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• W2163935490 abstract "Neutropenia is common to both Hermansky-Pudlak syndrome type 2 and canine cyclic hematopoiesis (CH) which are caused by mutations in the AP3B1 gene. The purpose of this study was to determine if pearl mice were neutropenic. Complete blood counts (CBCs) and bone marrow differential counts, colony forming unit (CFU) assay, bone marrow lineage negative (lin–), Sca+ and c-kit+ cells (LSK cells), bone marrow elastase, myeloperoxidase, and cathepsin G enzyme activity were compared in C57Bl6 (Bl/6) and pearl mice. Stress granulopoiesis was evaluated following 200 mg/kg cyclophosphamide or 1 mg/kg bortezomib administration and by limiting dilution bone marrow transplantation. The CBCs and CFUs were determined in Bl/6 and pearl mice following AMD3100 or granulocyte colony-stimulating factor (G-CSF) administration. Pearl mice were not neutropenic and did not have cyclic neutropenia. Bone marrow elastase, myeloperoxidase, and cathepsin G enzyme activity were similar in pearl and Bl/6 mice. The numbers of CFU-G, CFU-GEMM, and LSK cells were increased moderately in pearl mice. Stress granulopoiesis was similar in Bl/6 and pearl mice. CFU assays and CBCs performed on Bl/6 and pearl mice administered AMD3100 resulted in similar results. However, normal mice administered G-CSF had higher peripheral blood neutrophil counts and greater CFU numbers compared with pearl mice. Unlike patients with HPS-2 and dogs with CH, pearl mice did not have neutropenia or CH but had decreased hematopoietic progenitor cell and granulocyte mobilization in response to G-CSF. Neutropenia is common to both Hermansky-Pudlak syndrome type 2 and canine cyclic hematopoiesis (CH) which are caused by mutations in the AP3B1 gene. The purpose of this study was to determine if pearl mice were neutropenic. Complete blood counts (CBCs) and bone marrow differential counts, colony forming unit (CFU) assay, bone marrow lineage negative (lin–), Sca+ and c-kit+ cells (LSK cells), bone marrow elastase, myeloperoxidase, and cathepsin G enzyme activity were compared in C57Bl6 (Bl/6) and pearl mice. Stress granulopoiesis was evaluated following 200 mg/kg cyclophosphamide or 1 mg/kg bortezomib administration and by limiting dilution bone marrow transplantation. The CBCs and CFUs were determined in Bl/6 and pearl mice following AMD3100 or granulocyte colony-stimulating factor (G-CSF) administration. Pearl mice were not neutropenic and did not have cyclic neutropenia. Bone marrow elastase, myeloperoxidase, and cathepsin G enzyme activity were similar in pearl and Bl/6 mice. The numbers of CFU-G, CFU-GEMM, and LSK cells were increased moderately in pearl mice. Stress granulopoiesis was similar in Bl/6 and pearl mice. CFU assays and CBCs performed on Bl/6 and pearl mice administered AMD3100 resulted in similar results. However, normal mice administered G-CSF had higher peripheral blood neutrophil counts and greater CFU numbers compared with pearl mice. Unlike patients with HPS-2 and dogs with CH, pearl mice did not have neutropenia or CH but had decreased hematopoietic progenitor cell and granulocyte mobilization in response to G-CSF. AP-3 is a heterotetrameric protein complex involved in intracellular vesicle transport and sorting of cargo molecules to lysosome-related organelles. AP-3 is a member of a family of adaptor protein (AP) complexes that includes AP-1 to AP-4. AP complexes are widely distributed and highly conserved among eukaryotes. Molecular analysis has shown that mutations in the AP3B1 gene, which encodes the Beta3A subunit of the AP-3 complex, cause Hermansky-Pudlak syndrome 2 (HPS-2) in humans, cyclic hematopoiesis (CH) in gray collie dogs, and granule defects in pearl mice [1Benson K.F. Li F.Q. Person R.E. et al.Mutations associated with neutropenia in dogs and humans disrupt intracellular transport of neutrophil elastase.Nat Genet. 