Research in
Res Exp Med (1992) 192:245-255
Experimental Medicine 9 Springer-Verlag 1992
Detection of the granulocyte colony-stimulating factor receptor using biotinylated granulocyte colony-stimulating factor: presence of granulocyte colony-stimulating factor receptor on CD34-positive hematopoietic progenitor cells K. S h i m o d a t , S. O k a m u r a 2, N . H a r a d a 1, and Y. N i h o 1
1The First Department of Internal Medicine and ~Cancer Center, Faculty of Medicine, Kyushu University, Fukuoka 812, Japan Received October 12, 1991 / accepted April 12, 1992
Summary. Granulocyte colony-stimulating factor (G-CSF) was linked to NHSbiotin to yield biotinylated G-CSF (b-G-CSF), which retained the ability to stimulate colony formation by normal bone marrow (BM) cells in methylcellulose. The use of streptavidin-phycoerythrin conjugate in conjunction with flow cytometry demonstrated that the binding of biotinylated G-CSF to its receptor is saturable, competitive, and specific. A 100-fold molar excess of unlabeled GCSF almost completely inhibited the binding of the biotinylated G-CSF to the human leukemia cell line U937, which is known to possess the G-CSF receptor. G-CSF receptors were clearly detected by flow cytometry on adult human peripheral granulocytes and monocytes, but not on lymphocytes. Using this method, the expression of G-CSF receptors on hematopoietic progenitor cells in bone marrow and umbilical cord blood, detected as CD34-positive (CD34 +) cells, were examined. A small but significant number of CD34 + cells were detected among the bone marrow mononuclear cells and umbilical-cord-blood mononuclear cells (4.28% + 0.31%, 1.09% _+0.20%, respectively). The percentage of CD34 + BM mononuclear cells was significantly higher than for cord blood mononuclear cells ( P < 0.01). These CD34 + cells were then analyzed by biotinylated G-CSF binding. CD34 + cells from bone marrow contained 25.8% + 7.9% G-CSF receptor positive cells and those from cord blood possessed 29.2% ___7.0% of G-CSF receptor-positive cells. The difference was not statistically significant. Key words: G-CSF - G-CSF receptor - CD34 + cell - Flow cytometry Correspondence to. S. Okamura
246
Introduction G r a n u l o c y t e c o l o n y - s t i m u l a t i n g factor ( G - C S F ) is o n e o f the g l y c o p r o t e i n s p r o d u c e d from m a c r o p h a g e , f i b r o b l a s t , and e n d o t h e l i a l cells. It acts on n o r m a l b o n e m a r r o w to i n d u c e the p r o l i f e r a t i o n a n d d i f f e r e n t i a t i o n of n o r m a l b o n e m a r r o w g r a n u l o c y t i c p r e c u r s o r s [17, 31]. C S F s are b e l i e v e d to act t h r o u g h m e m b r a n e b o u n d r e c e p t o r s on the t a r g e t cells [1.21] a n d G - C S F r e c e p t o r s are r e p o r t e d to b e p r e s e n t on g r a n u l o c y t i c l i n e a g e f r o m the i m m a t u r e to m a t u r e stage, b u t absent on m a t u r e l y m p h o i d o r e r y t h r o i d cells [22]. P r e v i o u s l y , 125I-labeled G - C S F has b e e n u s e d to d e t e c t surface G - C S F r e c e p t o r s on cell-line cells o r e n r i c h e d fractions of m i x e d cell p o p u l a t i o n s [4, 15.27]. H o w e v e r , the r a d i o r e c e p t o r - a s s a y s y s t e m r e q u i r e s m a n y e x a m i n e d cells, m u c h t i m e , a n d it can o n l y a n a l y z e b u l k p o p u l a t i o n s . T h e flow c y t o m e t r i c m e t h o d for d e t e c t i n g G - C S F r e c e p t o r s using b i o t i n y l a t e d G - C S F , which we r e p o r t in this article, c o u l d o v e r c o m e t h e s e p r o b lems. C D 3 4 a n t i g e n was o r i g i n a l l y d e f i n e d by its r e a c t i v i t y with a n t i - M Y 1 0 m o n o c l o n a l a n t i b o d y [5, 32] a n d C D 3 4 - p o s i t i v e ( C D 3 4 - ) cells i n c l u d e h e m a t o p o i e t i c cells a b l e to f o r m c o l o n i e s in vitro [6, 9]. It has b e e n r e p o r t e d that C D 3 4 + cells c o n s t i t u t e a p p r o x i m a t e l y 1 - 4 % of n o r m a l h u m a n b o n e m a r r o w ( B M ) cells, which can be f o u n d in the p e r i p h e r a l b l o o d in c o n t r a s t to initial r e p o r t s [6, 9, 32]. In as m u c h as C D 3 4 + cells f o r m g r a n u l o c y t i c c o l o n i e s in s e m i s o l i d c u l t u r e [9] a n d i n c r e a s e t h e i r n u m b e r s in liquid c u l t u r e [10] in the p r e s e n c e of G - C S F , we s t u d i e d G - C S F r e c e p t o r s on B M a n d u m b i l i c a l c o r d b l o o d C D 3 4 + cells.
