A Specific Heptapeptide from a Phage Display Peptide Library Homes to Bone Marrow and Binds to Primitive Hematopoietic Stem Cells
http://www.100md.com
《干细胞学杂志》
a Division of Gene Therapy, Department of Medicine, University of Massachusetts Medical School,Worcester, Massachusetts, USA;
b Division of Hematology, Mayo Clinic, Rochester, Minnesota, USA;
c Department of Research, Roger Williams Medical Center, Providence, Rhode Island, USA;
d Dana-Farber Cancer Institute, Bostom, Massachusettes, USA;
e Division of Hematology, University of Washington, Seattle,Washington, USA
Key Words. Stem cells ? Engraftment ? Bone marrow transplant ? Cell adhesion molecules ? Bone marrow microenvironment
Correspondence: Pamela S. Becker, M.D., Ph.D., Division of Hematology, Box 357710, University of Washington, 1959 N.E. Pacific Street, HSB K-136, Seattle,WA 98195, USA. Telephone: 206-616-1589; Fax: 206-543-3560; e-mail: pbecker@u.washington.edu
ABSTRACT
Hematopoietic progenitors are capable of migration and homing, processes fundamental to embryonal development, circulation in the vasculature, and stem cell transplantation. Engraftment after bone marrow transplant may involve several adhesion receptors, each recruited sequentially in the movement from bloodstream through vascular walls to the bone marrow. Studies in 4 integrin–null mice indicated that migration of embryonic hematopoietic precursors to the fetal liver, bone marrow, and spleen was independent of these receptors but that maintenance of hematopoiesis by these organs was entirely dependent on 4-integrins . The very late antigen (VLA)-4 receptor plays the most critical role in engraftment after transplant, as demonstrated by the following observations: First, murine colony-forming unit spleen cells and reconstituting hematopoietic stem cells attached to the connecting segment-1 (CS-I) peptide of alternatively spliced fibronectin (Fn), and this binding was blocked by antibody to 4 ; second, human long-term bone marrow culture-initiating cells (LTBM-ICs) bound to Fn in the heparin-binding region (a function of 4), and these Fn peptides could block their adherence to irradiated stroma ; third, antibody to VLA-4 reduced bone marrow engraftment in mice ; and, fourth, blocking antibody to human VLA-4 reduced bone marrow engraftment of human CD34+ cells in fetal sheep . The VLA-4/vascular cellular adhesion molecule-1 (VCAM-1) pathway was found to be critical for homing to the bone marrow, whereas CD44 was involved in the localization of colony-forming unit–spleen in the bone marrow and spleen . Antibody blocking of VCAM-1 in vivo in E-selectin/P-selectin knockout mice suggested that all three receptors were required for optimal homing to the bone marrow . Moreover, human CD34+ cells were dependent on CXCR4 (receptor for stromal cell–derived factor-1) (SDF-1) for optimal bone marrow homing and engraftment in the nonobese diabetic/severe combined immunodeficiency model , indicating that chemotaxis to SDF-1 may be critical . VLA-4 was then shown to possess the dominant role over both selectins and ?2-integrins for engraftment by using antibody to 4-integrin with donor cells from selectin-null or ?2-integrin–null mice . These prior approaches to the study of surface molecules in homing and engraftment examined receptors known to be expressed by the hematopoietic progenitors or the bone marrow stroma.
We sought to discover novel adhesion mechanisms in bone marrow homing and engraftment using a phage display peptide library (PDPL) . The PDPL consists of an assembly of phages, each of which has fusions of a random peptide to a coat protein, with approximately five copies of each peptide per phage . The number of unique peptide sequences depends on peptide length. Because there are 20 coding amino acids, a random heptapeptide display library has 207 or 1.3 x109 unique sequences (clones). Phages that display specific peptides are selected by binding to selector or target molecules. Amplification of selected clones bearing peptides of interest is possible because the peptide sequence is coded by phage DNA. This process of selection and amplification is called biopanning and can be repeated several times to enrich the phage pool in favor of binding sequences. Individual clones may then be selected and characterized by DNA sequencing.
