Original Paper International Journal of Cell Cloning 8:357-367 (1990)

Human G-CSF Produced by Adherent Cells in the Presence of Human Recombinant GM-CSF Hiroshi Hara", Yoshihiro Okada', Tetsuo Kbhriya", Mitsuo Namiki", Mahito Misawa", Yokiko Ohe", Shunro Kai', Sumire Ando', Rie kkbabab, Masatoshi kbhsaki', Yoshihiro Yamamoto: Junichi Furuyama' *Division of Blood Transfusion, bThe Second Department of Internal Medicine, 'The Department of Genetics, Hyogo College of Medicine, Hyogo, Japan

Key Mbrds. GMCSF


Adherent cells


G r a n d ~ - m a c r o p h a g colony e

Abstract. Adherent cells (MCs) in blood from normal volunteers produced granulocyte-macrophage (GM) colony-stimulating activity (CSA) in the presence of 10 nglml of recombinant human granulocyte-macrophagecolony-stimulating factor (rhGM-CSF) in vitro. GM-CSA produced by adherent cells in the presence of GM-CSF reached a plateau level on day 6. Colonies stimulated by adherent cell-conditioned medium (MC-CM-GM-CSF), which had been harvested after 6 days of incubation of AdCs with rhGM-CSF, were granulocyte predominant. When phagocyte-depleted marrow mononuclear cells (PD-M-MNCs) were cultured with AdC-CM-GM-CSF and anti-rabbit serum against rhGM-CSF, 99%of the colonies on day 7 were exclusively composed of neutrophils. When 2 x lo4PD-M-MNCs were cultured in a medium containing MC-CM-GM-CSF, MC-CM-GM-CSF + antiGM-CSF, MC-CM-GM-CSF + anti-G-CSF, or MC-CM-GM-CSF both of the antibodies, the PD-M-MNCs formed (mean rt SD) 100 f 2.0%, 64.3 f 2.5%,38.6 f 0.4%, and 6.0 f 0.4% GM colonies, respectively. Furthermore, northern blot analysis revealed that AdCs incubated with 10 nglml of rhGM-CSF for 6 h expressed much more mRNA of G-CSF than those without the CSF. These data indicated that AdCs in blood produce G-CSF in the presence of GM-CSF.



Recent progress related to hemopoietic growth factors [l-61 makes it possible to obtain the factors as a recombinant product and to analyze the interaction of the factors and cells in hemopoietic organs. Recent research revealed that endothelid cells, stromal cells and macrophagesproduce some kinds of hemopoietic factors when the cells are incubated with lymphokines or monokines, such as tumor necro~




Correspondence: Hiroshi Hara, M.D., Division of Blood Transfusion, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663, Japan. Received February 28, 1990; provisionally accepted March 27, 1990; accepted for publication April 23, 1990. 0737-1454/90/$2.OO/O 0AlphaMed Press

Hara et al.


sis factor (TNF-a) [7, 81, interleukin (IL) 1 [9-131, and macrophage colonystimulating factor (M-CSF) [14, 151. For instance, endothelial cells stimulated by TNF-a produce granulocyte-macrophage CSF (GM-CSF). Canisrra er al. [16] also reported that macrophages stimulated by GM-CSF express the mRNA of the TNF-a gene which stimulates macrophages to produce granulocyte CSF (G-CSF) [7]. Furthermore, our collaborators [17] revealed that a large part of the action of GM-CSF in GM colony formation depends on the co-existence of monocytes. In the present paper, we describe the production of colony-stimulating activity (CSA) from monocytes stimulated by GM-CSF and the characteristics of the CSA.