Materials and methods
Biotin labeling of recombinant G-CSF Biotin labeling of recombinant G-CSF (rG-CSF) was carried out as follows. In a volume i ml, 250 fig of rG-CSF (Kirin Breweries. Tokyo. Japan) was diluted in 3.5 ml of 0.2 M sodium bicarbonate, pH 8.4, by gel-filtration through a PDI0 column (Pharmacia, Uppsala, Sweden) equilibrated with sodium bicarbonate buffer. Biotinvlation was carried out by addition of 1 rag/ ml biotinyl N-hydroxy succinimide ester (EOY Labs, San Mateo, Calif., USA) in dimethyl sulfoxide (DMSO, Sigma Chemicals, St. Louis. Mo., USA) to yield a final concentration of 0.25 mg/ml. After 4 h of shaking at room temperature, further reaction was carried out at 4~ overnight. Then the unbound reagent was removed using a PD10 column equilibrated with phosphate-buffered saline (PBS) supplemented with 0.1% BSA. Sodium azide was added to the recovered sample at a final concentration of 0.1%. Enzyme-linked immunosorbent assay [26] was used to determine whether the biotinylated form of G-CSF (b-G-CSF) contained biotin, b-G-CSF and unlabeled G-CSF were sequentially diluted in PBS containing 0.1% BSA, and 100 lal of these preparations (corresponding to 3.42500 ng/ml b-G-CSF and G-CSF, respectively) was placed in each well of a 96-welt microplate and incubated at 37~ for 2h to allow binding to the plate. After the plate had been washed with PBS containing 0.05% Tween 20 (PBS-T), it was then incubated with 5% skim milk in PBS for 2 h at room temperature to prevent non-specific binding and then washed again. To each well was added 100gl of streptavidin-horseradish peroxidase conjugate (Bethesda Research Laboratories, Gaithersburg, Mass., USA) and the plate was incubated for 2 h at room temperature. After washing. 75 gl of 3,3', 5,5'-tetramethyl benzidine (Katayama Chemicals, Osaka, Japan) in 0.1 M sodium acetate buffer, pH 5.5, and 25111 of 2 M H202 were added to the wells. After 20 rain, the coloring reaction was stopped by adding 25 gl of 2 M H2804. The optical density at 450nm was measured using a Titertek Multiskan plate reader (Flow Lab, McLean, Va., USA).
247
Biological assay An in vitro granulocyte-macrophage colony-forming unit (CFU-GM) assay was carried out to detect the growth-promoting activity of labeled G-CSF before adding sodium azide. The assay procedure has been reported in detail previously [23, 25, 29]. Briefly, 2 • 104 human BM ceils were cultured in 1 ml of Iscove's modified Dulbecco medium (IMDM) supplemented with 20% FCS, 0.88% methylcellulose, and 10 ng of labeled or unlabeled G-CSF in a 35-mm-diameter plastic petric dish (Nunc, Naperville, Ill., USA) at 37~ in a humidified atmosphere of 5% CO2 in air. On day 7, the numbers of C F U - G M colonies consisting of more than 20 cells were counted using an inverted microscope.
Cells and cell line preparations Three human leukemic cell lines, U937, K562, and Rail, provided by the Japanese Cancer Research Promotion Foundation (Tokyo, Japan), were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal-calf serum (FCS) in a humidified atmosphere of 5% CO2 in air at 37~ Human white-blood cells were obtained from healthy volunteers by hemolysing 100~tl whole blood using 3ml hemolysis buffer (155mM ammonium chloride, 10mM potassium hydrogen carbonate, and 0.1 mM disodium ethylenediaminetetraacetate in distilled water) and washed three times with PBS containing 1% FCS. BM cells were obtained from normal healthy volunteers who had given informed consent, and human umbilical cord blood cells were obtained from umbilical cord and placental tissues scheduled for disposal after delivery. Mononuclear cells (MNCs) from BM and umbilical cord blood were separated after density-gradient centrifugation [24].