PDPLs were previously used in a variety of applications , including epitope mapping of monoclonal antibodies , identification of peptide ligands for 5?1-integrin, and isolation of structural and functional mimics of the arginine-glycine-aspartic acid–binding site of integrins . Peptides identified by biopanning procedures are frequently involved in important functional interactions; for example, peptides mimicking cytokines may exhibit their functional activities . Also, peptide sequences often mimic the sequence of natural ligands such as peptides selected against anti-platelet antibodies that share sequence homology with glycoprotein (GP) IIb/IIIa and GPIb surface molecules . Biopanning can also be conducted in vivo, allowing selection of peptides that interact with tissues and cells in their native microenvironment. This approach was used to identify peptides that exhibit organ-specific homing for the kidney and brain and later for other organs and tumor vasculature in animals. Similar studies to select organ-homing peptides were initiated in humans .
For the present study, we used biopanning in vivo to identify a peptide that homes to the bone marrow. Surprisingly, the identical heptapeptide was isolated by biopanning in vitro on hematopoietic stem cells. A functional assay demonstrated its ability to interfere with early homing of hematopoietic progenitors.
MATERIALS AND METHODS
Biopanning In Vivo
The sequencing of single-phage colonies from the fifth round of biopanning in vivo (diagram of procedure in Fig. 1) revealed two predominant consensus sequences, STFTKSP and NHWASPR, constituting 50% and 28% of sequences, respectively. The remaining sequences were single or incomplete sequences, some of which contained motifs of the two predominant sequences.
Biopanning In Vitro Using FACS on Hematopoietic Stem Cells
Biopanning combined with FACS was repeated three times, yielding phage clones that attached to primitive Lin–Hoechstdullrhodaminedull stem cells. The results of sequencing showed that 85% of the single-phage colonies had the same sequence as the most predominant peptide isolated by biopanning in vivo, STFTKSP. The remaining sequences were single or incomplete.
Flow Cytometry Analysis of Phage-Binding Bone Marrow Cells
Flow cytometry analysis of the density-depleted bone marrow stained with specific phage, antibody to phage M13 protein, and secondary fluorescent-labeled antibody revealed a distinctive population constituting 1.10% of the density-depleted bone marrow cells. The following percentages of C57BL6 bone marrow cells exhibited costaining by both lineage marker and specific phage when gated on cells binding phage: Ter-119, 3%; YW25, 2%; Mac-1, 3%; CD45R, 2%; CD4, 0%; CD8, 0%; GR-1, 1%; and GPIIb/IIIa, 0%. These results indicate minimal expression of lineage markers by cells that bind the specific phage. In a separate set of experiments using BALB/c bone marrow, of the cells with attached specific phage, 36.6 ± 17.5% expressed Sca-1 (1.01 ± 0.14% for the IgG2a isotype control), 49.9 ± 15.5% expressed c-kit (1.64 ± 0.16% for the IgG2b isotype control), and 27.18 ± 4.05% expressed CD84 (0.99 ± 0.12% for the IgG1 isotype control). Moreover, 86% of the BALB/c lineage-depleted bone marrow cells that expressed c-kit also expressed CD84, and 57% of those that expressed Sca-1 also expressed CD84.
Organ Specificity of Bone Marrow Homing Phage
The organs sections were stained with FITC-labeled anti-phage M13 antibody, and staining was observed within the bone marrow and lungs (Fig. 3), as follows: 12.5% of the DAPI-counterstained cells from the bone marrow, 3% of the cells from the lung sections, 0.5% of the cells from a lymph node, and 0.5% of the cells from the thymus exhibited staining of bound phage. These numbers may represent an overestimation because of the appearance of staining of clusters of cells or adjacent cells. There was no staining for phage within the kidney, heart, brain, spleen, or liver.
Figure 3. BM and other cells were stained by immunofluorescence for phage to assess the specificity of organ homing. The left panels show the dual stain of the cell nuclei with 4',6'-diamidino-2-phenylindole (blue), indicating the location of cells and the staining with anti-phage monoclonal antibodies and phycoerythrin (red)–labeled secondary antibodies. As a control, the BM of the animal that was not injected with the phage is shown in the right panels. Representative cells from the BM, lung, and brain are shown. Abbreviation: BM, bone marrow.