2003; 35: 90-96Crossref PubMed Scopus (134) Google Scholar, 2Feng L. Seymour A.B. Jiang S. et al.The beta3A subunit gene (Ap3b1) of the AP-3 adaptor complex is altered in the mouse hypopigmentation mutant pearl, a model for Hermansky-Pudlak syndrome and night blindness.Hum Mol Genet. 1999; 8: 323-330Crossref PubMed Scopus (216) Google Scholar]. Beta3A is ubiquitously expressed in most cells. A related gene AP3B2, which encodes Beta3B (B-NAP), is expressed in neuronal cells [3Seong E. Wainer B.H. Hughes E.D. Saunders T.L. Burmeister M. Faundez V. Genetic analysis of the neuronal and ubiquitous AP-3 adaptor complexes reveals divergent functions in brain.Mol Biol Cell. 2005; 16: 128-140Crossref PubMed Scopus (63) Google Scholar]. A mutation in the AP3B1 gene typically results in observable phenotypes in cells with abundant lysosome-related organelles or intracellular granules such as cytotoxic T lymphocytes, melanocytes, natural killer cells, neutrophils, and platelets [4Fontana S. Parolini S. Vermi W. et al.Innate immunity defects in Hermansky-Pudlak type 2 syndrome.Blood. 2006; 107: 4857-4864Crossref PubMed Scopus (105) Google Scholar, 5Huizing M. Scher C.D. Strovel E. et al.Nonsense mutations in ADTB3A cause complete deficiency of the beta3A subunit of adaptor complex-3 and severe Hermansky-Pudlak syndrome type 2.Pediatr Res. 2002; 51: 150-158Crossref PubMed Scopus (117) Google Scholar, 6Badolato R. Parolini S. Novel insights from adaptor protein 3 complex deficiency.J Allergy Clin Immunol. 2007; 120 (quiz 742–733): 735-741Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar]. Because many of these cell types are derived from the hematopoietic stem cells (HSCs), and because lysosome-related organelles have important housekeeping functions for maintaining cellular homeostasis, alterations in the organelles' contents or formation could have a deleterious effect on the hematopoietic or immune systems [7Ohno H. Overview: Membrane traffic in multicellular systems: More than just a housekeeper.J Biochem. 2006; 139: 941-942Crossref PubMed Scopus (12) Google Scholar, 8Crotzer V.L. Blum J.S. Cytosol to lysosome transport of intracellular antigens during immune surveillance.Traffic. 2008; 9: 10-16Crossref PubMed Scopus (35) Google Scholar]. Cyclic neutropenia and approximately 50% of the cases of congenital neutropenia result from mutations in the ELANE (ELA2) gene, which encodes neutrophil elastase [9Dale D.C. Link D.C. The many causes of severe congenital neutropenia.New Engl J Med. 2009; 360: 3-5Crossref PubMed Scopus (62) Google Scholar]. Because Beta3A deficiency is associated with CH in gray collie dogs and congenital neutropenia in patients with HPS-2 [1Benson K.F. Li F.Q. Person R.E. et al.Mutations associated with neutropenia in dogs and humans disrupt intracellular transport of neutrophil elastase.Nat Genet. 2003; 35: 90-96Crossref PubMed Scopus (134) Google Scholar, 10Feng L. Novak E.K. Hartnell L.M. Bonifacino J.S. Collinson L.M. Swank R.T. The Hermansky-Pudlak syndrome 1 (HPS1) and HPS2 genes independently contribute to the production and function of platelet dense granules, melanosomes, and lysosomes.Blood. 2002; 99: 1651-1658Crossref PubMed Scopus (56) Google Scholar], it has been hypothesized that AP-3 functions as a chaperone to shuttle elastase from the site of biosynthesis in the endoplasmic reticulum (ER) and Golgi complex to its storage depot, the primary granules [1Benson K.F. Li F.Q. Person R.E. et al.Mutations associated with neutropenia in dogs and humans disrupt intracellular transport of neutrophil elastase.Nat Genet. 