Lakemia Rawarch Vol. 15, No. 9, pp. 819-826, 1991. Printedin &eat Britain.
0145-2124191 33.00 + .oo Pergamon Press plc
PRODUCTION OF MACROPHAGE COLONY-STIMULATING FACTOR BY HUMAN LYMPHOBLASTOID CELL LINES* CHIYUKI KAwAsAk&t$ SEIICHIOKAMURA,$SEIJIKONDO,t MUNEO YAMADA§
and
YOSHIYUKI NIHOt tl’he First Department of Internal Medicine; *Cancer Center, Faculty of Medicine, Kyushu University, Fukuoka; and BMorinaga Milk Industry Company Ltd, Kanagawa, Japan (Received 15 January 1991. Revision accepted 25 March 1991)
Abstract-Thein vitro production of macrophage colony-stimulating factor (M-CSF) was studied in 14 human lymphoblastoid cell lines and their lineages were ascertained by surface phenotype analysis. M-CSF gene transcripts were detected in a T-lymphocyte-derived cell line (CCRF-CEM) and 3 Blymphoblastoid cell lines (IM-9, BALL-l, and CCRF-SB) by Northern-blot analysis. The secretion of M-CSF protein into the culture supematant by each cell line was also studied using an enzymelinked immunosorbent assay for M-CSF. The 4 cell lines which expressed the M-CSF gene secreted considerable amounts of M-CSF into their culture supematants, while the 10 cell lines without MCSF gene expression did not do so. The cell lines which constitutively produced M-CSF were then subjected to Southern-blot analysis of the M-CSF gene structure, and all 14 cell lines were examined for infection by the Epstein-Barr virus. Neither structural changes nor amplification of the M-CSF gene were detected, and Epstein-Barr virus infection was found to be not directly related to M-CSF production. Key words: M-CSF,
nosorbent
lymphoblastoid assay, gene expression.
cell lines, Epstein-Barr
immu-
culture supernatants [l&12]. However, the production of M-CSF by lymphoblastoid cell lines without exogenous stimulation has not yet been clearly demonstrated [3,13]. In this article, we report that various human lymphoblastoid cell lines of either Tor B-cell origin can constitutively produce M-CSF.
INTRODUCTION
MACROPHAGE colony-stimulating CSF-1) is one of the hematopoietic
virus, c-fms, enzyme-linked
factor (M-CSF or regulators and its
action is necessary for macrophage progenitor cells to become mature macrophages [l]. Cloning of the cDNA of M-CSF has been performed by both Kawasaki et al. [2] and Wong et al. [3]. M-CSF is known to be produced by activated monocytes [ 11, endothelial cells [4], fibroblasts [5,6], and various tumor cell lines [7-91. Recently, Epstein-Barr virus (EBV)transformed B lymphocytes and a human T-leukemic cell line were reported to release M-CSF into their
MATERIALS
AND METHODS
Preparation of cells and cell culture supernatants
The following 14 lymphoblastoid cell lines were studied; RPM1 8226 [14], HS-Sultan [15], CCRF-SB [16], RAJI [17], BALL-l [18], RAMOS [19], WI-L2 NS [20], IM9 [21], CCRF-CEM [22], MT-1 [23], CCRF-HSB2 [24], TALL-l [ 181, MOLT-3 [25], and MOLT-4 [25]. All cell lines were provided by the Foundation for Promotion of Cancer Research (Tokyo, Japan). Cells were cultured at 37°C in RPM1 1640 medium supplemented with 10% fetal bovine serum (FBS), under a humidified 5% COr atmosphere. The cells were harvested in the logarithmic growth phase and analyzed by flow cytometry using a FACScan (Becton Dickinson, Mountain View, CA). The monoclonal antibodies used to determine the surface antigen profiles included Leu-Sb/CDZ, Leu-4/CD3, Leu-3a/CD4, Leu-l/ CD5, Leu-2a/CD8, Leu-12/CD19, Leu-16/CD20, HLADR, and PCA-1. They were purchased either from Becton Dickinson or from Coulter Immunology (Hialeah, FL). For the measurement of M-CSF levels in culture supernatants, the cells were cultured at a concentration of
* This work was partly supported by Grants-in-Aid for Cancer Research and Scientific Research (Nos 60770949, 02256106, 03670325 and 63015063) from the Ministry of Education, Science and Culture of Japan and from the Fukuoka Anti-Cancer Society. Abbreviations: M, macrophage; G, granulocyte; GM, granulocyte-macrophage; CSF, colony-stimulating factor; EBV, Epstein-Barr virus; FBS, fetal bovine serum; LPS, lipopolysaccharide; ELZSA, enzyme-linked immunosorbent assay; IL, interleukin. Correspondence to: Dr Seiichi Okamura, Cancer Center, Kyushu University Hospital, 3-l-l Maidashi, Higashi-ku, Fukuoka 812, Japan. 819