Immunofluorescent Staining of Bone Marrow–Homing Phage Binding to Lin–HoechstdullRhodaminedull Stem Cells
There were two patterns of staining: circumferential membrane and more dense, diffuse staining (Fig. 4).
Figure 4. The Lin–Hoechst 33342dull/rhodamine 123dull stem cells were examined by immunofluorescence to demonstrate binding of the specific phage. The upper panels show staining with DAPI. The lower first panel on the left represents a control with FITC-labeled secondary antibody without phage; the lower middle and right panels show staining with phage, antibody to phage M13 protein, and FITC-labeled secondary antibody. Abbreviations: Ab, antibody; DAPI, 4',6'-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate.
Bone Marrow Homing Assay of CFDA-SE–Labeled Lin–Sca-1+ Cells with Heptapeptide/Phage Displaying Heptapeptide Blockade
Large-event flow cytometry analysis demonstrated a 26% reduction in homing 3 hours after transplant in an animal injected with the synthetic heptapeptide compared with the control animal (data not shown). When the phage displaying the bone marrow homing heptapeptide was used instead of a free heptapeptide, there was a 41% reduction in homing 3 hours after transplant, whereas homing to the spleen was fully preserved at 109% of control. The difference was statistically significant for the difference in homing to the bone marrow compared with control (p = .007) but was not statistically significant for the spleen (p = .66; Fig. 5).
Figure 5. BM homing assay of carboxyfluorescein diacetate succinimidyl ester–labeled Lin–Sca-1+ cells was performed in the presence of the specific phage-displaying peptide. In the presence of the phage, there was diminished homing to the bone marrow (p = .007) but preserved homing to the spleen (p = .66) compared with the absence of phage in an in vivo homing assay. Abbreviation: BM, bone marrow.
Database Search Results
Although nucleotide database searches were not successful in identifying homologous sequences, a Genbank search using NCBI protein–protein blast BLASTP 2.1.3 revealed several sequences with varying degrees of homology (Table 1).
Table 1. Genbank search results using NCBI protein–protein blast BLASTP 2.1.3
Affinity Chromatography with the Heptapeptide of Murine Bone Marrow
Passage of Triton X-100–solubilized bone marrow membranes over an affinity column bearing attached synthetic heptapeptide resulted in elution of an 82-kDa polypeptide from the column with 1M NaCl (Fig. 6A).
Figure 6. Affinity chromatography and protein binding on blots of solubilized BM membranes were performed to determine the identity of the receptor for the specific phage. (A):Affinity chromatography of solubilized BM membranes with synthetic heptapeptide bound to the affinity column. The first lane shows the MW standards; the second, solubilized BM membranes; the third, the proteins of the supernatant of the Triton-extracted BM cells that did not bind to the affinity column (flow thru); the fourth, the proteins of the Triton-extracted BM cell pellet; and the fifth, the specific 82-kDa protein bound to the affinity column that eluted with 1M NaCl. (B): Western blot phage protein binding. Note that a doublet of MW 37/33 kDa was stained. Abbreviations: BM, bone marrow; MW, molecular weight.
Western-Type Blot of Membrane Receptors for Phage
Bone marrow cell-membrane proteins resolved on SDS-polyacrylamide gels were electrophoretically transferred to nitrocellulose. After incubation with specific phage and antibody to phage M13 and then development with anti-mouse IgG-conjugated alkaline phosphatase and substrates, staining revealed doublet protein bands of 37 and 33 kDa (Fig. 6B). It is unclear whether these bands represent proteolytic degradation products of the above-described 82-kDa protein or whether a different protein was obtained by binding of phage to SDS-solubilized bone marrow membranes rather than native Triton-solubilized bone marrow membranes.
DISCUSSION
This publication was made possible by NIH grants P01 DK50222, R01 DK27424, and R01 DK60084 from the National Institute for Diabetes and Digestive and Kidney Diseases, P01 HL56920, R01 HL63184, and R01 HL073749 from the National Heart Lung and Blood Institute, and P20 RR018757 from the National Center for Research Resources. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.
