Expression of Adhesion Molecules on Human Hematopoietic Progenitor Cells at Different Maturational Stages

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1 Expression of Adhesion Molecules on Human Hematopoietic Progenitor Cells at Different Maturational Stages Masanobu Kobayashi,a Masahiro Imamura,b Toshimitsu Uede," Keisuke Sakurada,b Shiro Maeda,b Hiroshi Iwasaki,b Yuuzou Tsuda,b Manabu Musashi,b Tamotsu Miyazakib "Laboratory of Pathology, Cancer Institute, Hokkaido University School of Medicine, Sapporo, Japan; bthe Third Department of Internal Medicine, Hokkaido University School of Medicine, Sapporo, Japan; 'Section of Immunopathogenesis, Institute of Immunological Science, Hokkaido University School of Medicine, Sapporo, Japan; Key Words. Hematopoietic progenitor cells Adhesion molecule Abstract. In this report we examined the expression of several adhesion molecules on human hematopoietic progenitor cells at different maturational stages. Human hematopoietic progenitor cell-enriched fractions were prepared from bone marrow cells by depleting lymphocytes and monocytes (CD2+, CD14' and CD19' cells). These cells were separated into adhesion molecule-positive and -negative cell populations by immunomagnetic separation methods and then assessed for their ability to form various colony forming cells (CFC). CD44 and CD49d were expressed on multipotent hematopoietic progenitor cells, or mixed colony forming units (CFU-Mix), erythroid burst forming units (BFU-E), granulocyte-macrophage CFU (CFU-GM) and erythroid CFU (CFU-E). Leu8 was expressed on CFU-Mix, BFU-E and some populations of CFU-GM, but not CFU-E. CDlla was expressed on some populations of CFU-Mix, CFU-GM and BFU-E. CD54 was expressed only on some populations of CFU-GM. These results suggest that Leu8, CD44, CD49d and CDlla appear to play important roles in the differentiation and proliferation of human hematopoietic progenitor cells at different maturational stages in the bone marrow microenvironment. Introduction The bone marrow microenvironment is essential for the proliferation and differentiation Correspondence: Dr. M. Kobayashi, Laboratory of Pathology, Cancer Institute, Hokkaido University School of Medicine, Nishi-7, Kita-15, Kita-ku, Sapporo, Japan 060. Received August 20, 1993; provisionally accepted October 21, 1993; accepted for publication December 23, OAlphaMed Press /94/$5.00/0 of hematopoietic stem and progenitor cells [ 11. Interactions between the bone marrow microenvironment and hematopoietic cells are thought to be mediated by the recognition and adherence through adhesion molecules expressed on hematopoietic cells [2, 31. Several investigators have reported that hematopoietic stem and progenitor cells can adhere to the extracellular matrix (ECM) [4-81, suggesting that the cells may express very late activation antigens (VLA). Tavassoli et al. reported that the homing of marrow colony forming units-spleen (CFU-S) and committed progenitor cells to marrow stroma is mediated by a surface membrane lectin with galactosyl and mannosyl specificities [9, 101. Miller et al. reported that murine hematopoietic progenitor cells express LFA-1 [ll]. Kansas et al. reported that primitive hematopoietic cells detected by flow cytometry express CDlla, CD44 and leukocyte adhesion molecule 1 [ 121. Recently, Lewinsohn et al. have reported that both colony forming unitsgranulocyte/macrophage (CFU-GM) and burst forming units-erythroid (BFU-E) express the homing-associated cell-adhesion molecule (H-CAM) (CD44) [13], and Papayannopoulou et al. have reported that CDl la is expressed on BFU-E [ 141. Thus, considerable numbers of reports concerning the expression of adhesion molecules have accumulated. However, it is not yet clear how the adhesion molecules are expressed on human hematopoietic progenitor cells at different maturational stages because of the very low frequency of hematopoietic stem and progenitor cells in bone marrow. In this study, we examined the expression of several adhesion molecules on human STEM CELLS 1994;12:

2 Kobayashi et al. 317 hematopoietic progenitor cells at different maturational stages by separating lymphocyte- and monocyte-depleted bone marrow mononuclear cells into adhesion molecule-negative and -positive cells using immunomagnetic separation methods which have been used for the purification of CD34' cells [15]. Materials and Methods Reagents Recombinant human interleukin 3 (rhil-3), rh erythropoietin (rhepo) and rh granulocyte colony stimulating factor (rhg-csf) were kindly provided by Kirin Brewery Co. Ltd. (Kanagawa, Japan). rh granulocyte-macrophage CSF (rhgm-csf) was purchased from Genzyme (Boston, MA). NU-TER (CD2), anti-lfa- 1 (SPV-L7), B4 (CD19), My4 (CD14), anti-leu8 antibody, anti-leu44 antibody (CD44), and anti-icam- 1 antibody (CD54) were purchased from Nichirei (Tokyo, Japan), Coulter Immunology (Hialeah, FL), Becton Dickinson (San Jose, CA), and British Bio-technology (Abington, UK). Anti-VLA4 antibody (CD49d) was supplied by Dr. C. Morimoto (Division of Tumor Immunology, Dana Farber Cancer Institute) [16]. Bovine serum albumin (BSA) and 2-mercaptoethanol (2-ME) were purchased from Sigma Chemical Co. (St. Louis, MO). Iscove's modified Dulbecco's medium (IMDM) was purchased from GIBCO Life Technologies Inc. (Grand Island, NY). Fetal calf serum (FCS) was purchased from Hyclone Laboratories Inc. (Logan, UT). Bone Marrow Cell Preparation Bone marrow was obtained from the iliac crest of normal volunteers after obtaining informed consent. Bone marrow mononuclear cells (BMMNC) were obtained by centrifugation on Ficoll-Conray (1.077). Bone marrow adherent cells (BMAC) were removed by the incubation of BMMNC for 1 h in plastic flasks twice. Bone marrow lymphocyte- and monocyte-depleted mononuclear cells (BMLMDC) were obtained by the depletion of CD2+, CD14+ and CD19' cells from bone marrow, non-adherent cells (BMNAC) using immunomagnetic separation methods described previously [ 151. Briefly, BMNAC were incubated with 2-4 pg (1 x lo7 cells) of antibodies (anti-cd2, anti-cd14 and anti-cd19 antibodies) for 30 min at 4 C with intermittent agitations, washed with cold phosphate buffered saline (PBS) supplemented with 0.1 % BSA, and then incubated with antimouse immunoglobulin G (IgG) coated immunobeads (Dynabeads'" M-450 Goat Antimouse IgG; Dynal Inc., Great Neck, NY) with a microbeads per cell ratio of 1O:l for 45 min at 37 C. After removing immunobeads rosetted cells, 5 x lo5 BMLMDC were incubated with 0.5 pg of antibodies including anti-leu8, anti-leu44, anti-lfa- 1, and anti-icam- 1 antibodies, and 1:300 diluted anti-vla4 antibody for 30 min at 4 C with intermittent agitations, washed with cold PBS supplemented with 0.1% BSA, and then incubated with Dynabeads'" M-450 Goat Antimouse IgG (Dynal Inc.) with a microbeads per cell ratio of 1O:l for 45 min at 37 C. Adhesion molecule-positive and -negative cells were separated by a magnet (Dynal MPC'"-1; Dynal Inc.) and then resuspended in 0.5 ml of IMDM. Less than 8.5% ( % for Leu8, % for CD44, % for CDlla, % for CD54, and % for CD49d) of positive cells were contained in the negative cell fraction, which was determined by immunohistochemical staining after removing the rosetted beads using DETACHaBEADa (Dynal MPC'"-1; Dynal Inc.). The numbers of positive cells obtained from 5 x lo5 BMLMDC were 0.78 f 0.16 x lo5 (Leu8), 2.0 f 1.41 x lo5 (CD44), 1.12 f 0.92 x lo5 (CD1 la), 0.54 f 0.38 x lo5 (CD54), and 1.06 f 0.28 x lo5 (CD49d). Colony Assay Colony assays were performed using a previously described method with minor modifications [ 151. Briefly, 0.15 ml of the cell suspensions were mixed in 3 ml of agar mixture containing IMDM, 30% FCS, 1% BSA, 1 x lo4 M 2-ME and 0.3% agar, and 400 ml of agar mixture was plated in six wells of leukocyte migration plates (Sterilin, Seltham, UK) (final numbers of colonies formed in each well represent the numbers of colony forming cells contained in antigen-positive and -negative populations of 2 x lo4 BM Lin- cells) and incubated at 37 C in a humidified atmosphere of 5% COz for 14 days. Each assay was done in the presence of rhil-3, rhgm-csf, rhg-csf and EPO at 100 U/ml, 100 ng/ml, 100 ng/ml and 2 U/ml, respectively. After 7 and 14 days, colony numbers were counted in situ using an inverted

