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Stem Cells, Vol. 16, No. 1, 49-53, January 1998
© 1998 AlphaMed Press

Difference between Expression of Adhesion Molecules on CD34+ Cells from Bone Marrow and G-CSF-Stimulated Peripheral Blood

N. Krögera, W. Zellera, H.T. Hassana, J. Dierlammb, A.R. Zandera

a Bone Marrow Transplantation Unit,
b Department of Oncology and Hematology, University Hospital Hamburg, Hamburg, Germany

Key Words. Adhesion molecules • Mobilization of CD34+ cells • Bone marrow

Correspondence: Dr. Nicolaus Kröger, Bone Marrow Transplantation Unit, Department of Oncology and Hematology, University Hospital Eppendorf, Martinistrasse 52, D-20246 Hamburg, Germany.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Three-color immunofluorescence cytometry was used to quantify the expression of different adhesion molecules on CD34+ cells of steady-state bone marrow (BM) and peripheral blood stem cells (PBSC) after mobilizing with G-CSF (10 µg/kg/body weight) in nine cancer patients undergoing high-dose chemotherapy with subsequent autologous blood stem cell rescue. The expression rate of each adhesion molecule on CD34+ cells showed great inter-individual variations. High expression (>50%) on CD34+ cells from PBSC and BM was found for CD58 (leukocyte function-associated antigen-3), CD31 (platelet-endothelial cell adhesion molecule-1), CD11a (leukocyte function-associated antigen-1) and CD49d (very late activation antigen-4); a moderate expression (20%-40%) was seen for CD49e (very late activation antigen-5), CD62L (leukocyte-endothelial cell adhesion molecule), CD54 (ICAM-1) and CD117 (c-kit).

c-kit, CD58, CD62L and CD49d were less expressed on CD34+ cells of PBSC than of BM, the difference being statistically significant for CD49d (p < 0.05). CD49e and CD37 were expressed more in PBSC than BM without being statistically significant. The mean fluorescence intensity for all adhesion molecules on CD34+ cells did not differ significantly between PBSC and BM. The significantly lower expression of CD49d on G-CSF-mobilized PBSCs might suggest that downregulation of this molecule may be involved in the process of peripheral stem cell mobilization.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
High-dose chemotherapy with support of mobilized peripheral blood stem cells (PBSC) is increasingly used in the treatment of patients with hematological and solid tumors [1]. A substantial number of CD34+ cells can be mobilized by chemotherapy, hematopoietic growth-factors or a combination of both. The mechanisms underlying the mobilization are still poorly understood [2]. Adhesion to bone marrow (BM) stroma is an important mechanism for the "homing" of hematopoietic stem cells in BM for hematopoietic regeneration after myeloablative therapy [3]. Adhesion molecules involved in the interaction between adhesive hematopoietic and stromal cells include integrins, selectins and immunoglobulin-like adhesion molecules [4].

Integrins
At least 17 distinct heterodimers are formed by the association of 13 different alpha units and 7 different beta units [5]. The integrins are generally distinguished according to their ß-chains into ß1 integrins (known as very late antigens [VLA]: VLA 1-6) and ß2 integrins (leucocyte integrins) such as leukocyte function-associated antigen-1 (LFA-1) (CD18/CD11a) and Mac-1 (CD18/CD11b). Integrins have been extensively studied in mature leukocytes and have been found to be crucial for a variety of immune functions as well as for transendothelial migration of leukocytes into inflammatory loci [6, 7].

Selectins
L-selectin (CD62L) and P-selectin (CD62P) are transmembranous glycoproteins that recognize carbohydrate residues on endothelial cells [4]. Selectins are involved in extravasation of leukocytes in that they bind leukocytes to endothelial cells [8]. CD34 is expressed on endothelial cells and may therefore play a role as ligand for L-selectin (CD62L) in stem cell adhesion to BM stroma [2]. It is thus of interest that after stem cell transplantation the number of CD62L+ hematopoietic progenitor cells (HPCs) correlates better with platelet recovery than with the total number of CD34+ cells [9].