REFERENCES
Arroyo AG,Yang JT, Rayburn H et al. 4 integrins regulate the proliferation/differentiation balance of multilineage hematopoietic progenitors in vivo. Immunity 1999;11:555–566.
Williams DA, Rios M, Stephens C et al. Fibronectin and VLA-4 in haematopoietic stem cell-microenvironment interactions. Nature 1991:352:438–441.
Verfaillie CM, McCarthy JB, McGlave PB. Differentiation of primitive human multipotent hematopoietic progenitors into single lineage clonogenic progenitors is accompanied by alterations in their interaction with fibronectin. J Exp Med 1991;174:693–703.
Papayannopoulou T, Craddock C, Nakamoto B et al. The VLA4/VCAM-1 adhesion pathway defines contrasting mechanisms of lodgment of transplanted murine hematopoietic progenitors between bone marrow and spleen. Proc Natl Acad Sci U S A 1995;92:9647–9651.
Zanjani ED, Flake AW,Almeida-Porada G et al. Homing of human cells in the fetal sheep model: modulation by antibodies activating or inhibiting very late activation antigen-4-dependent function. Blood 1999;94:2515–2522.
Vermeulen M, Le Pesteur F, Gagnerault MC et al. Role of adhesion molecules in the homing and mobilization of murine hematopoietic stem and progenitor cells. Blood 1998;92:894–900.
Frenette PS, Subbarao S, Mazo IB et al. Endothelial selectins and vascular cell adhesion molecule-1 promote hematopoietic progenitor homing to bone marrow. Proc Natl Acad Sci U S A 1998;95:14423–14428.
Peled A, Petit I, Kollet O et al. Dependence of human stem cell engraftment and repopulation of NOD/SCID mice on CXCR4. Science 1999;283:845–848.
Jo DY, Rafii S, Hamada T et al. Chemotaxis of primitive hematopoietic cells in response to stromal cell-derived factor 1. J Clin Invest 2000;105:101–111.
Papayannopoulou T, Priestley GV, Nakamoto B et al. Molecular pathways in bone marrow homing: dominant role of 4?1 over ?2-integrins and selectins. Blood 2001;98:2403–2411.
Mullaney B, Pallavicini MG. Protein-protein interactions in hematology and phage display. Exp Hematol 2001;29:1136–1146.
Smith GP. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 1985;228:1315–1317.
Tseng-Law J, Szalay P, Guillermo R et al. Identification of a peptide directed against the CD34 antibody, 9c5, by phage display and its use in hematopoietic stem cell selection. Exp Hematol 1999;27:936–945.
Pasqualini R, Koivunen E, Ruoslahti E. A peptide isolated from phage display libraries is a structural and functional mimic of an RGD-binding site on integrins. J Cell Biol 1995;130:1189–1196.
McConnell SJ, Dinh T, Le MH et al. Isolation of erythropoietin receptor agonist peptides using evolved phage libraries. J Biol Chem 1998;379:1279–1286.
Gevorkian G, Manoutcharian K, Govezensky T et al. Identification of mimotypes of platelet autoantigens associated with autoimmune thrombocytopenic purpura. J Autoimmun 2000;15:33–40.
Pasqualini R, Ruoslahti E. Organ targeting in vivo using phage display peptide libraries. Nature 1996;380:364–366.
Arap W, Pasqualini R, Ruoslahti E. Cancer treatment by targeted drug delivery to tumor vasculature in a mouse model. Science 1998;279:377–380.
Arap W, Lahdenranta J, Cardo-Vila M et al. Steps toward mapping the human vasculature by phage display. Nat Med 2002;8:121–127.
Spangrude GJ, Scollay R. A simplified method for enrichment of mouse hematopoietic stem cells. Exp Hematol 1990;18:920–926.
Reddy GP, McAuliffe CI, Pang L et al. Cytokine receptor repertoire and cytokine responsiveness of Ho(dull)/ Rh(dull) stem cells with differing potentials for G(1)/S phase progression. Exp Hematol 2002;30:792–800.
