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Blood, 1 January 2007, Vol. 109, No. 1, pp. 109-111. Prepublished online as a Blood First Edition Paper on August 24, 2006; DOI 10.1182/blood-2006-06-026427. This Article Abstract Full Text (PDF) Supplemental Figure All Versions of this Article: blood-2006-06-026427v1 109/1/109    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Priestley, G. V. Articles by Papayannopoulou, T. Search for Related Content PubMed PubMed Citation Articles by Priestley, G. V. Articles by Papayannopoulou, T. Related Collections Hematopoiesis and Stem Cells Stem Cells in Hematology Brief Reports Related Article in Blood Online Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  HEMATOPOIESIS Brief report Sustained alterations in biodistribution of stem/progenitor cells in Tie2Cre+4f/f mice are hematopoietic cell autonomous Gregory V. Priestley1, Tatiana Ulyanova1, and Thalia Papayannopoulou1, 1 Department of Medicine/Division of Hematology, University of Washington, Seattle    Abstract Top Abstract Introduction Materials and methods Results and discussion References   We have generated Tie2Cre+4f/f mice with documented 4-integrin ablation in hematopoietic and endothelial cells. A prominent feature in this model is a sustained, significant increase in circulating progenitors at levels higher than the levels seen with Tie2Cre+VCAM-1f/f mice. To test whether phenotypic differences are due to contributions by ligands other than VCAM-1 in bone marrow, or to 4-deficient endothelial cells or pericytes, we carried out transplantation experiments using these mice as donors or as recipients. Changes in progenitor biodistribution after transplantation were seen only with 4-deficient donor cells, suggesting that these cells were necessary and sufficient to reproduce the phenotype with no discernible contribution by 4-deficient nonhematopoietic cells. Because several similarities are seen after transplantation between our results and those with CXCR4–/– donor cells, the data suggest that VLA4/VCAM-1 and CXCR4/CXCL12 pathways contribute to a nonredundant, ongoing signaling required for bone marrow retention of progenitor cells during homeostasis.    Introduction Top Abstract Introduction Materials and methods Results and discussion References   The use of Cre-lox strategy to mediate conditional deletion of embryonic lethal genes in a tissue-restricted manner has enabled the investigation of their functional roles in adult life. A commonly used approach to delete chosen floxed genes in hematopoietic cells is the interbreeding with MxCre transgenic mice harboring an interferon-inducible transgene.1-5 Although efficient deletion in hematopoietic cells has been documented using this strategy, the extent of deletion in nonhematopoietic cells has not been rigorously assessed previously. The 4 integrin is expressed widely in hematopoietic and nonhematopoietic cells, such as endothelial cells6 or pericytes7 within the bone marrow (BM). In fact, its presence in endothelial cells is critical in tumor progression, in neoangiogenesis, and in developing blood vessels.6,7 Conditional deletion of 4 integrin in adults has led to sustained changes in biodistribution of stem/progenitor cells at steady-state hematopoiesis.5 A similar, albeit transient, picture has been described with the use of antifunctional 4-integrin antibodies in vivo.8 Using either strategy, it has not been clear to what extent the phenotype is solely attributed to deletion of 4 in hematopoietic cells or contribution by similarly affected cells in BM microenvironment, as has been recently advocated.9-11 To address this issue the following approaches were used. First, we generated mice in which the 4 integrin is ablated both in hematopoietic and in endothelial cells, by breeding 4 floxed mice (f/f) with Tie2Cre+ transgenic mice with the expected 4 ablation in hematopoietic and endothelial cells.12 Second, we carried out transplantation experiments using either donor cells from these mice or using them as recipients of normal cells. The results from these experiments provide definitive conclusions about the contribution of 4-integrin ablation only in hematopoietic cells to the phenotype seen at homeostasis.    