Fluorescence staining procedure Quantitative fluorescence analysis was performed using a FACScan flow cytometer (Becton Dickinson, Mountain View, Calif., USA). Analysis of G-CSF receptor expression on cells identified by forward- and right-angle light-scatter properties or by green-fluorescence intensity was performed using Consort-30 research software (Becton Dickinson). Detection of G-CSF receptors by flow cytometry was performed on U937 cells, shown previously to possess the presence of G-CSF receptors using human G-CSF 125I-labeled by the lactoperoxidase method [15]. All incubations were performed at 4~ in PBS containing 0.1% BSA and 0.1% sodium azide (binding buffer). To prepare cells for flow cytometry, 25 p3 of a cell suspension of 4 x 106/ml was added to a polystyrene round-bottomed R I A tube (Becton Dickinson) containing various amounts of b-G-CSF in a total volume of 100 I~1. In a competition binding assay, both b-G-CSF and a 100-fold excess of unlabeled G-CSF was added to 1 • 105 cells in a total volume of 100gl. After incubation for 30min at 4~ the cells were washed twice with ice-cold binding buffer and incubated with 10 ng of streptavidin-phycoerythrin (PE) conjugate (Becton Dickinson) for 30 min at 4~ The cells were then washed, and resuspended in binding buffer for flow-cytometric analysis. The background level of fluorescence was established by incubating cells with the second-step streptavidin-PE reagent alone. The cut-off point was set to include 97% of those cells treated with streptavidin-PE alone. The fraction of cells that were shifted to a greater fluorescence intensity after specific ligand binding was taken to represent the percentage of positive cells. For double-staining experiments of G-CSF receptors and CD34 antigens, 5 • 105 cells were incubated with 145 ng of b-G-CSF and 500 ng of HPCA-1 (Becton Dickinson) for 30 min at 4~ After washing, 50ng streptavidin-PE and 500rig of goat anti-mouse immunoglobulin fluorescein conjugate (GAMIg-FITC, Becton Dickinson) were added and then incubated for 30min at 4~
Statistical analysis The ratio of CD34-positive and G-CSF receptor-positive cells were expressed as mean _+ SD, and a comparison of these ratios between BM MNCs and cord-blood MNCs was made by Student's t-test.
248 1.00.8 0.6 Fig. 1, Detection of biotinylated G-CSF. The reaction between b-G-CSF and streptavidin-HRP was dependent on the amount of b-G-CSF added to the well (o 0). By contrast, no reaction was observed between unlabeled G-CSF and streptavidin-HRP (o e)
C) 0.4
0.2 0
10~
101
10 2 b-GCSF
103
10 4
(ng/ml)
Table 1. Effect of biotinylated G-CSF on CFU-GM colony formation CSF used
Number of CFU-GM formed"
Biotinylated G-CSF G-CSF without labeling Medium only
24 _+4.3 20 _+3.8 0
a Per 2 x 104 bone marrow cells plated
Results
Reaction between b- G- CSF an d streptavidin In order to confirm whether b-G-CSF contained biotin capable of binding to streptavidin, we placed serially diluted b-G-CSF in the wells and observed the biotin-streptavidin reaction (Fig. 1). The b-G-CSF and streptavidin-HRP reaction was proportioned to the amount of b-G-CSF added to the well, although unlabeled G-CSF did not react with streptavidin.
Biological activity of labeled G-CSF b-G-CSF retained its ability to stimulate colony formation by normal BM cells in methylcellulose, as shown in Table 1. By this colony formation assay, b-G-CSF appeared to possess full biological activity.