Immunoglobulin-Like Adhesion Molecules
N-CAM (CD56), ICAM-1 (CD54), vascular cell adhesion molecule (CD106), and platelet-endothelial cell adhesion molecule-1 (CD31) have been found to be counterparts of integrins on endothelial cells. The expression of immunoglobulins is increased at sites of inflammation suggesting a role of lymphocyte influx [10]. CD58 (LFA-3) is the ligand for CD2 and might be involved in interaction between T cells and HPC [11]. Several adhesion molecules have been found to be expressed on CD34+ cells: CD44, CD53, CD56, CD31, VLA-4 (CD29/CD49d), VLA-5 (CD29/CD49e), LFA-1 (CD18/CD11a) and Mac-1 (CD18/CD11b) [9, 12-22).

Since it was shown that adhesion molecules are expressed during differentiation of HPCs, regulation of adhesion molecules on these cells has been assumed to play a key role in their mobilization and homing [12].

The aim of the present study is to investigate the potential differences in expression of different adhesion molecules on CD34+ cells of BM before mobilization and of peripheral blood (PB) cells after priming with G-CSF, thus to elucidate possible mechanisms of stem cell mobilization.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patients
Nine cancer patients (four male and five female) were studied: three with breast cancer, three with Hodgkin's disease and three with high-grade non-Hodgkin's lymphoma. The median age was 42 years (range 24 to 54). The breast cancer patients were treated with an adjuvant therapy consisting of four cycles of induction chemotherapy (epirubicin/ cyclophosphamide) followed by high-dose chemotherapy. The Hodgkin's disease and high-grade non-Hodgkin's lymphoma patients received two cycles of conventional and then high-dose chemotherapy. Induction chemotherapy for the lymphoma patients consisted either of dexamethasone, BCNU, melphalan and cytosine-arabinoside (Ara C) or adriamycin, methyl-prednisolone, Ara C and cisplatinum. All patients achieved at least a partial response before undergoing high dose chemotherapy. None of the patients had BM involvement. All patients had a hematopoietic engraftment with a leucocyte count >1.0/nl after a median of 11 days ( Table 1).


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  		Table 1.
 
Methods
Samples of BM before mobilization and PB after mobilization were obtained from each patient. The mobilization procedure consisted of 10 µg/kg/body weight (BW) G-CSF (Filgrastim, Amgen, Inc.; Thousand Oaks, CA) daily s.c. After four days of G-CSF injections, leukapheresis procedures were performed on two to three consecutive days as an outpatient procedure with continuous blood flow separator (Cobe Spectra; Denver, CO).

PBSC Collection and Cryopreservation Procedure
The collection of PBSC was performed with a Cobe Spectra using a 250 ml volume (vol) collection chamber. Up to 10 liters of blood per apheresis were processed at a flow rate of 50 to 70 ml/min. A mean vol of 250 ml was collected. This cell suspension was concentrated to a final vol of 50 ml and mixed with the same vol of minimal essential medium containing 20% dimethylsulfoxide. The final 100 ml of harvested product were transferred into freezing bags and frozen to –100°C with a computer-controlled cryopreservation device. The frozen cells were transferred in liquid phase of nitrogen and stored at –196°C. Mononuclear cells (MNC) of BM were separated by Ficoll-Hypaque and stored frozen at –8°C.

Antibodies and Flow Cytometry Analysis
We focused on the integrins CD49e as alpha-chain of VLA-5 (CD29/CD49e), CD49d as alpha-chain of VLA-4 (CD29/CD49d) and CD11a as alpha-chain of LFA-1 (CD11a/CD18). In the immunoglobulin family we analyzed CD54 (ICAM-1) as ligand of LFA-1 [10] and CD58 as ligand of LFA-3 and CD31 (platelet-endothelial cell adhesion molecule-1). Within the selectin family we analyzed CD62L (L-selectin) and CD117 (c-kit).

Fluorescein isothiocyanate (FITC)-labeled IgG and phycoerythrin (PE)-labeled IgG2a (Becton-Dickinson; San Jose, CA) were used as controls. PE-labeled CD58 (Becton-Dickinson), CD54 (Becton-Dickinson), CD117 (Immunotech; Germany), CD62L (Becton-Dickinson) and FITC-labeled CD49e (Immunotech), CD49d (Immunotech), CD11a (Immunotech), CD 31 (Pharmingen; San Diego, CA) as well as PE-Cy5-labeled CD34 (Immunotech) were applied.