2003; 35: 90-96Crossref PubMed Scopus (134) Google Scholar, 11Horwitz M. Benson K.F. Duan Z. Li F.Q. Person R.E. Hereditary neutropenia: Dogs explain human neutrophil elastase mutations.Trends Mol Med. 2004; 10: 163-170Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 12Horwitz M.S. Duan Z. Korkmaz B. Lee H.H. Mealiffe M.E. Salipante S.J. Neutrophil elastase in cyclic and severe congenital neutropenia.Blood. 2007; 109: 1817-1824Crossref PubMed Scopus (183) Google Scholar]. Primary granule protein biosynthesis is tightly regulated and occurs primarily in neutrophilic promyelocytes. It has been proposed that ELA2 mutations result in elastase misfolding in promyelocytes, causing increased ER stress, induction of the unfolded protein response, and apoptosis of myeloid progenitor cells, which results in decreased expansion of neutrophil precursors and cyclic or chronic neutropenia [13Kollner I. Sodeik B. Schreek S. et al.Mutations in neutrophil elastase causing congenital neutropenia lead to cytoplasmic protein accumulation and induction of the unfolded protein response.Blood. 2006; 108: 493-500Crossref PubMed Scopus (157) Google Scholar, 14Xia J. Link D.C. Severe congenital neutropenia and the unfolded protein response.Curr Opin Hematol. 2008; 15: 1-7Crossref PubMed Scopus (43) Google Scholar, 15Grenda D.S. Murakami M. Ghatak J. et al.Mutations of the ELA2 gene found in patients with severe congenital neutropenia induce the unfolded protein response and cellular apoptosis.Blood. 2007; 110: 4179-4187Crossref PubMed Scopus (149) Google Scholar]. Abnormalities in platelet dense granules, melanosomes, and natural killer cell cytotoxic granules have been well described in the pearl mouse, patients with HPS-2, and dogs with CH [2Feng L. Seymour A.B. Jiang S. et al.The beta3A subunit gene (Ap3b1) of the AP-3 adaptor complex is altered in the mouse hypopigmentation mutant pearl, a model for Hermansky-Pudlak syndrome and night blindness.Hum Mol Genet. 1999; 8: 323-330Crossref PubMed Scopus (216) Google Scholar, 10Feng L. Novak E.K. Hartnell L.M. Bonifacino J.S. Collinson L.M. Swank R.T. The Hermansky-Pudlak syndrome 1 (HPS1) and HPS2 genes independently contribute to the production and function of platelet dense granules, melanosomes, and lysosomes.Blood. 2002; 99: 1651-1658Crossref PubMed Scopus (56) Google Scholar, 16Zhen L. Jiang S. Feng L. et al.Abnormal expression and subcellular distribution of subunit proteins of the AP-3 adaptor complex lead to platelet storage pool deficiency in the pearl mouse.Blood. 1999; 94: 146-155Crossref PubMed Google Scholar, 17Trail P.A. Yang T.J. Canine cyclic hematopoiesis: alterations in T lymphocyte subpopulations in peripheral blood, lymph nodes, and thymus of gray collie dogs.Clin Immunol Immunopathol. 1986; 41: 216-226Crossref PubMed Scopus (1) Google Scholar, 18Yang T.J. Pathobiology of canine cyclic hematopoiesis (review).In Vivo. 1987; 1: 297-302PubMed Google Scholar, 19Lothrop Jr., C.D. Candler R.V. Pratt H.L. Urso I.M. Jones J.B. Carroll R.C. Characterization of platelet function in cyclic hematopoietic dogs.Exp Hematol. 1991; 19: 916-922PubMed Google Scholar]. However, it was not known whether cyclic or chronic neutropenia occur in pearl mice with mutations in the AP3B1 gene. The purpose of this study was to determine whether pearl mice have CH or chronic neutropenia. The results demonstrate that pearl mice do not have congenital neutropenia or cyclic neutropenia, but do have decreased hematopoietic progenitor cell and granulocyte mobilization. C57BL/6J (Bl/6), B6Pin.C3-Ap3b1pe/J (pearl), and NOD.CB17-Prkdcscid/J (NOD/SCID) mouse strains were purchased from Jackson Laboratories (Bar Harbor, ME, USA). Mice were housed in ventilated racks in a barrier facility. Male