Materials and Methods GM-CSF Bacterially synthesized recombinant human (rh) GM-CSF obtained from Sumitomo Pharmaceuticals Co. (Osaka, Japan) was used in the present investigation. High performance liquid chromatography (HPLC) showed that the sample of GM-CSF used contained more than 95% rhGM-CSE The limulus test revealed that endotoxin was less than 1 ng/mg protein of the sample. The specific activity was 1 X lo8U/mg; 50 U are defined as the amount of material inducing 50% of maximal proliferation of human chronic myeloid leukemic cells. G-CSF rhG-CSF synthesized by CHO cells was obtained from Kirin-Amgen (Maebashi, Gumma, Japan). Mononuclear Cells (MNCs),Adherent Cells (AdCs), andNon-Adherent Cells (NAdCs) Buffy-coat samples (100 ml) were collected with informed conSent from healthy volunteers using a blood processor HM-30 (Haemonetics, Natick, MA). MNCs were obtained from the buffycoat samples using density separation techniques with Ficoll-sodium metrizoate solution (Lymphoprep; Nyegaad & Co., Oslo, Norway) as described by Boyum [18]. These were separated into AdCs and NAdCs according to the method described by Kumagai et al. [19]. Briefly, 1 X lo6I d of MNCs were incubated in 100 mm X 20 mm plastic dishes (Corning, via Iwaki Glass, Tokyo, Japan) which were coated with fetal calf serum (FCS; Irving Scientific Co., Santa Ana, CA) containing Iscove’s modified Dulbecco’s medium (IMDM; Flow Laboratories, Inc., Rockville, MD), and 10% FCS and were incubated overnight in a humidified atmosphere of 5% C o t in air at 37°C. After 24 h incubation, NAdCs were harvested after the dishes had been gently rotated. After washings and incubation of AdCs with Ca-free phosphate-buffered saline (Wako Junyaku Co., Osaka, Japan) containing 0.2% EDTA-2Na for 2 h, the detached AdCs were harvested [19]. Samples of AdCs accounted for more than 90% of monocytes as a-naphthyl butylate esterase-positive cells were used as AdCs in the present experiments. Analysis of the AdCs with a flow cytometer, Spectrum III (Ortho Diagnostics Japan, Tokyo, Japan), revealed that AdCs were composed of 92.9% (91.1-94.6%)macrophages as Leu M3-positive cells (CD14; Becton-Dickinson Overseas, Tokyo, Japan), 4.1% (3.943%) B cells as B1’ cells (CD20; Coulter Electronics, Hialeah, FL) and 3.0%(2.5-4.6%) null cells. T cells as OKTll+ cells (CD2; Ortho Diagnostics Japan) were not detected.

G-CSF Produced by Adherent Cells with GM-CSF


Conditioned Medium (CM) MNCs, AdCs, and NAdCs from blood were incubated for 6 days with various concentrations of rhGM-CSF or rhG-CSF at a concentration of 1 X 106/mlunder the same conditions described before unless otherwise specified. The supernatant was harvested after centrifuging at 4,500 g for 20 min and stored at -80°C as CM in small aliquots until use.

M u m Cells Marrow MNCs (M-MNCs) were obtained from healthy volunteers with their informed consent; they were harvested using density separation techniques [MI. M-MNCs were incubated at a concentration of 1 x lo6 cells/ml with an equal volume of silica particle suspension (Nihonkohtai Kenkyujo, Takasaki, Gumma, Japan) to deplete phagocytes. Phagocyte-depleted M-MNCs (PD-M-MNCs) were harvested from the interface after centrifugation of the M-MNCs using the density separation techniques [ H I . PD-M-MNCs were used as target cells of some CSF and CM. Briefly, 2 x lo4PD-M-MNCs were cultured in IMDM containing 0.8%methylcellulose, 10% FCS, and 10% volume of CM with or without antibodies against rhGM-CSF and/or rhG-CSF under the same conditions previously described. Cell clusters containing more than 20 cells were scored as colonies. Cell Analysis of Individual Colonies Individual colonies were picked up with capillaries (Becton-Dickinson Overseas) and smear samples wece prepared, using a Cytospin-typeapparatus developed by Dr. Takahi Nagasawa [20] (Osaka City University, Osaka, Japan). The samples of individual colonies were subjected to triple staining according to the method described by Phillips et al. [21]. Cells stained with a-naphthyl butyrate esterase were scored as macrophages, those with naphthol AS-D chloroacetate as neutrophils, and those with lux01 fast blue as eosinophils. Antisera Against G-CSF or GM-CSF Rabbit antisera against rhG-CSF and rhGM-CSF were obtained from Kirin-Amgen and Sumitomo Pharmaceuticals Co., Ltd. (Osaka, Japan), respectively. The specificity of both antisera were confirmed by GM colony formation in the presence of M-CSF (Cetus, Emeryville, CA), rhG-CSF and rhGM-CSF. Cellular RNA of AdCs Total cellular RNA was extracted according to the method previously described [22]. AdCs incubated with or without rhGM-CSF or rhG-CSF for 6 h were lysed in guanidium isothiocyanate, and total celluar RNA was recovered by centrifugation through cesium chloride. RNA samples (15 pg) were then electrophoresed on a 1.2% agarose gel with 6%formaldehyde and blotted onto nitrocellulose membranes (Bio-Rad, Richmond, VA). G-and GM-CSF messages were detected by specific cDNA probes (generously provided by Kenichi Arai, DNAX, Palo Alto, CA). Hybridization was performed for 20 hat 60°C in a solution containing 1 M NaCI, 1% sodium dodecyl sulfate (SDS), 10% dextran sulfate, 100 pglml salmon sperm DNA and 1 X lo6 cpm/ml of the cDNA nick translated. The membrane was washed with 2X standard saline citrate (SSC)/I% SDS for 1 h at 65°C and 0.1 x SSC at morn temperature for 1 h (1X SSC = 0.15 M Na citrate, pH 7.0). The blot was then dried and exposed to RX X-ray film (Fuji Photo Film Co., Ltd., Minami Ashigara, Kanagawa, Japan) with an intensifying screen. Statistical Analysis Significant levels for comparison between treatment groups were determined using the Student’s t test.