After thawing, 1 x 106 MNC were incubated for 30 min at 4°C in the dark with PE-Cy5 conjugated monoclonal antibody (mAb) anti-CD34, and FITC- and PE-conjugated antibodies of different adhesion molecules. The cells were analyzed with a three-color FACScan flow cytometer (Becton-Dickinson) and LYSIS II software. Forward scatter characteristics versus CD45 fluorescence dot plot were used to discriminate between hematopoietic cell population and erythrocytes or debris. The CD34+ cell population was gated in a fluorescence (FL-3) versus side scatter characteristics dot plot. The adhesion molecule expression on CD34+ cells was examined on cells acquired in this FL-3/side scatter gate. The percentage of positive cells was corrected for negative control. At least 1,000 events in the CD34+ gate were counted.

Statistical Analysis
For analysis, WINSTAT (Kalmia Co., Inc.; Cambridge, MA) software was used. For the analyzed non-normal values, data were summarized by means and standard error of the mean. Differences were determined according to the Student's t-test for paired samples. A p value lower than alpha = 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
There were considerable interindividual differences of adhesion molecule expression on CD34+ cells of PBSC and BM. Of the ß1-integrins, CD49e was less expressed than CD49d with a slightly higher expression in PBSC than in BM. CD49d expression was found in 70.8% of the CD34+ cells in BM, but in only 43.6% of the CD34+ cells in PBSC (p = 0.012). The mean ß2-integrin CD11a expression was 38.7% in PBSC and 31.1% in BM CD34+ cells. Selectin CD62L (leukocyte-endothelial cell adhesion molecule) was weakly expressed on CD34+ cells in PBSC (13.8%) and BM (16.4%) without significant differences ( Table 2, Fig. 1).


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  		Table 2. Expression of adhesion molecule in BM before mobilization and PB after stimulation with G-CSF
 


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  		Figure 1. Comparison between median expression (%) and SE of adhesion molecules in bone marrow (BM) and leukapheresed product (LP).

 
The immunoglobulin CD58 was higher, but not significantly, expressed on CD34+ cells in BM (90.6%) than on PBSC (84.8%). CD31 was found to be more highly expressed on PBSC (81.1%) than on BM CD34+ cells (65.6%). A lower expression rate was found for CD54 with similar results in BM (19.5%) and PBSC (21.9%) ( Fig. 2). The expression rate of CD117 (c-kit), which is the ligand of the stem cell factor (SCF), was slightly higher expressed on BM than on PBSC CD34+ cells (21.6% versus 18.1%).



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  		Figure 2. Comparison between mean expression and SE of the immunoglobulins CD54, CD58, CD31 and CD117 (c-kit) on CD34+ cells in bone marrow (BM) and leukapheresed product (LP).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present work confirmed the results of other groups showing that the adhesion molecules on CD34+ cells in BM and mobilized PB cells are expressed at different levels and intensity. We found a lower expression of CD117, CD62L, CD49d and CD58 on G-CSF-mobilized CD34+ cells than on steady-state CD34 cells of BM. The differences, however, were only statistically significant for CD49d. Turner et al. [22] and Dercksen et al. [9] also demonstrated a lower expression on mobilized CD34+ cells in PB for CD49d and CD58. Turner et al. also found a significantly lower expression for CD49e in PB, but only concerning the expression rate and not with regard to the fluorescence intensity. The importance of CD49d in mobilizing stem cells is supported by animal experiments indicating that the blocking of CD49d with mAb mobilizes stem cells into PB [23]. Recently, a functional adherent assay showed that a higher expression of CD49d was found on adherent CD34+ cells, but that the addition of blocking antibodies for alpha-4 integrins did not inhibit adhesion [24]. In the present analysis no significant difference was detected between expression of CD11a. Recently Möhle et al. [24] found a significantly higher expression of CD11a on CD34+ cells in BM than in PBSC. The difference between their results and ours might be explained by timing of BM aspiration and the kind of mobilizing regime: in our study, samples from BM were obtained before stimulation, and the same mobilization regime was used in all patients. A possible role of CD11a and CD18 during mobilization was described by Gunji et al. [25], who used anti-CD11a and anti-CD18 antibodies in a coculture system of CD34+CD33– cells with stromal layers. They found a significantly decreased number of generated colony forming units compared with a control.