Flow cytornetry Detection of G-CSF receptors by flow cytometry was p e r f o r m e d using U937 cells, which we had previously shown to possess G-CSF receptors using human G-CSF labeled with 125I by the lactoperoxidase method [15]. Saturation binding with b-G-CSF was achieved in less than 30 min, as shown in Fig. 2A. This incubation period was therefore used for the subsequent experiments. Figure 2B shows
249
Fig. 2A, B. Binding curve of biotinylated G-CSF to U937 cells as measured by flow cytometry. A Kinetics of binding of b-G-CSF to U937 cells, b-G-CSF was added to 1 • 105 U937 cells for the indicated times followed by two washes and a final incubation with 10 ng streptavidin-PE for 30rain. All reactions were performed at 4~ After washing, the cells were analyzed for their positivity ratio. B Binding curve of b-G-CSF to U937 cells. Results are expressed as ng b-G-CSF added per 1 • 105 U937 cells versus the percentage of positive cells. Incubation between U937 cells and b-G-CSF was carried out for 30 min
Fig.3A. Expression of G-CSF receptors on U937 cells and B K562 cells as measured by flow cytometry. Three overlapping histograms of cells treated with b-G-CSF followd by streptavidin-PE (1), in the presence of a 100-fold molar excess of unlabeled G-CSF with b-GCSF (2), and streptavidin-PE alone (3). The ordinate indicates cell number per channel, and the abscissa indicates the fluorescence intensity in arbitrary units
t h e d o s e - d e p e n d e n t b i n d i n g o f b - G - C S F to U937 cells. S a t u r a t i o n was a c h i e v e d using 25 ng of b - G - C S F with 1 • 105 cells after 30 rain of i n c u b a t i o n at 4~ F i g u r e 3 A shows t h e f l u o r e s c e n c e profile o f U937 cells in t h e a b s e n c e a n d p r e s e n c e o f a 100-fold excess o f u n l a b e l e d G - C S F u n d e r the e s t a b l i s h e d c o n d i t i o n s . S t a i n e d cells (profile 1 in Fig. 3 A ) s h o w e d a b o u t a 30-fold i n c r e a s e o f f l u o r e s c e n c e intensity c o m p a r e d with t h e c o n t r o l cells, which w e r e s t a i n e d o n l y b y s t r e p t a v i d i n - P E (profile 3 in Fig. 3 A ) . T h e c o m p e t i t i v e b l o c k i n g o f the m a j o r i t y o f t h e staining in
250
Fig. 4A-D. G-CSF receptors on normal adult peripheral white blood ceils. A Dot plot of forward- versus right-angle light scattering by peripheral white-blood cells. Three windows (B, C, and D), indicating granulocytes, monocytes and lymphocytes, were used for analysis of fluorescence data. B-D Cells were incubated with b-G-CSF ( ) or. as controls, with buffer only ( ......... ) or with b-G-CSF in the presence of a 100-fold excess of unlabeled G-CSF ( - - - ) . After 30rain of incubation at 4~ the cells were washed, incubated for 30min at 4~ with streptavidin-PE, washed again, and analyzed bv flow cytometry. Fluorescence was analyzed in the windows for forward- and right-angle light-scatter indicated by the boxes in A the presence of excess unlabeled G - C S F (profile 2 in Fig. 3 A ) d e m o n s t r a t e d the specificity of the m e t h o d . In contrast to U937 cells. G - C S F receptors were not detected on K562 or Raji cells (Fig. 3B), as also observed by r a d i o r e c e p t o r assay [15].
G-CSF receptors on aduh peripheral white-blood cells' G - C S F receptors on n o r m a l adult peripheral white blood cells were also analyzed. The stained cells were analyzed in the gated area of granulocytes (Fig. 4B), m o n o c y t e s (Fig. 4C), or l y m p h o c y t e s (Fig. 4 D ) in electronic screen windows for forward- and right-angle light scatter, indicated by the boxes in Fig. 4 A . T h e resulting fluorescence profiles, shown in Fig. 4B and 4C, indicated that granulocytes and m o n o c y t e s expressed G - C S F receptors and that most of the fluores-
251
Fig. $. SSC and FSC gates for CD34-positive cells (case 3 in Table 1). CD34-positive cells had a relatively narrow range of SSC and a relatively wide range of FSC
Table 2. Positivity ratio of CD34 antigen and G-CSF receptor on CD34-positive cells Case
Sample
CD34 +
G-CSF receptor
1 2 3 4 5 6 7 8 9
BM BM BM CB CB CB CB CB CB
3.94 4.55 4.35 1.21 1.10 1.28 0.81 0.88 1.26
25.8 18.0 33.7 31.3 25.7 41.0 19.8 27.8 29.4
Mean
BM CB
4.28 _+ 0.31 1.09 _+ 0.20
25.8 + 7.9 29.2 + 7.0
(%)
(%)
BM, Bone marrow; CB, cord blood
cence was a b r o g a t e d in the p r e s e n c e o f excess u n l a b l e d G - C S F with b - G - C S F . B y c o n t r a s t , no G - C S F r e c e p t o r was d e t e c t e d on l y m p h o c y t e s (Fig. 4 D ) in t h e " D " w i n d o w o f Fig. 4 A . G - C S F receptors on C D 3 4 + cells