and female 12–26-week-old mice were used for experimental procedures. All animal procedures were approved by the Auburn University and University of Alabama at Birmingham Institutional Animal Care and Use Committees. Bl/6 mice (n = 3) and pearl mice (n = 3) were given a sublethal dose of ketamine and xylazine. The tip of the tail (∼3 mm) was cut off with a razor. The tail was then placed into phosphate-buffered saline (PBS) at 37°C. Bleed times were then measured. Bleeding was considered terminated when the stream of blood into the PBS ceased. At 15 minutes, timing of the bleed was ended in mice that had not stopped bleeding. Peripheral blood (50 to 100 μL) from femoral and tail vein bleeds was collected with 1-mL syringes with zero-volume swaged 27-gauge needles that had been heparin preflushed into ethylenediaminetetraacetic acid–coated Eppendorf tubes. Complete blood counts and differential counts were performed using a Heska Veterinary Hematology Analyzer (Heska, Denver, CO, USA). Bone marrow was harvested by flushing both femurs with PBS containing 0.1% bovine serum albumin. Manual bone marrow differential counts were performed on Wright-stained cytospin preparations. A minimum of 500 cells was counted to determine the bone marrow differential count. Bone marrow was harvested from femurs by flushing as described earlier. Cells were then washed twice with PBS. Washed cells were resuspended in PBS supplemented with 0.5% bovine serum albumin and 2 mM ethylenediaminetetraacetic acid for antibody staining. Bone marrow cells were incubated with purified anti–CD16/CD32 monoclonal antibody 2.4G2 to block Fc binding (BD Bioscience, San Diego, CA, USA). The cells were then stained with FITC-conjugated anti–Sca-1 Ab, phycoerythrin-conjugated anti–c-kit Ab, and allophycocyanin (APC)-conjugated Abs to CD3e, CD11b, CD45R/B220, Ly-76 (erythroid), Ly-6G, Ly-6C (gr-1), or APC-conjugated isotype-matched control antibodies (BD Bioscience) for 20 min at 4○C in the dark. After washing twice with PBS, cells were fixed with BD Cytofix buffer (BD Bioscience). Cells were analyzed on a MoFlo flow cytometer (Beckman Coulter, Fullerton, CA, USA). For measurement of lin–Sca-1+c-kit+ (LSK) cells, the percentage of c-kit+ Sca-1+ cells was analyzed on electronically gated lin– cells. Bone marrow mononuclear cells (BMMCs) were plated in 3.0 mL methylcellulose media (MethoCult M3334 for erythroid progenitors, M3534 for granulocyte and macrophage progenitors; Stem Cell Technologies, Vancouver, Canada) supplemented with human erythropoietin for erythroid progenitors (CFU-E and BFU-E) or murine stem cell factor, murine interleukin (IL) 3 and human IL-6 for quantification of granulocyte and macrophage progenitors (CFU-GM, CFU-M, CFU-G). Cultures were plated: 2 × 105 cells per 35-mm dish for erythroid progenitors or 2 × 104 cells per 35-mm dish for granulocytic progenitors, in duplicate and placed in a humidified chamber with 5% CO2 at 37°C. Colony-forming unit-erythroid (CFU-E) were enumerated after 2–3 days of culture, and mature burst-forming unit–erythroid (BFU-E) were detected after 3–4 days of culture. Granulocyte and macrophage progenitor colony formation was counted after 12 days. For determination of granulocyte colony-stimulating factor (G-CSF) dose response, bone marrow cells (2 × 105) from Bl/6 (n = 2) and pearl (n = 2) mice were placed in methylcellulose medium (R&D Systems, Minneapolis, MN; 1244354) without cytokines or G-CSF (0, 2.5, 5.0, 10.0, and 25.0 ng/mL). Colonies were counted after 6 days. Cyclophosphamide (Sigma-Aldrich, St. Louis, MO, USA) was reconstituted in sterile water and given as a single intraperitoneal injection at a dose of 200 mg/kg [20Grenda D.S. Johnson S.E. Mayer J.R. et al.Mice expressing a neutrophil elastase mutation derived from patients with severe congenital neutropenia have normal granulopoiesis.Blood. 