3 318 Adhesion Molecules on Hematopoietic Progenitors microscope, Colony forming units-erythroid (CFU-E) contained more than 8 hemoglobin-containing cells. CFU-GM contained more than 40 granulocytes and/or macrophages. BFU-E contained more than 100 hemoglobin-containing cells or consisted of more than 3 clusters of hemoglobin-containing cells. Colony forming units-mix (CFU-Mix) contained both nonerythroid cells and hemoglobin-containing cells. The gels were placed on glass microscope slides, dried, fixed in methanol and then stained with May-Griinwald Giemsa to confirm colony types. Statistical Analysis Experiments were done using four different bone marrow cells. Statistical analysis was done by Student's t-test. Percentages of colonies formed in adhesion molecule-negative cell populations were compared with percentages of adhesion molecule-positive cells in negative cell populations. Results Figure 1 shows percent formation of CFU-E, BFU-E, CFU-GM and CFU-Mix in adhesion molecule-positive and -negative cell populations compared with the total numbers of colony forming cells (CFC) in unfractionated cell populations. All CFU-Mix colonies were formed in a Leu8+ cell fraction, and the ratio of colonies formed in a Leu% cell fraction increased along with the differentiation indicating that the expression of Leu8 was specific for immature progenitor cells (Fig. 1A). Almost all colonies derived from CFC at different maturational stages were formed in a CD44' cell fraction and a CD49d' fraction indicating that CD44 and CD49d antigens were expressed throughout all maturational stages (Fig. 1B and Fig. 1E). Colonies derived from CFU-Mix, BFU-E and CFU-GM were formed in both CDl la+ and CD1 la- cell fractions, whereas almost all colonies derived from CFU-E were formed in a CD1 la- cell fraction, indicating that the expression of CDlla was lost at a maturational stage of CFU-E (Fig. 1C). Almost all colonies derived from CFU-Mix, BFU-E and CFU-E were formed in a CD54- cell fraction, whereas colonies derived from CFU-GM were formed in both CD54- and CD54' cell fractions (Fig. 1D). Discussion In this study, we show that CD44 and CD49d were expressed on human hematopoietic progenitor cells at all maturational stages tested from CFU-Mix to CFU-E. Leu8, CDlla and CD54 were expressed on human hematopoietic progenitor cells at specific maturational stages. Leu8 was expressed on immature progenitor cells and lost on CFU-E. CDl la was lost on CFU-E. The expression of CD54 was transient and observed only on CFU-GM. Although our methods do not allow us to measure the intensity of the expression of adhesion molecules, results obtained in this study clearly show that the expression of adhesion molecules changes along with the differentiation of human hematopoietic progenitor cells (Fig. 2). It has been reported that erythroid colony forming cells bind strongly whereas CFU-GM bind only weakly to fibronectin [6, 71. In addition, day-12 CFU-S cells have been reported to bind to fibronectin by recognizing the CS-1 peptide of the alternatively spliced non-type I11 connecting segment of human plasma fibronectin [8]. These results suggest that erythroid colony forming cells and CFU-S express CD49d (VLA4). Our result that both CFU-GM and BFU-E express CD49d appears to be inconsistent with the previous studies [6, 71. This inconsistency may be explained by the different methods used for the examination between ours and theirs, or by the difference of target molecules of CD49d expressed on CFU-GM and BFU-E. Alternatively, CD49d expressed on erythroid progenitor cells may be activated. These mechanisms controlling the binding activity through the activation of adhesion molecules have been reported for several adhesion molecules, such as LFA-1. Recently, a cdna clone that increases cell adhesion to components of the ECM without any changes in the levels of expression of adhesion molecules has been reported [ 171. Recent fluorescence-activated cell sorting (FACS) analyses have revealed that CD34' progenitor cells express CD49d, which suggests that both BFU-E and CFU-GM express CD49d and support our results [18, 191. CD44 recognized by anti-leu44 antibody has been reported to be a lymphocyte surface antigen which mediates the interaction between lymphocytes and mucosal high-walled endothelial venule (HEV) [20]. This molecule has also been termed H-CAM and is identical to Pgp-1 [21].