Cerny J, Dooner M, McAuliffe C et al. Homing of purified murine lymphohematopoietic stem cells: a cytokine-induced defect. J Hematother Stem Cell Res 2002;11:913–922.
Cofano F, Moore SK, Tanaka S et al. Affinity purification, peptide analysis, and cDNA sequence of the mouse inter-feron gamma receptor. J Biol Chem 1990;265:4064–4071.
Scharenberg CW, Harkey MA, Torok-Storb B. The ABCG2 transporter is an efficient Hoechst 33342 efflux pump and is preferentially expressed by immature human hematopoietic progenitors. Blood 2002;99:507–512.
Wolf NS, Kone A, Priestley GV et al. In vivo and in vitro characterization of long-term repopulating primitive hematopoietic cells isolated by sequential Hoechst 33342-rhodamine 123 FACS selection. Exp Hematol 1993;21:614–622.
Brown CB, Feiner L, Lu MM et al. Plexin A2 and semiphorin signaling during cardiac neural crest development. Development 2001;128:3071–3080.
de la Fuente MA, Pizcueta P, Nadal M et al. CD84 leukocyte antigen is a new member of the Ig superfamily. Blood 1997; 90:2398–2405.
Zaiss M, Hirtreiter C, Rehli M et al. CD84 expression on human hematopoietic progenitor cells. Exp Hematol 2003; 31:798–805.
Martin M, Romero X, de la Fuente MA et al. CD84 functions as a homophilic adhesion molecule and enhances IFN-gamma secretion: adhesion is mediated by Ig-like domain 1. J Immunol 2001;167:3668–3676.
Palou E, Pirotto F, Sole J et al. Genomic characterization of CD84 reveals the existence of five isoforms differing in their cytoplasmic domains. Tissue Antigens 2000;55:118–127.
Ellerby HM,Arap W, Ellerby LM et al. Anti-cancer activity of targeted pro-apoptotic peptides. Nat Med 1999;5:1032–1038.
Grifman M, Trepel M, Speece P et al. Incorporation of tumor-targeting peptides into recombinant adenoviral-associated viral capsids. Mol Ther 2001;3:964–975.(Grzegorz S. Nowakowskia,b)
b Division of Hematology, Mayo Clinic, Rochester, Minnesota, USA;
c Department of Research, Roger Williams Medical Center, Providence, Rhode Island, USA;
d Dana-Farber Cancer Institute, Bostom, Massachusettes, USA;
e Division of Hematology, University of Washington, Seattle,Washington, USA
Key Words. Stem cells ? Engraftment ? Bone marrow transplant ? Cell adhesion molecules ? Bone marrow microenvironment
Correspondence: Pamela S. Becker, M.D., Ph.D., Division of Hematology, Box 357710, University of Washington, 1959 N.E. Pacific Street, HSB K-136, Seattle,WA 98195, USA. Telephone: 206-616-1589; Fax: 206-543-3560; e-mail: pbecker@u.washington.edu
ABSTRACT
Hematopoietic progenitors are capable of migration and homing, processes fundamental to embryonal development, circulation in the vasculature, and stem cell transplantation. Engraftment after bone marrow transplant may involve several adhesion receptors, each recruited sequentially in the movement from bloodstream through vascular walls to the bone marrow. Studies in 4 integrin–null mice indicated that migration of embryonic hematopoietic precursors to the fetal liver, bone marrow, and spleen was independent of these receptors but that maintenance of hematopoiesis by these organs was entirely dependent on 4-integrins . The very late antigen (VLA)-4 receptor plays the most critical role in engraftment after transplant, as demonstrated by the following observations: First, murine colony-forming unit spleen cells and reconstituting hematopoietic stem cells attached to the connecting segment-1 (CS-I) peptide of alternatively spliced fibronectin (Fn), and this binding was blocked by antibody to 4 ; second, human long-term bone marrow culture-initiating cells (LTBM-ICs) bound to Fn in the heparin-binding region (a function of 4), and these Fn peptides could block their adherence to irradiated stroma ; third, antibody to VLA-4 reduced bone marrow engraftment in mice ; and, fourth, blocking antibody to human VLA-4 reduced bone marrow engraftment of human CD34+ cells in fetal sheep . The VLA-4/vascular cellular adhesion molecule-1 (VCAM-1) pathway was found to be critical for homing to the bone marrow, whereas CD44 was involved in the localization of colony-forming unit–spleen in the bone marrow and spleen . Antibody blocking of VCAM-1 in vivo in E-selectin/P-selectin knockout mice suggested that all three receptors were required for optimal homing to the bone marrow . Moreover, human CD34+ cells were dependent on CXCR4 (receptor for stromal cell–derived factor-1) (SDF-1) for optimal bone marrow homing and engraftment in the nonobese diabetic/severe combined immunodeficiency model , indicating that chemotaxis to SDF-1 may be critical . VLA-4 was then shown to possess the dominant role over both selectins and ?2-integrins for engraftment by using antibody to 4-integrin with donor cells from selectin-null or ?2-integrin–null mice . These prior approaches to the study of surface molecules in homing and engraftment examined receptors known to be expressed by the hematopoietic progenitors or the bone marrow stroma.