Materials and methods Top Abstract Introduction Materials and methods Results and discussion References   Mice (C57Bl/6 x 129) mice homozygous for the floxed 4 allele (4f/f) were crossed to C57Bl/6 mice expressing Cre under the control of the Tie2 promoter to generate Tie2Cre+4f/f mice. Genotyping was performed by polymerase chain reaction (PCR) using the following primers: F-1: 5'-CGGGATCAGAAAGAATCCA-3'; F-2: 5'-CCACCTGGTGTATGAAAGC-3'; Rev: 5'-GATCACATACAGTTGCAAGC-3' for discrimination of wild-type (WT), f, and alleles, respectively. Tie2Cre+/VCAM-1f/f mice were previously described.13 Antibodies Directly conjugated antibodies from BD Biosciences (San Diego, CA) were B220, CD3, CD4, CD8, CD45, TER119, and GR1. Directly conjugated CD49d antibody was from Southern Biotechnology (Birmingham, AL). BM or blood cells stained with these antibodies were analyzed on a FACSCalibur (BD Biosciences) using CellQuest software (BD Biosciences). Clonogenic progenitor assays Colony-forming unit in culture (CFU-C) assays were performed from BM, spleen, or peripheral blood (PB) samples using a methylcellulose mixture (Methocult GF; StemCell Technologies, Vancouver, BC, Canada), as described previously.5 Transplantation experiments BM cells (0.5, 2.5, or 5.0 x 106/recipient) from 4-deficient or from normal mice were infused into lethally irradiated normal or 4-deficient recipients (1150 cGy whole body irradiation with a 137Cs source). Recipients were analyzed at multiple points after transplantation. Donor reconstitution was verified by fluorescence-activated cell sorting (FACS) or PCR or both. BM stromal endothelial cell cultures Endothelial cells were procured from cleansed, flushed, and minced long and pelvic bones and cultured as described.13 For generating BM-derived fibroblasts the cells were cultured in DMEM with 20% FCS for 4 to 7 weeks. Contaminating macrophages were removed by depletion of CD45+ cells, whereas endothelial cells were identified by several endothelial-specific markers.13 Reverse transcription-PCR (RT-PCR) was performed as described.13 Primers for 4: forward, 5'-TGTGGAAGGCTGGATTCTTT-3', reverse, 5'-CGGGTCTTCTGAACAGGATT-3'; VWF: forward, 5'-AGAGGAGAGTACATCTGGGAG-3', reverse, 5'-AGAACACCTGCACTGCATGGC-3'. Statistical analysis All statistical analyses were performed with a 2-tailed Student t test.    Results and discussion Top Abstract Introduction Materials and methods Results and discussion References   Tie2Cre+4f/f mice are viable and healthy with no apparent ill effects when observed for more than a year. Phenotypically all circulating PB or BM cells were virtually 4– (Figure 1A-B), as were BM endothelial cells, whereas 4 deletion in bone fibroblasts/mesenchymal cells was partial at best (Figure 1B). Detailed studies on hematopoiesis disclosed that white blood cell (WBC) counts were above control levels (Figure 1C) with no deviation in lineage-specific contributions (data not shown). Circulating progenitors were several-fold higher than controls (Figure 1C) and this difference was maintained through 16 months of postnatal life (Figure S1A, available on the Blood website; see the Supplemental Figure link at the top of the online article). Overall the hematopoietic phenotype was similar to that previously described for Tie2Cre+VCAM-1f/f mice.13 However, circulating progenitors in Tie2Cre+4f/f were higher (Figure 1C). The fact that 4 integrin has additional ligands other than VCAM-1, (ie, fibronectin, osteopontin, VWF, ADAMS28, 4-chain itself) could explain the more efficient release of 4/ progenitors. Alternatively, one may suggest that efficient excision