2002; 100: 3221-3228Crossref PubMed Scopus (55) Google Scholar]. The CBCs were determined before injection and on 3, 5, 7, and 10 days after cyclophosphamide administration. Isogenic pearl and Bl/6 mice were administered bortezomib (1 mg/kg subcutaneously). The percent of bone marrow granulocytes and the percent of B cells in peripheral blood were determined by flow cytometry on days 2, 3, 4, and 5 after injection as described earlier. Bone marrow cells were incubated with anti-Mouse Ly-6G PE (551461; BD Pharmingen, San Diego, CA, USA) for 20 minutes at 25°C. The percent of granulocytes was determined as the percent of Ly-6G positive cells. Peripheral blood (50–100 μL) was incubated with anti-Mouse CD19 Alexa Fluor 647 (557684; BD Pharmingen) for 30 min at 25°C. Blood was then incubated with red blood cell lysis buffer (00-4300-54; eBioscience, San Diego, CA, USA) for 10 min at 25°C. Antibody and lysis buffer was then discarded, and cells were washed once in 1% rat gamma globulin in PBS. Cells were then resuspended in 500 μL of 1% rat gamma globulin in PBS. The percentage of B cells was determined as the percentage of CD19 positive cells. Bone marrow cells were obtained from the long bones of 3–6-month-old isogenic Bl/6 and pearl mice. Increasing numbers of bone marrow cells were administered by tail vein injection to 2–6-month-old NOD/SCID recipient mice that received 3 Gy total body irradiation 2–4 hours before transplantation. The percent donor cell engraftment in spleen and bone marrow was determined after 4–5 weeks by flow cytometry. Bone marrow and spleen cells (1 × 106 cells) were placed in 400 μL of red blood cell lysis buffer (Purogene, Minneapolis, MN, USA) for 5 min. Lysis buffer was then removed from the cells, and 100 μL of 10% mouse serum (015-000-120; Jackson ImmunoResearch, West Grove, PA, USA) in PBS was added to the cells. Bone marrow and spleen cells were then incubated with anti-Mouse CD45.2/Ly5.2 (553772; BD Pharmingen) and anti-mouse CD45.1/Ly5.1 (553776; BD Pharmingen) for 20 minutes at 25°C. The antibodies were then removed from the cells. The cells were resuspended in 500 μL of 10% mouse serum in PBS. The percent of donor cells was determined as the number of Ly 5.2 positive cells. The percent of recipient cells was determined as the number of Ly 5.1 positive cells. 7-actinomycin D (51-68981E; BD Pharmingen) was added to exclude dead cells. The number of donor cells from different lineages was estimated by analyzing the side scatter pattern of CD45.2/Ly5.2 positive cells. Cells were classified as lymphocytes, monocytes, granulocytes, or myeloid cells based on the side scatter pattern. Bone marrow mononuclear cells (1 × 106) were lysed in radioimmunoprecipitation assay buffer containing protease inhibitors. Cell lysates were subfractionated on discontinuous Percoll gradients as described previously [21Meng R Bridgman R Toivio-Kinnucan M et al.Neutrophil elastase-processing defect in cyclic hematopoietic dogs.Exp Hematol. 2010; 38: 104-115Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar]. Elastase, myeloperoxidase, and cathepsin G enzyme activity were determined using labeled peptide substrates for elastase (N-methoxysuccinyl-ala-ala-pro-val P-nitroanilide M4765; Sigma-Aldrich), for myeloperoxidase (MPO) (3,3′,5,5′-tetramethylbenzidine 0440; Sigma-Aldrich), and for cathepsin G (N-succinyl-ala-ala-pro-phe pNA S7388; Sigma-Aldrich). Specificity was determined using specific inhibitors for elastase, MPO, and cathepsin G. Granulocyte colony-stimulating factor (100 μg/kg; Filgrastim, Amgen, Thousand Oaks, CA, USA) was administered via intraperitoneal