B M m o n o n u c l e a r cells ( M N C s ) a n d c o r d - b l o o d M N C s w e r e s t a i n e d with b - G CSF and HPCA-1 followed by streptavidin-PE and GAMIg-FITC secondary. F i g u r e 5 shows t h e l i g h t - s c a t t e r i n g p r o p e r t i e s of C D 3 4 § cells. C D 3 4 § cells h a d a r e l a t i v e l y n a r r o w r a n g e o f side s c a t t e r i n g (SSC) a n d a r e l a t i v e l y wide r a n g e o f f o r w a r d s c a t t e r i n g ( F S C ) . W e set the g a t e using t h e s e r a n g e s of SSC a n d F S C
252 100-
~6 c~
E c 0~ >
k
D
Res Exp Med (1992) 192:245-255
Experimental Medicine 9 Springer-Verlag 1992
Detection of the granulocyte colony-stimulating factor receptor using biotinylated granulocyte colony-stimulating factor: presence of granulocyte colony-stimulating factor receptor on CD34-positive hematopoietic progenitor cells K. S h i m o d a t , S. O k a m u r a 2, N . H a r a d a 1, and Y. N i h o 1
1The First Department of Internal Medicine and ~Cancer Center, Faculty of Medicine, Kyushu University, Fukuoka 812, Japan Received October 12, 1991 / accepted April 12, 1992
Summary. Granulocyte colony-stimulating factor (G-CSF) was linked to NHSbiotin to yield biotinylated G-CSF (b-G-CSF), which retained the ability to stimulate colony formation by normal bone marrow (BM) cells in methylcellulose. The use of streptavidin-phycoerythrin conjugate in conjunction with flow cytometry demonstrated that the binding of biotinylated G-CSF to its receptor is saturable, competitive, and specific. A 100-fold molar excess of unlabeled GCSF almost completely inhibited the binding of the biotinylated G-CSF to the human leukemia cell line U937, which is known to possess the G-CSF receptor. G-CSF receptors were clearly detected by flow cytometry on adult human peripheral granulocytes and monocytes, but not on lymphocytes. Using this method, the expression of G-CSF receptors on hematopoietic progenitor cells in bone marrow and umbilical cord blood, detected as CD34-positive (CD34 +) cells, were examined. A small but significant number of CD34 + cells were detected among the bone marrow mononuclear cells and umbilical-cord-blood mononuclear cells (4.28% + 0.31%, 1.09% _+0.20%, respectively). The percentage of CD34 + BM mononuclear cells was significantly higher than for cord blood mononuclear cells ( P < 0.01). These CD34 + cells were then analyzed by biotinylated G-CSF binding. CD34 + cells from bone marrow contained 25.8% + 7.9% G-CSF receptor positive cells and those from cord blood possessed 29.2% ___7.0% of G-CSF receptor-positive cells. The difference was not statistically significant. Key words: G-CSF - G-CSF receptor - CD34 + cell - Flow cytometry Correspondence to. S. Okamura
246
Introduction G r a n u l o c y t e c o l o n y - s t i m u l a t i n g factor ( G - C S F ) is o n e o f the g l y c o p r o t e i n s p r o d u c e d from m a c r o p h a g e , f i b r o b l a s t , and e n d o t h e l i a l cells. It acts on n o r m a l b o n e m a r r o w to i n d u c e the p r o l i f e r a t i o n a n d d i f f e r e n t i a t i o n of n o r m a l b o n e m a r r o w g r a n u l o c y t i c p r e c u r s o r s [17, 31]. C S F s are b e l i e v e d to act t h r o u g h m e m b r a n e b o u n d r e c e p t o r s on the t a r g e t cells [1.21] a n d G - C S F r e c e p t o r s are r e p o r t e d to b e p r e s e n t on g r a n u l o c y t i c l i n e a g e f r o m the i m m a t u r e to m a t u r e stage, b u t absent on m a t u r e l y m p h o i d o r e r y t h r o i d cells [22]. P r e v i o u s l y , 125I-labeled G - C S F has b e e n u s e d to d e t e c t surface G - C S F r e c e p t o r s on cell-line cells o r e n r i c h e d fractions of m i x e d cell p o p u l a t i o n s [4, 15.27]. H o w e v e r , the r a d i o r e c e p t o r - a s s a y s y s t e m r e q u i r e s m a n y e x a m i n e d cells, m u c h t i m e , a n d it can o n l y a n a l y z e b u l k p o p u l a t i o n s . T h e flow c y t o m e t r i c m e t h o d for d e t e c t i n g G - C S F r e c e p t o r s using b i o t i n y l a t e d G - C S F , which we r e p o r t in this article, c o u l d o v e r c o m e t h e s e p r o b lems. C D 3 4 a n t i g e n was o r i g i n a l l y d e f i n e d by its r e a c t i v i t y with a n t i - M Y 1 0 m o n o c l o n a l a n t i b o d y [5, 32] a n d C D 3 4 - p o s i t i v e ( C D 3 4 - ) cells i n c l u d e h e m a t o p o i e t i c cells a b l e to f o r m c o l o n i e s in vitro [6, 9]. It has b e e n r e p o r t e d that C D 3 4 + cells c o n s t i t u t e a p p r o x i m a t e l y 1 - 4 % of n o r m a l h u m a n b o n e m a r r o w ( B M ) cells, which can be f o u n d in the p e r i p h e r a l b l o o d in c o n t r a s t to initial r e p o r t s [6, 9, 32]. In as m u c h as C D 3 4 + cells f o r m g r a n u l o c y t i c c o l o n i e s in s e m i s o l i d c u l t u r e [9] a n d i n c r e a s e t h e i r n u m b e r s in liquid c u l t u r e [10] in the p r e s e n c e of G - C S F , we s t u d i e d G - C S F r e c e p t o r s on B M a n d u m b i l i c a l c o r d b l o o d C D 3 4 + cells.