Ham et al.


Results lime-Course Studies on CSA in AdC-CM Figure 1 shows that CSA in AdC-CM incubated with or without 10 ng/ml of rhGM-CSF gradually increased and reached a peak on day 6. CSA produced in the AdC-CM without any CSA gradually increased and stimulated GM colonyforming units (CFU-gm) in 2 x lo4 PD-M-MNCs to form 120 f 11 (mean f SD) GM colonies. CSA of AdC-CM with 10 n g / d of rhGM-CSF reached a plateau on day 6, and this stimulated 530 f 18 (mean f SD of 3 experiments) GM colonies from 2 x lo4 PD-M-MNCs. litration Studies of GM-CSFon CSA in AdC-CM, m C - C M and N-AdC-CM Figure 2 shows that CSA was produced in AdC-CM, MNC-CM, and NAdCCM incubated with various concentrations of rhGM-CSF (Fig. 2A) or rhG-CSF (Fig. 2B). When PD-M-MNCs were cultured in medium containing 10% (v/v) of each CM to assay CSA in the CM, AdC-CM with 10 n g / d of rhGM-CSF (AdCCM-GM-CSF) exhibited the highest CSA. CSA in the AdC-CM-GM-CSF reached a peak which was significantly higher than that in MNC-CM and NAdC-CM with rhGM-CSF. The AdC-CM with rhG-CSF exhibited approximately the same CSA of 1 ng/ml of rhGM-CSF. CSA in NAdC-CM incubated with 10 ng/ml of rhGMCSF was less than that in NAdC-CM with rhG-CSF. When 1 x 10Vml of AdCs, MNCS, and NAdCs from blood were incubated with various concentrations of rhGM-CSF, CSA in AdC-CM had increased and reached a peak at 10 ng/ml of rhGM-CSF, as shown in Figure 2A. CSA in NAdC-CM also increased gradually, but remained the lowest among them. As demonstrated in Figure 2B, AdCs endogenously produced some CSA, but PD-MNCs did not produce any detectable CSA. CSA detected in PD-MNC-CM was due to rhGM-CSF or rhG-CSF added to the CM. Cellular Component in GM Colonies Analysis of the cellular components in an individual colony revealed that 66% and 75% of the GM colonies in the presence of AdC-CM (with and without 10 ng/ml of rhGM-CSF, respectively) were composed solely of neutrophils (Table I). Both neutrophils and macrophages were present in 18% and 25% of the GM colonies formed under stimulation of AdC-CM with or without rhGM-CSF, respectively. When PD-M-MNCs were cultured with AdC-CM-GM-CSF anti-rabbit serum against rhGM-CSF, 99% of the GM colonies were exclusively composed of neutrophils. The results from morphological analysis were very similar to those found in the presence of 10 ng/ml of rhG-CSF. Figure 3 shows that antisera against rhG-CSF (anti-rhG-CSF) and rhGMCSF (anti-rhGM-CSF) completely abolished CSA of rhG-CSF and rhGM-CSF,


G-CSF Produced by Adherent Cells with GM-CSF


Fig. 1. Representative results of time-course studies on GM-CSA in 1 X 106/mladherent cell- (in circulating MNCs) conditioned medium (MC-CM) harvested after incubation for various days. The culture medium contained 10%of MC-CM. Closed and open circles indicate the numbers of GM colonies stimulated by MC-CM incubated with and without 10 nglml of rhGM-CSF, respectively. Vertical bars indicate f SD.