We sought to discover novel adhesion mechanisms in bone marrow homing and engraftment using a phage display peptide library (PDPL) . The PDPL consists of an assembly of phages, each of which has fusions of a random peptide to a coat protein, with approximately five copies of each peptide per phage . The number of unique peptide sequences depends on peptide length. Because there are 20 coding amino acids, a random heptapeptide display library has 207 or 1.3 x109 unique sequences (clones). Phages that display specific peptides are selected by binding to selector or target molecules. Amplification of selected clones bearing peptides of interest is possible because the peptide sequence is coded by phage DNA. This process of selection and amplification is called biopanning and can be repeated several times to enrich the phage pool in favor of binding sequences. Individual clones may then be selected and characterized by DNA sequencing.
PDPLs were previously used in a variety of applications , including epitope mapping of monoclonal antibodies , identification of peptide ligands for 5?1-integrin, and isolation of structural and functional mimics of the arginine-glycine-aspartic acid–binding site of integrins . Peptides identified by biopanning procedures are frequently involved in important functional interactions; for example, peptides mimicking cytokines may exhibit their functional activities . Also, peptide sequences often mimic the sequence of natural ligands such as peptides selected against anti-platelet antibodies that share sequence homology with glycoprotein (GP) IIb/IIIa and GPIb surface molecules . Biopanning can also be conducted in vivo, allowing selection of peptides that interact with tissues and cells in their native microenvironment. This approach was used to identify peptides that exhibit organ-specific homing for the kidney and brain and later for other organs and tumor vasculature in animals. Similar studies to select organ-homing peptides were initiated in humans .
For the present study, we used biopanning in vivo to identify a peptide that homes to the bone marrow. Surprisingly, the identical heptapeptide was isolated by biopanning in vitro on hematopoietic stem cells. A functional assay demonstrated its ability to interfere with early homing of hematopoietic progenitors.
MATERIALS AND METHODS
Biopanning In Vivo
The sequencing of single-phage colonies from the fifth round of biopanning in vivo (diagram of procedure in Fig. 1) revealed two predominant consensus sequences, STFTKSP and NHWASPR, constituting 50% and 28% of sequences, respectively. The remaining sequences were single or incomplete sequences, some of which contained motifs of the two predominant sequences.
Biopanning In Vitro Using FACS on Hematopoietic Stem Cells
Biopanning combined with FACS was repeated three times, yielding phage clones that attached to primitive Lin–Hoechstdullrhodaminedull stem cells. The results of sequencing showed that 85% of the single-phage colonies had the same sequence as the most predominant peptide isolated by biopanning in vivo, STFTKSP. The remaining sequences were single or incomplete.