Materials and methods
Biotin labeling of recombinant G-CSF Biotin labeling of recombinant G-CSF (rG-CSF) was carried out as follows. In a volume i ml, 250 fig of rG-CSF (Kirin Breweries. Tokyo. Japan) was diluted in 3.5 ml of 0.2 M sodium bicarbonate, pH 8.4, by gel-filtration through a PDI0 column (Pharmacia, Uppsala, Sweden) equilibrated with sodium bicarbonate buffer. Biotinvlation was carried out by addition of 1 rag/ ml biotinyl N-hydroxy succinimide ester (EOY Labs, San Mateo, Calif., USA) in dimethyl sulfoxide (DMSO, Sigma Chemicals, St. Louis. Mo., USA) to yield a final concentration of 0.25 mg/ml. After 4 h of shaking at room temperature, further reaction was carried out at 4~ overnight. Then the unbound reagent was removed using a PD10 column equilibrated with phosphate-buffered saline (PBS) supplemented with 0.1% BSA. Sodium azide was added to the recovered sample at a final concentration of 0.1%. Enzyme-linked immunosorbent assay [26] was used to determine whether the biotinylated form of G-CSF (b-G-CSF) contained biotin, b-G-CSF and unlabeled G-CSF were sequentially diluted in PBS containing 0.1% BSA, and 100 lal of these preparations (corresponding to 3.42500 ng/ml b-G-CSF and G-CSF, respectively) was placed in each well of a 96-welt microplate and incubated at 37~ for 2h to allow binding to the plate. After the plate had been washed with PBS containing 0.05% Tween 20 (PBS-T), it was then incubated with 5% skim milk in PBS for 2 h at room temperature to prevent non-specific binding and then washed again. To each well was added 100gl of streptavidin-horseradish peroxidase conjugate (Bethesda Research Laboratories, Gaithersburg, Mass., USA) and the plate was incubated for 2 h at room temperature. After washing. 75 gl of 3,3', 5,5'-tetramethyl benzidine (Katayama Chemicals, Osaka, Japan) in 0.1 M sodium acetate buffer, pH 5.5, and 25111 of 2 M H202 were added to the wells. After 20 rain, the coloring reaction was stopped by adding 25 gl of 2 M H2804. The optical density at 450nm was measured using a Titertek Multiskan plate reader (Flow Lab, McLean, Va., USA).
247
Biological assay An in vitro granulocyte-macrophage colony-forming unit (CFU-GM) assay was carried out to detect the growth-promoting activity of labeled G-CSF before adding sodium azide. The assay procedure has been reported in detail previously [23, 25, 29]. Briefly, 2 • 104 human BM ceils were cultured in 1 ml of Iscove's modified Dulbecco medium (IMDM) supplemented with 20% FCS, 0.88% methylcellulose, and 10 ng of labeled or unlabeled G-CSF in a 35-mm-diameter plastic petric dish (Nunc, Naperville, Ill., USA) at 37~ in a humidified atmosphere of 5% CO2 in air. On day 7, the numbers of C F U - G M colonies consisting of more than 20 cells were counted using an inverted microscope.
Cells and cell line preparations Three human leukemic cell lines, U937, K562, and Rail, provided by the Japanese Cancer Research Promotion Foundation (Tokyo, Japan), were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal-calf serum (FCS) in a humidified atmosphere of 5% CO2 in air at 37~ Human white-blood cells were obtained from healthy volunteers by hemolysing 100~tl whole blood using 3ml hemolysis buffer (155mM ammonium chloride, 10mM potassium hydrogen carbonate, and 0.1 mM disodium ethylenediaminetetraacetate in distilled water) and washed three times with PBS containing 1% FCS. BM cells were obtained from normal healthy volunteers who had given informed consent, and human umbilical cord blood cells were obtained from umbilical cord and placental tissues scheduled for disposal after delivery. Mononuclear cells (MNCs) from BM and umbilical cord blood were separated after density-gradient centrifugation [24].