respectively. AdC-CM was harvested after incubation with 10 ng/ml of rhGM-CSF and 10% of the CM was added to the culture medium for GM-colony formation. Then, 2 x lo4PD-M-MNCs formed 1.590 f 24,756 f 13, and 623 f 14, (mean f SD) GM colonies on day 7. The numbers of GM colonies were used as a control in each experiment, and the data was expressed as % of control. When the PD-M-MNCs were cultured with 10 ng/ml of rhG-CSF, 41.9 f 1.0% (mean f SD) GM colonies were formed. Although the addition of anti-rhGM-CSFto the rhG-CSF had no significantinfluence on GM colony formation,the addition of anti-rhG-CSF to the CSF suppressed GM colony formation to 1.8 f 0.4% (mean f SD; p < 0.001). When the PD-M-MNCs were cultured with 10 ng/ml of rhGM-CSF, GM colony formation came to 38.6 f 0.4% (mean f SD). Addition of anti-rhG-CSF to the rhGM-CSF had no significant influence an GM colony formation, but addition of anti-rhGM-CSF to the rhGM-CSF reduced it to 0% (p < 0.001). When PD-M-MNCs were cultured in the medium containing 10%AdC-CM-GM-CSF and anti-rhGMCSFor anti-rhG-CSF;GM colony brmation significantly decreased to 64.3 f 2.5% (mean f SD;p c 0.01) and 38.6 f 0.4% (mean f SD;p C 0.01), respectively. The addition of both antisera to the culture medium distinctly suppressed GM colony formation to 6.0 f 0.4% (mean f SD; p < 0.001).

Fig. 2. A) Formationof GM colonies derived from PD-M-MNCs in the presence of 10%AdC-CM (-0-0-), MNC-CM (-0-0-) and PD-MNC-CM (---0---0---) stimulated with various concentrationsof rhGM-CSF in the culture medium. B) Formation of GM colonies derived from PD-M-MNCs in the presence of 10%of MC-CM (-O--O-), MNC-CM (-0-0-) and PD-MNC-CM (---0---0---) stimulated with various concentrations of rhG-CSF in the culture medium.

a\ h)


G-CSF Produced by Adherent Cells with GM-CSF


Table I. Morphological analysis of cells in each colony cultured with AdC-CM with or without rhGM-CSF Cells in each colony (%) Conditioned medium AdC-CM without CSF AdC-CM with rhGM-CSF MC-CM with rhGM-CSF anti-GM-CSF rhG-CSF' rhGM-CSFb


No. of colonies analyzed





16 95 87

75 66 99

25 18 1

0 12 0

0 4 0

52 78

93 54

7 30

0 8

0 8

'PD-M-MNCs were cultured in the medium containing 10 ng/ml of rhG-CSF. bPD-M-MNC~were cultured in the medium containing 10 ng/ml of rhGM-CSF. After 6 days incubation of AdCs at 1 X 106/ml with or without rhGM-CSF (10 ng/ml), MC-CM was harvested. n = neutrophil; nm = neutrophil-macrophage; m = macrophage; e = eosinophil

Northern Blot Analysis Northern blot analysis revealed that AdCs incubated with 10 ng/ml of rhGM-CSF for 6 h expressed more mRNA of the G-CSF gene than those without the CSF, and that AdCs incubated with 10 ng/ml of rhG-CSF for 6 h expressed no increase of mRNA of G-CSF gene.

Discussion Generally, GM-CSF is considered to be one of the important hemopoietic growth factors [23-261that can induce proliferation and differentiationof not only GM progenitors, but also progenitors of erythroid [26], megakaryocyte [27, 281 and multi-lineages [29,30]. Recent reports have also revealed that GM-CSF stimulates monocytes to secrete M-CSF [31] and TNF-a [16], and neutrophils to enhance phagocytosis of bacteria [32] and production of superoxide anion [33]. The existence of macrophage-producingCSA was suggested more than 10 years ago [34-361. Recent reports have revealed that macrophages stimulated by IL-I [9-Ill, GM-CSF [14-161, TNF-a [7, 81, or CSF-1 [l5] produce or secrete GM-CSF, TNF-a, G-CSF, and interferon, respectively. Furthermore, macrophages produce IL-1 [37,38]. In order to elucidate the influence of IL-1 by macrophages on CSA production of macrophages stimulated with rhGM-CSF, IL-la and IL-10 in AdC-CM with rhGMCSA were assayed. The concentrationsof IL-la and IL-10 were less than 1 ng/ml of AdC-CM (data not shown) and insufficient to stimulate macrophages producing detectable CSA. In this paper, we described the characteristics of the CSA produced by AdCs in the presence of rhGM-CSF. In order to identify the CSA-producing cells, AdC, NAdC, and MNC from


Hara et al.