of 4 integrin from endothelial cells or other microenvironmental cells in this model may have a contributing role.11 View larger version (17K): [in this window] [in a new window]   Figure 1. Deletion of 4 gene in hematopoietic and nonhematopoietic cells in Tie2Cre+4f/f mice. (A) Surface expression of 4 integrin on circulating WBCs in Tie2Cre+4f/f (open histogram, thick line) and control (filled histogram) mice. FACS analysis with PS/2, anti-4 antibody showed that in Tie2Cre+4f/f mice, 3.1% ± 0.2% (n = 20) of circulating WBCs and 2.6% ± 0.6% (n = 4) of BM cells (not shown) are 4+. Isotype-matched immunoglobulin (open histogram, dashed line) served as a negative control. (B) Deletion of 4 integrin in endothelial cells (E), fibroblasts (F), and hematopoietic BM cells (H) at the genomic level (left panel) and at the mRNA level (right panel). Note only partial deletion of 4 gene in fibroblasts. (C) Deletion of 4 integrin results in increase in WBCs and in circulating hematopoietic progenitors. Circulating CFU-C levels in Tie2Cre+a4f/f mice (2446 ± 256, n = 17) are significantly increased compared with control levels (338 ± 113, n = 9, P < .01). Circulating WBC and CFU-C levels in Tie2Cre+VCAM-1f/f mice are shown for comparison. The asterisk indicates significant difference over controls, P < .01; , significant difference over Tie2Cre+VCAM-1f/f mice, P < .05. Error bars indicate standard error of the mean (SEM).   To test these possibilities, transplantation experiments were carried out. We transplanted 4-deficient BM cells (Figure 2A) into lethally irradiated Cre(–) littermates (C57B1/6 x 129) and assessed the levels of circulating progenitors after complete hematopoietic reconstitution. The 4+/+ cells given to 4+/+ or to 4/ lethally irradiated recipients were also followed similarly (Figure 2B). Although 4+/+ cells given to 4+/+ or 4/ recipients did not show increased levels of circulating progenitors after transplantation, 4-deficient cells transplanted to normal recipients had significantly increased numbers of circulating progenitors up to 29 weeks after transplantation, compared to their concurrent controls. Thus, 4/ hematopoietic cells transplanted to a normal BM environment are necessary and sufficient to provide a picture similar to one seen in animals that did not receive a transplant (Figure S1B). Alternatively, 4 deletion in endothelial cells or pericytes7 in BM does not seem to contribute in any measurable way to the altered biodistribution of hematopoietic progenitor cells at steady state. View larger version (26K): [in this window] [in a new window]   Figure 2. Posttransplantation increase in circulating progenitors is seen only with 4 integrin–deficient donor cells. (A) Transplantation experiments were performed by injecting 4-deficient () or 4-sufficient (+/+ or +/; ) BM cells into normal recipients (2.5-5.0 million BM cells were transplanted into 4-7 mice/group), and (B) by injecting 4+/+ cells into normal () or 4-deficient () recipients. (One half million BM cells were transplanted to 5 mice/group.) A significant increase in circulating hematopoietic progenitor cells is seen only with 4/ donor cells and is maintained up to 29 weeks after transplantation; at that time, 2.9% ± 0.3% of BM cells and 3.0% ± 0.5% of WBCs were 4+ in mice given transplants (Figure S1B). No increase in circulating progenitors above control levels is seen when normal cells are transplanted into 4-deficient recipients. The asterisk indicates a significant difference between transplanting 4-deficient and 4-sufficient donor cells. Error bars indicate SEM.   