injection into Bl/6 mice (n = 8) and pearl mice (n = 8) daily for 5 days. The mice were sacrificed 5 hours after injection on day 5. Complete blood cell counts were performed on all mice on day 0 and again on day 5 just before sacrifice. Peripheral blood and bone marrow cells were prepared as described earlier. Fifty microliters of peripheral blood or 2 × 104 bone marrow cells were placed in methylcellulose medium (StemCell Technologies, Vancouver, Canada) containing insulin, transferrin, stem cell factor, IL-3, IL-6, and erythropoietin, and after 7 days the number of CFUs was determined. AMD3100 (5 mg/kg; A5602; Sigma-Aldrich) was administered via subcutaneous injection to Bl/6 mice (n = 3) and pearl mice (n = 3) 1 hour before sacrifice. The number of peripheral blood CFUs was determined after 7 days of culture. The bleeding times were measured for Bl/6 and pearl mice to verify that the pearl mice used in these studies had the characteristic qualitative platelet defect that resulted in prolonged bleeding times. The bleeding time for the Bl/6 mice was 132 ± 85 seconds, but the pearl mice bled for more than 15 min. The CBC was determined daily for 14 days to determine whether pearl mice were neutropenic. A second group of mice was bled every other day for 30 days for CBC analysis. Based on peripheral cell counts, neutropenia or cyclic neutropenia was not observed in the pearl mice (Fig. 1 and Table 1). However, hematologic parameter analysis indicated a mild but significant increase (p < 0.05) in the total white blood cell count, lymphocytes, monocytes, and granulocytes in pearl mice compared with BL/6 mice (Table 1).Table 1Pearl mice have significantly (p < 0.05) higher WBC, granulocyte, lymphocyte, and monocyte counts and significantly lower RBC, Hgb level, and hematocrit levelsHematologic parameterBl/6PearlWBC count13.7 ± 0.2916.2 ± 0.3∗p < 0.05.Lymphocytes9.97 ± 1.8311.27 ± 1.90∗p < 0.05.Granulocytes1.77 ± 0.422.45 ± 0.90∗p < 0.05.Monocytes1.96 ± 0.332.56 ± 0.48∗p < 0.05.RBC count × 106/μL10.6 ± 0.7110.1 ± 1.1∗p < 0.05.Plt count × 103/μL604.5 ± 134.6662.1 ± 136.4Hgb level, g/dL16.1 ± 0.115.3 ± 0.2∗p < 0.05.Hct (%)47.3 ± 3.1645.2 ± 4.60∗p < 0.05.Percentage of WBC count Lymphocytes (%)72.7 ± 0.369.4 ± 0.5 Monocytes (%)14.1 ± 0.115.5 ± 0.1 Granulocytes (%)13.2 ± 0.215.1 ± 0.4Hct = hematocrit; Hgb = hemoglobin; RBC = red blood cell; WBC = white blood cell.Blood samples were taken every other day for 30 days from individual mice.∗ p < 0.05. Open table in a new tab Hct = hematocrit; Hgb = hemoglobin; RBC = red blood cell; WBC = white blood cell. Blood samples were taken every other day for 30 days from individual mice. Femoral bone marrow cellularity of Bl/6 (1.69 × 107 ± 9.9 × 106) and pearl mice (1.26 × 107 ± 2.9 × 106) were not significantly different (p = 0.34). Bone marrow differential counts (500 cells) in pearl (n = 7) and Bl/6 (n = 8) mice demonstrated a significant (p < 0.05) increase in the number of lymphoid and lymphoid-like cells in the pearl mice (65.1 ± 19.2) compared with the Bl/6 mice (41.2 ± 11.8; Table 2). The myeloid and erythroid cell counts and the myeloid:erythroid ratio were not significantly different in the Bl/6 and pearl mice.Table 2Bone marrow differential counts in pearl and Bl/6 miceMouseNormal (n)Pearl (n)MeanSDMeanSDBlasts/promyelocytes12.254.3315.294.19% Blasts/promyelocytes2.450.873.060.84Myelocytes39.59.6727.867.29% Myelocytes7.91.935.571.46Metamyelocytes7923.1391.4318.99% Metamyelocytes15.84.6318.293.8Bands/neutrophils148.525.86126.8634.81% Band/neutrophils29.75.1725.376.96Eosinophils14.256.1814.716.85% Eosinophils2.851.242.941.37Erythroid165.2539.37158.4357.37% Erythroid33.057.8731.6911.47Lymphoid41.2511.7865.1419.22% Lymphoid8.252.3613.033.84Myeloid:Erythroid