint, somewhere between gabrb1 and tec, and thereby with the potential to directly interrupt the coding sequence of 10 genes.14Nagle DL Kozak CA Mano H Chapman VM Bucan M Physical mapping of the Tec and Gabrb1 loci reveals that the Wsh mutation on mouse chromosome 5 is associated with an inversion.Hum Mol Genet. 1995; 4: 2073-2079Crossref PubMed Scopus (48) Google Scholar The 3′ breakpoint had been localized approximately 72 kb upstream of Kit, extinguishing the influence of a locus control region important for mast cell Kit expression.15Berrozpe G Timokhina I Yukl S Tajima Y Ono M Zelenetz AD Besmer P The W(sh), W(57), and Ph Kit expression mutations define tissue-specific control elements located between -23 and -154 kb upstream of Kit.Blood. 1999; 94: 2658-2666PubMed Google Scholar, 16Berrozpe G Agosti V Tucker C Blanpain C Manova K Besmer P A distant upstream locus control region is critical for expression of the Kit receptor gene in mast cells.Mol Cell Biol. 2006; 26: 5850-5860Crossref PubMed Scopus (34) Google Scholar PCR analysis with primers flanking this region amplified product from wild-type genomic DNA, but not Wsh template (Figure 6, A and B), thus confirming these findings. Three nested reverse primers for sequencing immediately downstream of the predicted 3′ breakpoint were used to perform a “DNA walking” reaction using wild-type or Wsh template. Sequencing of a single 500-bp product amplified from Wsh, but not wild-type, genomic DNA revealed that the entire fragment was homologous to an inverted intron of corin, a gene residing within the region proposed to contain the 5′ breakpoint. PCR using several pairs of primers designed to flank the predicted 3′ breakpoint amplified the expected products from Wsh but not wild-type template (eg, Figure 6A). Sequence analysis confirmed the exact site of the 3′ breakpoint at Ensembl (release 49) position 75,903,511(+) between bases C and T, 67.5 kB upstream of the Kit start sequence, with an insertion of the bases TC (Figure 6B). Based on the sequence brought to the 3′ breakpoint from corin, the predicted site of the 5′ breakpoint resides at Ensembl position 72,790,584(−) between bases C and G (reverse strand). This position lies between exons 5 and 6 of corin. Multiple attempts to amplify a PCR product using primers expected to flank the 5′ endpoint were unsuccessful, raising concerns about potential loss or addition of genetic material at that breakpoint. To delimit the extent of such anoma
Referência(s)