Flow Cytometry Analysis of Phage-Binding Bone Marrow Cells
Flow cytometry analysis of the density-depleted bone marrow stained with specific phage, antibody to phage M13 protein, and secondary fluorescent-labeled antibody revealed a distinctive population constituting 1.10% of the density-depleted bone marrow cells. The following percentages of C57BL6 bone marrow cells exhibited costaining by both lineage marker and specific phage when gated on cells binding phage: Ter-119, 3%; YW25, 2%; Mac-1, 3%; CD45R, 2%; CD4, 0%; CD8, 0%; GR-1, 1%; and GPIIb/IIIa, 0%. These results indicate minimal expression of lineage markers by cells that bind the specific phage. In a separate set of experiments using BALB/c bone marrow, of the cells with attached specific phage, 36.6 ± 17.5% expressed Sca-1 (1.01 ± 0.14% for the IgG2a isotype control), 49.9 ± 15.5% expressed c-kit (1.64 ± 0.16% for the IgG2b isotype control), and 27.18 ± 4.05% expressed CD84 (0.99 ± 0.12% for the IgG1 isotype control). Moreover, 86% of the BALB/c lineage-depleted bone marrow cells that expressed c-kit also expressed CD84, and 57% of those that expressed Sca-1 also expressed CD84.
Organ Specificity of Bone Marrow Homing Phage
The organs sections were stained with FITC-labeled anti-phage M13 antibody, and staining was observed within the bone marrow and lungs (Fig. 3), as follows: 12.5% of the DAPI-counterstained cells from the bone marrow, 3% of the cells from the lung sections, 0.5% of the cells from a lymph node, and 0.5% of the cells from the thymus exhibited staining of bound phage. These numbers may represent an overestimation because of the appearance of staining of clusters of cells or adjacent cells. There was no staining for phage within the kidney, heart, brain, spleen, or liver.
Figure 3. BM and other cells were stained by immunofluorescence for phage to assess the specificity of organ homing. The left panels show the dual stain of the cell nuclei with 4',6'-diamidino-2-phenylindole (blue), indicating the location of cells and the staining with anti-phage monoclonal antibodies and phycoerythrin (red)–labeled secondary antibodies. As a control, the BM of the animal that was not injected with the phage is shown in the right panels. Representative cells from the BM, lung, and brain are shown. Abbreviation: BM, bone marrow.
Immunofluorescent Staining of Bone Marrow–Homing Phage Binding to Lin–HoechstdullRhodaminedull Stem Cells
There were two patterns of staining: circumferential membrane and more dense, diffuse staining (Fig. 4).
Figure 4. The Lin–Hoechst 33342dull/rhodamine 123dull stem cells were examined by immunofluorescence to demonstrate binding of the specific phage. The upper panels show staining with DAPI. The lower first panel on the left represents a control with FITC-labeled secondary antibody without phage; the lower middle and right panels show staining with phage, antibody to phage M13 protein, and FITC-labeled secondary antibody. Abbreviations: Ab, antibody; DAPI, 4',6'-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate.
Bone Marrow Homing Assay of CFDA-SE–Labeled Lin–Sca-1+ Cells with Heptapeptide/Phage Displaying Heptapeptide Blockade
Large-event flow cytometry analysis demonstrated a 26% reduction in homing 3 hours after transplant in an animal injected with the synthetic heptapeptide compared with the control animal (data not shown). When the phage displaying the bone marrow homing heptapeptide was used instead of a free heptapeptide, there was a 41% reduction in homing 3 hours after transplant, whereas homing to the spleen was fully preserved at 109% of control. The difference was statistically significant for the difference in homing to the bone marrow compared with control (p = .007) but was not statistically significant for the spleen (p = .66; Fig. 5).
Figure 5. BM homing assay of carboxyfluorescein diacetate succinimidyl ester–labeled Lin–Sca-1+ cells was performed in the presence of the specific phage-displaying peptide. In the presence of the phage, there was diminished homing to the bone marrow (p = .007) but preserved homing to the spleen (p = .66) compared with the absence of phage in an in vivo homing assay. Abbreviation: BM, bone marrow.
Database Search Results
Although nucleotide database searches were not successful in identifying homologous sequences, a Genbank search using NCBI protein–protein blast BLASTP 2.1.3 revealed several sequences with varying degrees of homology (Table 1).