ratio1.981.031.980.87There were no significant differences in myeloid and erythroid progenitors between Bl/6 and pearl mice. The number of bone marrow lymphoid cells was significantly (p < 0.05) increased in pearl mice. Open table in a new tab There were no significant differences in myeloid and erythroid progenitors between Bl/6 and pearl mice. The number of bone marrow lymphoid cells was significantly (p < 0.05) increased in pearl mice. Colony-forming assays were performed using total BMMCs from BL/6 and pearl mice to determine whether there were differences in the number of committed hematopoietic progenitors in the bone marrow in pearl and BL6 mice (Table 3). The in vitro colony-forming unit granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM) was significantly (p < 0.05) increased in the pearl (41.9 ± 6.0; n = 6) compared with BL/6 (33.8 ± 4.3; n = 8) mice. The colony forming unit-granulocyte (CFU-G) was also increased in pearl (13.6 ± 3.3, n = 6) compared with BL/6 mice (7.0 ± 0.5; n = 8). The number of CFU-GEMM and CFU-G in the spleen was similarly increased in the pearl mice compared with the Bl/6 mice. The bone marrow colony forming unit-erythroid (CFU-E) was decreased in pearl mice (3.5 ± 1.1 to 6.4 ± 0.9, pearl to BL/6, respectively), but the reverse was observed in the spleen. The number of BFU-E was similar in the pearl and Bl/6 mice.Table 3Pearl mice have significantly increased (p < 0.05) CFU-GEMM and CFU-G but decreased (p < 0.05) CFU-E from bone marrowBone MarrowSpleen#C57Bl/6J#Ap3b1pe/J#C57Bl/6J#Ap3b1pe/JCFU-E86.4 ± 0.963.5 ± 1.1∗p < 0.05.84.6 ± 0.867.2 ± 1.1BFU-E824.1 ± 4.1621.4 ± 1.489.1 ± 1.9613.7 ± 3.3CFU-G87.0 ± 0.5613.6 ± 3.3∗p < 0.05.72.5 ± 0.665.6 ± 0.7∗p < 0.05.CFU-M87.6 ± 0.668.7 ± 1.671.9 ± 0.362.5 ± 0.5CFU833.8 ± 4.3641.9 ± 6.0∗p < 0.05.85.8 ± 1.0611.1 ± 1.4∗p < 0.05.Total843.2 ± 3.6664.2 ± 9.1∗p < 0.05.89.7 ± 1.7619.2 ± 1.1∗p < 0.05.BFU-E = burst-forming unit–erythroid; CFU-E = colony-forming unit–erythroid; CFU-G = colony-forming unit–granulocyte; CFU-GEMM = colony-forming unit–granulocyte, erythroid, macrophage, and megakaryocyte.Colony assays were performed in vitro from total bone marrow mononuclear cells as described in the Methods section; 2 × 105 cells per 35-mm dish were plated for erythroid progenitors; and 2 × 104 cells per 35-mm dish were plated for granulocytic progenitors.∗ p < 0.05. Open table in a new tab BFU-E = burst-forming unit–erythroid; CFU-E = colony-forming unit–erythroid; CFU-G = colony-forming unit–granulocyte; CFU-GEMM = colony-forming unit–granulocyte, erythroid, macrophage, and megakaryocyte. Colony assays were performed in vitro from total bone marrow mononuclear cells as described in the Methods section; 2 × 105 cells per 35-mm dish were plated for erythroid progenitors; and 2 × 104 cells per 35-mm dish were plated for granulocytic progenitors. Colony-forming assays were performed using bone marrow cells from BL/6 (n = 2) and pearl (n = 2) mice to determine whether there were differences in the number of colonies formed after administration of G-CSF in vitro. Pearl mice bone marrow cells formed significantly more colonies at all doses of G-CSF (5, 10, and 25 ng/mL) except for 2.5 ng/mL (Fig. 2). Pearl mice bone marrow cells also formed significantly more colonies without the addition of G-CSF. Flow cytometric analysis was performed to compare the number of early hematopoietic stem cells in pearl (n = 4) and normal BL/6 (n = 4) mice. Based on the analysis of LSK cells, the percentage of early HSCs in pearl mice was significantly (p = 0.04) increased in pearl (4.75 ± 0.95) compared with Bl/6 (3.18 ± 0.39; Fig. 3). Stress granulopoiesis was compared in Bl/6 (n = 8) and pearl mice (n = 8) that were administered the myelosuppressive agent cyclophosphamide [20Grenda D.S. Johnson S.E. Mayer J.R. et al.Mice expressing a neutrophil elastase mutation derived from patients with severe congenital neutropenia have normal granulopoiesis.Blood. 