4 Kobayashi et al. 319 A Leu8 0 positivefractiw 1 w negativetraction CFU-E BFU-E CFU-GM CFU-Mix CFU-E BFU-E CFU-GM CFU-Mix D CD54 positivefraction CFU-E BFU-E CFU-GM CFU-Mix CFU-E BFU-E CFU-GM CFU-Mix CFU-E BFU-E CFU-GM CFU-Mix Fig. 1. Expression of adhesion molecules. Bone marrow lymphocyte- and monocyte-depleted mononuclear cells were separated into adhesion molecule-positive or -negative cell fractions for A) Leu& B) CD44, C) CD1 la, D) CD54 and E) CD49d. The cells in each fraction were evaluated for the numbers of colony forming cells according to the methods described in Materials and Methods. Results are shown as the mean f SD of percents of total colonies formed in unseparated cells in four experiments. *p c 0.01 as compared with percentages of adhesion molecule-positive cells in negative cell populations. **p c 0.05 as compared with percentages of adhesion molecule-positive cells in negative cell populations. Recently, Miyake et al. reported that monoclonal antibodies to Pgp-l/CD44 block lymphohemopoiesis in murine long-term bone marrow cultures and suggested that CD44 might play a role in the interaction between bone marrow stromal cells and hematopoietic stem cells [22]. Reuss et al. reported that CD34' cells expressed CD44 [19]. Our results, that CD44 was expressed on both immature and mature progenitor cells, are consistent with these reports. Leu8 recognizes the human homolog of murine homing receptor recognized by the MEL-14 antibody [23]. It is

5 3 20 Adhesion Molecules on Hematopoietic Progenitors CD44 CD49d Leu8 CDlla CD54 CFU-GM- CFU-Mix-BFU-E-CFU-E Fig. 2. Summary of adhesion molecule expression on human hemopoietic progenitor cells. interesting to note that the Mel-14 antigen is a mammalian lectin with carbohydrate specificity for mannose-6-phosphate [24], because Tuvussoli has claimed that the homing of marrow CFU-S and committed progenitor cells to marrow stroma is mediated by a surface membrane lectin with galactosyl and mannosyl specificities [9, 101. Our results suggest that Leu8, CD44, CD49d and CD 1 1 a appear to play distinct roles in the differentiation and proliferation of human hematopoietic progenitor cells at different maturational stages in the bone marrow microenvironment. Acknowledgments We wish to thank Dr. C. Morinzoto (Division of Tumor Immunology, Dana-Farber Cancer Institute) for anti-vla4 antibody and Kirin Brewery Co. Ltd. for EPO, G-CSF and IL-3. We also wish to thank Ms. M. Yunome for preparing the manuscript. References 1 Wintrobe MM. Clinical Hematology, 7th Ed., Philadelphia: Lea & Febiger, Gordon MY. Adhesive properties of haemopoietic stem cells. Brit J Haematol 1988;68: Liesveld JL, Winslow JM, Kempski MC, Ryan DH, Brennan JK, Abboud CK. Adhesive interactions of normal and leukemic human CD34' myeloid progenitors: role of marrow stromal, fibroblast, and cytomatrix components. Exp Hematol 1991; 19: Gordon MY, Rilley GP, Clarke D. Heparan sulfate is necessary for adhesive interactions between human early hematopoietic progenitor cells and the extracellular matrix of the marrow microenvironment. Leukemia 1988;2: Campbell AD, Long MW, Wicha MS. Developmental regulation of granulocytic cell binding to hemonectin. Blood 1990;76: Tsai S, Patel V, Beaumont E, Lodish HF, Nathan DG, Sieff CA. Differential binding of erythroid and myeloid progenitors to fibroblasts and fibronectin. Blood 1987;69: Coulombel L, Vuillet MH, Leory C, Tchernia G. Lineage- and stage-specific adhesion of human hematopoietic progenitor cells to extracellular matrices from marrow fibroblasts. Blood 1988;71: Williams DA, Rios M, Stephens C, Patel VP. Fibronectin and VLA-4 in hematopoietic stem cell-microenvironment interactions. Nature 1991;352: Aizawa S, Tavassoli M. Interactions of murine granulocyte-macrophage progenitors and supporting stroma involves a recognition mechanism with galactosyl and mannosyl specificities. J Clin Invest 1987;80: Tavassoli M, Hardy CL. Molecular basis of homing of intravenously transplanted stem cells to the marrow. Blood 1990;76: Miller BA, Antognetti G, Springer TA. Identification of cell surface antigens on murine hematopoietic stem cells. J Immunol 1985; 134: Kansas GS, Muirhead MJ, Dailey MO. Expression of the CDll/CD18, leukocyte adhesion molecule 1, and CD44 adhesion molecules during normal myeloid and erythroid differentiation in humans. Blood 1990;76: Lewinsohn DM, Nagler A, Ginzton N, Greenberg P, Butcher EC. Hematopoietic progenitor cell expression of the H-CAM (CD44) homing-associated adhesion molecule. Blood : Papayannopoulou T, Brice M. Integrin expression profiles during erythroid differentiation. Blood 1992;79: Kobayashi M, Imamura M, Gotohda Y, Maeda S, Iwasaki H, Sakurada K, Kasai M, Hapel AJ, Miyazaki T. Synergistic effects of interleukin- 18 and interleukin-3 on the expansion of human hematopoietic progenitor cells in liquid cultures. Blood 1991 ;78: Matsuyama T, Yamada A, Kay J, Yamada KM, Akiyama SK, Scholossman SF, Morimoto C. Activation of CD4 cells by fibronectin and anti-cd3 antibody: a synergistic effect mediated

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