Table 1. Genbank search results using NCBI protein–protein blast BLASTP 2.1.3
Affinity Chromatography with the Heptapeptide of Murine Bone Marrow
Passage of Triton X-100–solubilized bone marrow membranes over an affinity column bearing attached synthetic heptapeptide resulted in elution of an 82-kDa polypeptide from the column with 1M NaCl (Fig. 6A).
Figure 6. Affinity chromatography and protein binding on blots of solubilized BM membranes were performed to determine the identity of the receptor for the specific phage. (A):Affinity chromatography of solubilized BM membranes with synthetic heptapeptide bound to the affinity column. The first lane shows the MW standards; the second, solubilized BM membranes; the third, the proteins of the supernatant of the Triton-extracted BM cells that did not bind to the affinity column (flow thru); the fourth, the proteins of the Triton-extracted BM cell pellet; and the fifth, the specific 82-kDa protein bound to the affinity column that eluted with 1M NaCl. (B): Western blot phage protein binding. Note that a doublet of MW 37/33 kDa was stained. Abbreviations: BM, bone marrow; MW, molecular weight.
Western-Type Blot of Membrane Receptors for Phage
Bone marrow cell-membrane proteins resolved on SDS-polyacrylamide gels were electrophoretically transferred to nitrocellulose. After incubation with specific phage and antibody to phage M13 and then development with anti-mouse IgG-conjugated alkaline phosphatase and substrates, staining revealed doublet protein bands of 37 and 33 kDa (Fig. 6B). It is unclear whether these bands represent proteolytic degradation products of the above-described 82-kDa protein or whether a different protein was obtained by binding of phage to SDS-solubilized bone marrow membranes rather than native Triton-solubilized bone marrow membranes.
DISCUSSION
This publication was made possible by NIH grants P01 DK50222, R01 DK27424, and R01 DK60084 from the National Institute for Diabetes and Digestive and Kidney Diseases, P01 HL56920, R01 HL63184, and R01 HL073749 from the National Heart Lung and Blood Institute, and P20 RR018757 from the National Center for Research Resources. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.
REFERENCES
Arroyo AG,Yang JT, Rayburn H et al. 4 integrins regulate the proliferation/differentiation balance of multilineage hematopoietic progenitors in vivo. Immunity 1999;11:555–566.
Williams DA, Rios M, Stephens C et al. Fibronectin and VLA-4 in haematopoietic stem cell-microenvironment interactions. Nature 1991:352:438–441.
Verfaillie CM, McCarthy JB, McGlave PB. Differentiation of primitive human multipotent hematopoietic progenitors into single lineage clonogenic progenitors is accompanied by alterations in their interaction with fibronectin. J Exp Med 1991;174:693–703.
Papayannopoulou T, Craddock C, Nakamoto B et al. The VLA4/VCAM-1 adhesion pathway defines contrasting mechanisms of lodgment of transplanted murine hematopoietic progenitors between bone marrow and spleen. Proc Natl Acad Sci U S A 1995;92:9647–9651.
Zanjani ED, Flake AW,Almeida-Porada G et al. Homing of human cells in the fetal sheep model: modulation by antibodies activating or inhibiting very late activation antigen-4-dependent function. Blood 1999;94:2515–2522.
Vermeulen M, Le Pesteur F, Gagnerault MC et al. Role of adhesion molecules in the homing and mobilization of murine hematopoietic stem and progenitor cells. Blood 1998;92:894–900.
Frenette PS, Subbarao S, Mazo IB et al. Endothelial selectins and vascular cell adhesion molecule-1 promote hematopoietic progenitor homing to bone marrow. Proc Natl Acad Sci U S A 1998;95:14423–14428.
Peled A, Petit I, Kollet O et al. Dependence of human stem cell engraftment and repopulation of NOD/SCID mice on CXCR4. Science 1999;283:845–848.
Jo DY, Rafii S, Hamada T et al. Chemotaxis of primitive hematopoietic cells in response to stromal cell-derived factor 1. J Clin Invest 2000;105:101–111.
Papayannopoulou T, Priestley GV, Nakamoto B et al. Molecular pathways in bone marrow homing: dominant role of 4?1 over ?2-integrins and selectins. Blood 2001;98:2403–2411.
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