2002; 100: 3221-3228Crossref PubMed Scopus (55) Google Scholar]. The recovery from neutropenia was indistinguishable in wild type and pearl mice, suggesting that granulopoiesis in pearl mice is similar to Bl/6 mice, even with moderate hematopoietic stress (Fig. 4). To determine whether granulopoiesis in pearl mice was more sensitive to ER stress, pearl and Bl/6 mice were administered 1 mg/kg of bortezomib. The percent of bone marrow granulocytes and peripheral blood B cells was determined by flow cytometry before and on days 2, 3, 4, and 5 after bortezomib administration. There was no statistically significant difference in bone marrow granulocytes between Bl/6 and pearl mice on any day (Fig. 5A). The B cells percentage decreased in both Bl/6 and pearl mice (Fig. 5B). There was no significant (p < 0.05) difference between Bl/6 and pearl mice B cells on any day except day 5. Sublethally irradiated (3 Gy) NOD/SCID mice were transplanted with increasing doses of bone marrow cells from Bl/6 or pearl mice. The percent donor cell engraftment at 4–5 weeks was determined by flow cytometry by measuring the number of Ly5.2 (Bl/6 or pearl) positive cells in bone marrow and spleen of recipient mice. There was no significant (p < 0.05) difference in the percent donor engraftment at 1 × 105 and 1 × 106 donor cell dose (Table 4). At 1 × 104 donor cells, neither Bl/6 nor pearl mice showed percent donor engraftment above background. Secondary transplants with bone marrow (1 × 106 cells) obtained from primary recipients 4 weeks after transplantation demonstrated similar engraftment in normal and pearl mice (data not shown). There was no significant difference (p < 0.05) between Bl/6 and pearl mice transplanted cells in the number of lymphocytes, monocytes, granulocytes, or myeloid cells (Fig. 6 and Table 5).Table 4Bl/6 and pearl mice show similar engraftmentDonorNo. of miceNo. of cellsBM or spleenAverage % donorBlack 6131 × 106BM45.37 ± 18.61Pearl151 × 106BM42.99 ± 14.92Black 6131 × 106Spleen62.11 ± 24.66Pearl141 × 106Spleen59.49 ± 22.07Black 671 × 105BM13.02 ± 15.91Pearl121 × 105BM10.29 ± 12.76Black 671 × 105Spleen12.23 ± 12.76Pearl121 × 105Spleen10.77 ± 10.29Black 6121 × 104BM1.1 ± 0.88Pearl51 × 104BM0.02 ± 0.04Black 6121 × 104Spleen1.8 ± 1.16Pearl51 × 104Spleen0 ± 0BM = bone marrow.Limiting dilution bone marrow transplantation into NOD/SCID mice show no significant difference (p < 0.05) between Bl6 and pearl mice in engraftment in bone marrow or spleen after 4 weeks. Open table in a new tab Table 5Bone marrow differential counts in pearl and Bl/6 mice treated with granulocyte colony-stimulating factorMouseNormal (n = 5)Pearl (n=6)MeanSDMeanSDBlasts/promyelocytes83.546.51.05% Blasts/promyelocytes1.60%0.011.30%0Myelocytes23.86.6122.834.02% Myelocytes4.76%0.014.57%0.01Metamyelocytes89.815.82110.6728.99% Metamyelocytes17.96%1.2922.13%0.06Bands/neutrophils273.822.26254.177.3% Band/neutrophils54.76%0.0450.85%0.06Eosinophils17.47.317.178.57% Eosinophils3.48%0.013.43%0.02Erythroid7.66.1931.8328.87% Erythroid1.52%0.016.37%0.06Lymphoid79.623.4156.839.45% Lymphoid15.92%0.0511.37%0.02Myeloid:Erythroid ratio136.56173.6122.2516.84The number of band cells and neutrophils were increased in both Bl/6 and pearl mice. There were no s" @default.
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• W2163935490 date "2013-10-01" @default.
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• W2163935490 title "Decreased hematopoietic progenitor cell mobilization in pearl mice" @default.
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