Fluorescence staining procedure Quantitative fluorescence analysis was performed using a FACScan flow cytometer (Becton Dickinson, Mountain View, Calif., USA). Analysis of G-CSF receptor expression on cells identified by forward- and right-angle light-scatter properties or by green-fluorescence intensity was performed using Consort-30 research software (Becton Dickinson). Detection of G-CSF receptors by flow cytometry was performed on U937 cells, shown previously to possess the presence of G-CSF receptors using human G-CSF 125I-labeled by the lactoperoxidase method [15]. All incubations were performed at 4~ in PBS containing 0.1% BSA and 0.1% sodium azide (binding buffer). To prepare cells for flow cytometry, 25 p3 of a cell suspension of 4 x 106/ml was added to a polystyrene round-bottomed R I A tube (Becton Dickinson) containing various amounts of b-G-CSF in a total volume of 100 I~1. In a competition binding assay, both b-G-CSF and a 100-fold excess of unlabeled G-CSF was added to 1 • 105 cells in a total volume of 100gl. After incubation for 30min at 4~ the cells were washed twice with ice-cold binding buffer and incubated with 10 ng of streptavidin-phycoerythrin (PE) conjugate (Becton Dickinson) for 30 min at 4~ The cells were then washed, and resuspended in binding buffer for flow-cytometric analysis. The background level of fluorescence was established by incubating cells with the second-step streptavidin-PE reagent alone. The cut-off point was set to include 97% of those cells treated with streptavidin-PE alone. The fraction of cells that were shifted to a greater fluorescence intensity after specific ligand binding was taken to represent the percentage of positive cells. For double-staining experiments of G-CSF receptors and CD34 antigens, 5 • 105 cells were incubated with 145 ng of b-G-CSF and 500 ng of HPCA-1 (Becton Dickinson) for 30 min at 4~ After washing, 50ng streptavidin-PE and 500rig of goat anti-mouse immunoglobulin fluorescein conjugate (GAMIg-FITC, Becton Dickinson) were added and then incubated for 30min at 4~
Statistical analysis The ratio of CD34-positive and G-CSF receptor-positive cells were expressed as mean _+ SD, and a comparison of these ratios between BM MNCs and cord-blood MNCs was made by Student's t-test.
248 1.00.8 0.6 Fig. 1, Detection of biotinylated G-CSF. The reaction between b-G-CSF and streptavidin-HRP was dependent on the amount of b-G-CSF added to the well (o 0). By contrast, no reaction was observed between unlabeled G-CSF and streptavidin-HRP (o e)
C) 0.4
0.2 0
10~
101
10 2 b-GCSF
103
10 4
(ng/ml)
Table 1. Effect of biotinylated G-CSF on CFU-GM colony formation CSF used
Number of CFU-GM formed"
Biotinylated G-CSF G-CSF without labeling Medium only
24 _+4.3 20 _+3.8 0
a Per 2 x 104 bone marrow cells plated
Results
Reaction between b- G- CSF an d streptavidin In order to confirm whether b-G-CSF contained biotin capable of binding to streptavidin, we placed serially diluted b-G-CSF in the wells and observed the biotin-streptavidin reaction (Fig. 1). The b-G-CSF and streptavidin-HRP reaction was proportioned to the amount of b-G-CSF added to the well, although unlabeled G-CSF did not react with streptavidin.
Biological activity of labeled G-CSF b-G-CSF retained its ability to stimulate colony formation by normal BM cells in methylcellulose, as shown in Table 1. By this colony formation assay, b-G-CSF appeared to possess full biological activity.