The data presented here and the information we recently published14 display several common features to those described15 with transplantation of CXCR4–/– fetal cells: (1) high numbers of circulating progenitor cells starting at 2 weeks after transplantation and continuing for at least 16 weeks; (2) rescue of engraftment defects when higher numbers of donor cells are used; and (3) the presence of a significant number of long-term repopulating cells in circulation, as documented by transplantation experiments using blood.5,15 The enhanced release of progenitors from BM to blood seen in both cases is cell autonomous and would suggest that the 2 pathways, 4/VCAM-1 or CXCR4/CXCL12 (SDF-1), have a nonredundant influence on the retention of progenitors in BM at steady-state hematopoiesis. Although alterations in progenitor biodistribution were seen previously in other genetically deficient models, that is, with Rac2 GTPase–/–4 or CaR–/– cells,16 a picture recapitulating after transplantation the phenotype seen in animals that did not receive a transplant has not been documented in these cases. A dramatic increase was seen after transplantation with Rac2–/– Rac1/ doubly deficient donor cells, but this was transient, lasting 4 weeks after transplantation,17 unlike our results. Surprisingly, transplantation of MxCre+ß1f/f cells and their ablation after transplantation, theoretically affecting several ß1-integrin heterodimers, did not lead to any alterations in stem/progenitor biodistribution,2 inviting the speculation that ß1 integrins expressed in nonhematopoietic cells or niche cells are more important.9,10 Although ß1/ß7-deficient animals display a phenotype (in BM and PB) similar to our Tie2Cre+4f/f mice, this phenotype, unlike the one in our mice, reverts to normal with aging as a result of putative compensatory changes.11 Whatever the case, our results clearly show that active engagement of 4 integrin in hematopoietic cells plays an important role in physiologic progenitor retention within BM.    Acknowledgments   This work was supported by National Institutes of Health grant HL046557.    Footnotes   Submitted June 1, 2006; accepted August 9, 2006. Prepublished online as Blood First Edition Paper, August 24, 2006 DOI: 10.1182/blood-2006-06-026427 Contribution: G.V.P. performed experiments and analyzed and evaluated the data; T.U. performed experiments (PCR); and T.P. directed research and wrote the manuscript. The online version of this article contains a data supplement. 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. Conflict-of-interest disclosure: The authors declare no competing financial interests. Correspondence: Thalia Papayannopoulou,University of Washington, Division of Hematology, Box 357710, Seattle, WA 98195; e-mail: thalp{at}u.washington.edu.    References Top Abstract Introduction Materials and methods Results and discussion References   Leuker CE, Labow M, Muller W, Wagner N. Neonatally induced inactivation of the vascular cell adhesion molecule 1 gene impairs B cell localization and T cell-dependent humoral immune response. J Exp Med 2001; 193:755–768.[Abstract/Free Full Text] Brakebusch C, Fillatreau S, Potocnik AJ, et al. Beta1 integrin is not essential for hematopoiesis but is necessary for the T cell-dependent IgM antibody response. Immunity 2002; 16:465–477.[CrossRef][Medline] [Order article via Infotrieve] Mikkola HK, Klintman J, Yang H, et al. Haematopoietic stem cells retain long-term repopulating activity and multipotency in the absence of stem-cell leukaemia SCL/tal-1 gene. Nature 2003; 421:547–551.[CrossRef][Medline] [Order article via Infotrieve] Gu Y, Filippi MD, Cancelas JA, et al. Hematopoietic cell regulation by Rac1 and Rac2 guanosine triphosphatases. Science 2003; 302:445–449.[Abstract/Free Full Text] Scott LM, Priestley GV, Papayannopoulou T. Deletion of alpha4 integrins from adult hematopoietic cells reveals roles in homeostasis, regeneration, and homing. Mol Cell Biol 2003; 23:9349–9360.[Abstract/Free Full Text] Jin H, Su J, Garmy-Susini B, Kleeman J, Varner J. Integrin alpha4beta1 promotes monocyte trafficking and angiogenesis in tumors. Cancer Res 2006; 66:2146–2152.