Fig. 3. Influence of the MC-CM and antiserum against rhGM-CSF and rhG-CSF on GM colony formation. PD-M-MNCs at a concentration of 2 X 104/mlwere cultured in 1.0 ml of CM containing IMDM, 0.1 ml of FCS and (1) 10 ng of rhG-CSF + rabbit serum (R-serum), (2) 10 ng of rhG-CSF + rabbit antiserum against rhGM-CSF (anti-GMCSF), (3) 10 ng of rhG-CSF rabbit antiserum against rhG-CSF (anti-G-CSF), (4) 10 ng of rhGM-CSF + rabbit-serum, (5) 10 ng of rhGM-CSF + anti-GM-CSF, (6) 10 ng of rhGM-CSF + anti-G-CSF, (7)100 pl of MC-CM with rhG-CSF + rabbit serum, (8) 100 pl of MC-CM with rhG-CSF + anti-GM-CSF, (9) 100pl of AdC-CM with rhG-CSF + anti-G-CSF, (10) 100 pI of MC-CM with rhGM-CSF rabbit serum, (11) 100 pl of MC-CM with rhGM-CSF anti-GM-CSF, (12) 100 pl of MC-CM with rhGM-CSF + anti-G-CSF, and (13) 100 pl of AdC-CM with rhGM-CSF anti-GM-CSF + anti-G-CSF. When MC-CM with 10 ng/ml of rhGM-CSF was added to the CM for 2 X lo4 PD-M-MNCs formed 1,590 f 24,756 f 13, and 623 k 14 GM colonies (mean f SD; quadruplicate plates of 3 experiments). The numbers were expressed 100% in each experiment and used as a control. *Numbers in parentheses indicate the concentration of rhG-CSF or rhGM-CSF calculated from the concentration of rhG-CSF or rhGM-CSF added to the medium for MC-CM.



+ +

blood were incubated with rhGM-CSF, and their supernatant CM was harvested after incubation for different periods. The highest CSA was always found in AdC-CM and the lowest in NAdC-CM. The present results also support the hypothesis that AdCs in blood produce another type of CSA in the presence of rhGM-CSF. Morphological analysis of individual colonies revealed that the percentages of neutrophil, neutrophil-macrophage,macrophage and eosinophil colonies stimulated by AdC-CM containing 10 ng/ml of rhGM-CSF were similar to those directly

G-CSF Produced by Adherent Cells with GM-CSF


stimulated by rhG-CSF, rather than those stimulated by rhGM-CSF. Also, the percentages of morphological types in the colonies stimulated by AdC-CM with rhGM-CSF were similar to those of rhG-CSF when rabbit antiserum against rhGM-CSF was added to the AdC-CM. The data strongly suggest that the CSA in AdC-CM produced in the presence of rhGM-CSF was due to G-CSF. Our experiments on the effect of antisera against CSFs in GM colony formation revealed that the antisera against rhGM-CSF and rhG-CSF almost completely suppressed GM colony formation on day 7 by rhGM-CSF and rhG-CSF, respectively. Although CSA in AdC-CM produced in the presence of rhGM-CSF was partly suppressed by anti-rhGM-CSF or anti-rhG-CSF, the CSA was almost completely abolished by both antisera, and the GM colony formation was reduced to 6.0 f 0.4%. Furthermore, AdCs incubated with rhGM-CSF for only 6 h expressed more mFWA of G-CSF gene than those without rhGM-CSF. The present results completely agree with the hypothesis that mononuclear AdCs stimulated by rhGM-CSF produce G-CSF in the CM not only at the protein level, but also at the mRNA level. Recent in vivo studies of primates [38], patients with acquired immunodeficiency syndrome (AIDS)[39] and patients with myelodysplasticsyndrome [a] revealed that administration of rhGM-CSF induces a significant increment of leukocytes in blood. The present results suggest that not only a direct effect of rhGM-CSF, but also of rhG-CSF produced by monocytes (macrophages)exposed to rhGM-CSF may play an important role in neutrophil production in vivo.

Acknowledgments We gratefully acknowledge the supply of rhG-CSF and antiserum against rhG-CSF from Kirin-Amgen (Tokyo, Japan), rhGM-CSF and antiserum against rhGM-CSF from Sumitomo Pharmaceutical Co., Ltd. (Osaka, Japan), cDNA of rhGM-CSF and rhG-CSF provided by Dr. Kenichi Arai (DNAX Research Institute, Palo Alto, CA)and IL-1and rabbit antiserum against IL-1received from Yoshikatsu Hirai (Ohtsuka Pharmaceutical Co., Ltd., Osaka, Japan). This work was supported in part by a grant (60480286)and a Grant-in-Aid for clinical research of hemopoietic stem cells from the Japanese Ministry of Education.

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Human G-CSF produced by adherent cells in the presence of human recombinant GM-CSF.

Adherent cells (AdCs) in blood from normal volunteers produced granulocyte-macrophage (GM) colony-stimulating activity (CSA) in the presence of 10 ng/...
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