Flow cytornetry Detection of G-CSF receptors by flow cytometry was p e r f o r m e d using U937 cells, which we had previously shown to possess G-CSF receptors using human G-CSF labeled with 125I by the lactoperoxidase method [15]. Saturation binding with b-G-CSF was achieved in less than 30 min, as shown in Fig. 2A. This incubation period was therefore used for the subsequent experiments. Figure 2B shows
249
Fig. 2A, B. Binding curve of biotinylated G-CSF to U937 cells as measured by flow cytometry. A Kinetics of binding of b-G-CSF to U937 cells, b-G-CSF was added to 1 • 105 U937 cells for the indicated times followed by two washes and a final incubation with 10 ng streptavidin-PE for 30rain. All reactions were performed at 4~ After washing, the cells were analyzed for their positivity ratio. B Binding curve of b-G-CSF to U937 cells. Results are expressed as ng b-G-CSF added per 1 • 105 U937 cells versus the percentage of positive cells. Incubation between U937 cells and b-G-CSF was carried out for 30 min
Fig.3A. Expression of G-CSF receptors on U937 cells and B K562 cells as measured by flow cytometry. Three overlapping histograms of cells treated with b-G-CSF followd by streptavidin-PE (1), in the presence of a 100-fold molar excess of unlabeled G-CSF with b-GCSF (2), and streptavidin-PE alone (3). The ordinate indicates cell number per channel, and the abscissa indicates the fluorescence intensity in arbitrary units
t h e d o s e - d e p e n d e n t b i n d i n g o f b - G - C S F to U937 cells. S a t u r a t i o n was a c h i e v e d using 25 ng of b - G - C S F with 1 • 105 cells after 30 rain of i n c u b a t i o n at 4~ F i g u r e 3 A shows t h e f l u o r e s c e n c e profile o f U937 cells in t h e a b s e n c e a n d p r e s e n c e o f a 100-fold excess o f u n l a b e l e d G - C S F u n d e r the e s t a b l i s h e d c o n d i t i o n s . S t a i n e d cells (profile 1 in Fig. 3 A ) s h o w e d a b o u t a 30-fold i n c r e a s e o f f l u o r e s c e n c e intensity c o m p a r e d with t h e c o n t r o l cells, which w e r e s t a i n e d o n l y b y s t r e p t a v i d i n - P E (profile 3 in Fig. 3 A ) . T h e c o m p e t i t i v e b l o c k i n g o f the m a j o r i t y o f t h e staining in
250
Fig. 4A-D. G-CSF receptors on normal adult peripheral white blood ceils. A Dot plot of forward- versus right-angle light scattering by peripheral white-blood cells. Three windows (B, C, and D), indicating granulocytes, monocytes and lymphocytes, were used for analysis of fluorescence data. B-D Cells were incubated with b-G-CSF ( ) or. as controls, with buffer only ( ......... ) or with b-G-CSF in the presence of a 100-fold excess of unlabeled G-CSF ( - - - ) . After 30rain of incubation at 4~ the cells were washed, incubated for 30min at 4~ with streptavidin-PE, washed again, and analyzed bv flow cytometry. Fluorescence was analyzed in the windows for forward- and right-angle light-scatter indicated by the boxes in A the presence of excess unlabeled G - C S F (profile 2 in Fig. 3 A ) d e m o n s t r a t e d the specificity of the m e t h o d . In contrast to U937 cells. G - C S F receptors were not detected on K562 or Raji cells (Fig. 3B), as also observed by r a d i o r e c e p t o r assay [15].
G-CSF receptors on aduh peripheral white-blood cells' G - C S F receptors on n o r m a l adult peripheral white blood cells were also analyzed. The stained cells were analyzed in the gated area of granulocytes (Fig. 4B), m o n o c y t e s (Fig. 4C), or l y m p h o c y t e s (Fig. 4 D ) in electronic screen windows for forward- and right-angle light scatter, indicated by the boxes in Fig. 4 A . T h e resulting fluorescence profiles, shown in Fig. 4B and 4C, indicated that granulocytes and m o n o c y t e s expressed G - C S F receptors and that most of the fluores-
251
Fig. $. SSC and FSC gates for CD34-positive cells (case 3 in Table 1). CD34-positive cells had a relatively narrow range of SSC and a relatively wide range of FSC
Table 2. Positivity ratio of CD34 antigen and G-CSF receptor on CD34-positive cells Case
Sample
CD34 +
G-CSF receptor
1 2 3 4 5 6 7 8 9
BM BM BM CB CB CB CB CB CB
3.94 4.55 4.35 1.21 1.10 1.28 0.81 0.88 1.26
25.8 18.0 33.7 31.3 25.7 41.0 19.8 27.8 29.4
Mean
BM CB
4.28 _+ 0.31 1.09 _+ 0.20
25.8 + 7.9 29.2 + 7.0
(%)
(%)
BM, Bone marrow; CB, cord blood
cence was a b r o g a t e d in the p r e s e n c e o f excess u n l a b l e d G - C S F with b - G - C S F . B y c o n t r a s t , no G - C S F r e c e p t o r was d e t e c t e d on l y m p h o c y t e s (Fig. 4 D ) in t h e " D " w i n d o w o f Fig. 4 A . G - C S F receptors on C D 3 4 + cells
B M m o n o n u c l e a r cells ( M N C s ) a n d c o r d - b l o o d M N C s w e r e s t a i n e d with b - G CSF and HPCA-1 followed by streptavidin-PE and GAMIg-FITC secondary. F i g u r e 5 shows t h e l i g h t - s c a t t e r i n g p r o p e r t i e s of C D 3 4 § cells. C D 3 4 § cells h a d a r e l a t i v e l y n a r r o w r a n g e o f side s c a t t e r i n g (SSC) a n d a r e l a t i v e l y wide r a n g e o f f o r w a r d s c a t t e r i n g ( F S C ) . W e set the g a t e using t h e s e r a n g e s of SSC a n d F S C
252 100-
~6 c~
E c 0~ >
k
D