The weaker expression of CD49d or CD58 might facilitate the migration of BM HPCs into PB, but the role of G-CSF in this regulative process remains unclear. Unfortunately, samples of BM after stimulation with G-CSF were not available. The fact that stimulation with G-CSF does not induce an immediate increase of CD34+ cells suggests a more complex interaction of several cytokines in the BM microenvironment. In this respect, a candidate cytokine could be CD117: in our study CD117 expression on G-CSF- stimulated CD34+ cells was only marginally weaker than in CD34+ cells of BM. To et al. [26] found a higher expression of CD117 on CD34+ cells in BM than in PB after stimulation with G-CSF or chemotherapy. Two investigations have demonstrated a pivotal role of SCF in stem cell mobilization; Cynshi et al. [27] showed that G-CSF is less effective in mobilizing progenitor cells in manipulated mice with defective c-kit receptors than in healthy mice. It has also been demonstrated that CD34+CD117– cells contained fewer assayable progenitor cells in long-term BM cultures than CD34+CD117+ cells [28]. It is possible that this lower expression of CD117 also reflects the lineage commitment of the cells [17].

Another possible mechanism of stem cell mobilization could be alterations of the sinusoidal endothelium or the BM stroma as could be shown for chemotherapy [29, 30]. Moore [31] postulated that the mobilization of progenitor cells from the BM results from increased cell proliferation leading to an egress from BM to PB.

We conclude that regulative expression of adhesion molecules on CD34+ cells is likely to be involved in mobilizing stem cells from BM into PB after treatment with G-CSF. The cytokines of the BM microenvironment and the kinetic of adhesion molecule expression on CD34+ cells during mobilization remain to be determined for a better understanding of the mechanisms responsible for mobilization of HPCs.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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  10. Marlin SD, Springer TA. Purified intercellular adhesion-molecule-1 (ICAM1) is a ligand for lymphocyte function associated antigen-1 (LFA-1). Cell 1987;59:813-819.

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  14. Lewinsohn DM, Nagler A, Ginzton N et al. Hematopoietic progenitor cell expression of the H-CAM (CD44) homing associated adhesion molecule. Blood 1990;75:589-595.[Abstract/Free Full Text]

  15. Kansas GS, Muirhead MJ, Dailey MO. Expression of the CD11/CD18 leucocyte adhesion molecule 1, and CD44 adhesion molecules during normal myeloid and erythroid differentiation in humans. Blood 1990;76:2483-2492.[Abstract/Free Full Text]

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  17. Gunji Y, Nakamura M, Osawa H et al. Human primitive hematopoietic progenitor cells are more enriched in KIT-low cells than KIT-high cells. Blood 1993;82:3283-3289.[Abstract/Free Full Text]

  18. Ryan DH, Nuccie BL, Abboud CN et al. Vascular cell adhesion molecules-1 and the integrin VLA-4 mediate adhesion of human B cell precursors to cultured bone marrow adherent cells. J Clin Invest 1991;88:995-1004.

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  20. Möhle R, Haas R, Hunstein W. Expression of adhesion molecules and c-kit on CD34+ hematopoietic progenitor cells— comparison of cytokine-mobilized blood stem cells with normal bone marrow and peripheral blood. J Hematother 1993;2:483-489.[Medline]

  21. Saeland S, Duvert V, Caux C et al. Distribution of surface-membrane molecules on bone marrow and cord blood CD34+ hematopoietic cells. Exp Hematol 1992;20:24-33.[Medline]

  22. Turner ML, McIlwaine K, Anthony RS et al. Differential expression of cell adhesion molecules by human hematopoietic progenitor cells from bone marrow and mobilized adult peripheral blood. STEM CELLS 1995;13:311-316.[Abstract]

  23. Papayannopoulou T, Nakamoto B. Peripheralization of hematopoietic progenitors in primates treated with anti-VLA-4 integrin. Proc Natl Acad Sci USA 1993;90:9374-9378.[Abstract/Free Full Text]

  24. Möhle R, Murea S, Kirsch M et al. Differential expression of L-selectin, VLA-4, and LFA-1 on CD34+ progenitor cells from bone marrow and peripheral blood during G-CSF-enhanced recovery. Exp Hematol 1995;23:1535-1542.[Medline]

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  31. Moore MAS. Clinical implications of positive and negative hematopoietic stem cell regulators. Blood 1991;78:1-19.[Free Full Text]

accepted for publication on September 25, 1997.




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