[Abstract/Free Full Text] Grazioli A, Alves CS, Konstantopoulos K, Yang JT. Defective blood vessel development and pericyte/pvSMC distribution in alpha4 integrin-deficient mouse embryos. Dev Biol 2006; 293:165–177.[CrossRef][Medline] [Order article via Infotrieve] Papayannopoulou T and Nakamoto B. Peripheralization of hemopoietic progenitors in primates treated with anti-VLA4 integrin. Proc Natl Acad Sci U S A 1993; 90:9374–9378.[Abstract/Free Full Text] Potocnik AJ, Brakebusch C, Fassler R. Fetal and adult hematopoietic stem cells require beta1 integrin function for colonizing fetal liver, spleen, and bone marrow. Immunity 2000; 12:653–663.[CrossRef][Medline] [Order article via Infotrieve] Suda T, Arai F, Hirao A. Hematopoietic stem cells and their niche. Trends Immunol 2005; 26:426–433.[CrossRef][Medline] [Order article via Infotrieve] Bungartz G, Stiller S, Bauer M, et al. Adult murine hematopoiesis can proceed without ß1 and ß7 integrins Blood Prepublished May 30, 2006, as DOI 10.1182/blood-2005-10-007658 (Now available as Blood. 2006;108:1857-1864). Motoike T, Loughna S, Perens E, et al. Universal GFP reporter for the study of vascular development. Genesis 2000; 28:75–81.[CrossRef][Medline] [Order article via Infotrieve] Ulyanova T, Scott LM, Priestley GV, et al. VCAM-1 expression in adult hematopoietic and nonhematopoietic cells is controlled by tissue-inductive signals and reflects their developmental origin. Blood 2005; 106:86–94.[Abstract/Free Full Text] Priestley GV, Scott LM, Ulyanova T, Papayannopoulou T. Lack of alpha4 integrin expression in stem cells restricts competitive function and self-renewal activity. Blood 2006; 107:2959–2967.[Abstract/Free Full Text] Foudi A, Jarrier P, Zhang Y, et al. Reduced retention of radioprotective hematopoietic cells within the bone marrow microenvironment in CXCR4–/– chimeric mice. Blood 2006; 107:2243–2251.[Abstract/Free Full Text] Adams GB, Chabner KT, Alley IR, et al. Stem cell engraftment at the endosteal niche is specified by the calcium-sensing receptor. Nature 2006; 439:599–603.[CrossRef][Medline] [Order article via Infotrieve] Cancelas JA, Lee AW, Prabhakar R, Stringer KF, Zheng Y, Williams DA. Rac GTPases differentially integrate signals regulating hematopoietic stem cell localization. Nat Med 2005; 11:886–891.[CrossRef][Medline] [Order article via Infotrieve] CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? Related Article in Blood Online: Adult murine hematopoiesis can proceed without 1 and 7 integrins Gerd Bungartz, Sebastian Stiller, Martina Bauer, Werner Müller, Angela Schippers, Norbert Wagner, Reinhard Fässler, and Cord Brakebusch Blood 2006 108: 1857-1864. [Abstract] [Full Text] [PDF] This article has been cited by other articles: J. Grassinger, D. N. Haylock, M. J. Storan, G. O. Haines, B. Williams, G. A. Whitty, A. R. Vinson, C. L. Be, S. Li, E. S. Sorensen, et al. Thrombin-cleaved osteopontin regulates hemopoietic stem and progenitor cell functions through interactions with {alpha}9{beta}1 and {alpha}4{beta}1 integrins Blood, July 2, 2009; 114(1): 49 - 59. [Abstract] [Full Text] [PDF] E. G. Houston Jr., R. Nechanitzky, and P. J. Fink Cutting Edge: Contact with Secondary Lymphoid Organs Drives Postthymic T Cell Maturation J. Immunol., October 15, 2008; 181(8): 5213 - 5217. [Abstract] [Full Text] [PDF] R. Silva, G. D'Amico, K. M. Hodivala-Dilke, and L. E. 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[Abstract] [Full Text] [PDF] This Article Abstract Full Text (PDF) Supplemental Figure All Versions of this Article: blood-2006-06-026427v1 109/1/109    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Priestley, G. V. Articles by Papayannopoulou, T. Search for Related Content PubMed PubMed Citation Articles by Priestley, G. V. Articles by Papayannopoulou, T. Related Collections Hematopoiesis and Stem Cells Stem Cells in Hematology Brief Reports Related Article in Blood Online Social Bookmarking                   What's this?

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