C. KAWASAKI et al.
820
5 x lo* cells/ml for 3 days and cell-free supernatants were obtained by centrifugation. Human monocytes were separated from the peripheral mononuclear cells obtained from a healthy donor who gave informed consent by adherence to the plastic culture dish. The monocytes were resuspended in RPM1 1640 medium with 10% FBS at 5 X lo5 cells/ml and cultured with 100 ng/ ml of bacterial lipopolysaccharide (LPS; Difco, Detroit, MI) for 2 h. Then cells were harvested by centrifugation to obtain the total RNA for use as a positive control in the Northern-blot analysis. Non-adherent and unstimulated human mononuclear cells, including T and B cells, were used as the negative control for the Northern-blot analysis. Northern-blot analysis
The procedure used for the Northern-blot analysis of human CSF genes has been reported previously [26,27]. Total cellular RNA was isolated from cultured cells by the acid-guanidinium thiocyanate-phenol method [28]. After glyoxalation, 20-ug samples of total RNA were size-fractionated by agarose gel electrophoresis and transferred to nylon membranes (Hybound-N+, Amersham, Arlington Heights, IL). The membranes were then hybridized with the cDNA probes according to the method of Meinkoth et al. [29]. The probes used for the analysis were the cDNA encoding M-CSF [3], kindly provided by the Genetics Institute (Cambridge, MA) and c-fms [30], which was purchased from Oncogene Science Inc. (North Mineola, NY). Radiolabeling of the cDNA probes was performed according to the method of Feinberg et al. [31] and the membranes were washed and autoradiographed with an intensifying screen. Measurement of M-CSF nosorbent assay (ELISA)
levels by enzyme-linked
immu-
Quantitation of M-CSF was performed by ELISA as previously reported [32]. In brief, the assay was based on the dual antibody immunometric sandwich principle, using horse and rabbit polyclonal antibodies against human urinary CSF. The minimum detectable level of M-CSF was 50 pg/ml . Southern-blot analysis
Chromosomal DNA was isolated from cultured lymphoblastoid cell lines and peripheral mononuclear cells by phenol extraction [33] and then digested with the restriction enzymes Hind III or Barn HI (Toyobo, Osaka, Japan). Samples of digested DNA (15 ug) were then subjected to electrophoresis on 0.8% agarose gel and transferred to Hybond-N+ membranes. The membranes were subsequently hybridized with the cDNA probe for M-CSF and autoradiographed as described above. Detection of Epstein-Barr virus infections
Infection of the cell lines with Epstein-Barr virus (EBV) was assessed using an EBV detection kit purchased from Enzo Diagnostics Inc. (New York, NY). In brief, cells fixed on a slide glass were incubated with the biotinylated DNA probe for the EBV genome and heated to 92°C for 2 min. The slide was then slowly cooled to room temperature, allowing the DNA probe to anneal with the viral DNA. Avidin-biotinylated horseradish peroxidase was then added to the slide and incubation was performed for 15 min. After being washed, the slide was soaked for 10 min at room temperature in a chromogenic substrate solution consisting of aminoethylcarbazole and hydrogen peroxide
in acetate buffer, and then counterstained with fast green. EBV-infected cells were then detected by microscopy as those containing a red-stained substance in their nuclei.
RESULTS Surface phenotypes
of the cell lines
The lineages of the cell lines were ascertained by surface phenotype analysis (Table 1). Eight out of the 14 cell lines (RPM1 8226, HS-Sultan, CCRF-SB, RAJI, BALL-l, RAMOS, WI-L2 NS, and IM-9) were determined to be of B-cell lineage, because the expression of both CD19 and CD20 was detected, except in the case of RPM1 8226, which showed PCA-1 expression. The other 6 cell line cells were determined to be of T-cell origin. TALL-l, MOLT-4, MOLT-3, and CCRF-CEM showed CD2-positivity. MT-l showed the surface phenotype for helper T cells and CCRF-HSB2 expressed CD5, a marker for pan-T lymphocyte. EBV infection of the cell lines The presence of EBV infection in the 14 lymphoblastoid cell lines is also shown in Table 1. Viral genomes were detected as red dots in the nuclei of 5 cell lines, including RPM18226, HS-Sultan, CCRFSB, RAJI, and IM-9. Northern-blot
analysis
Transcripts of the M-CSF gene were detected in 4 cell lines (CCRF-SB, BALL-l, IM-9, and CCRFCEM) out of the 14 cell lines examined (Fig. 1). The size of the transcripts was 4.0 kb, which was the same as the transcript detected in LPS-stimulated and other transcripts were not macrophages, detected. No M-CSF transcripts were detected in unstimulated normal human lymphocytes. In addition to M-CSF gene expression, we investigated the expression of the c-fms oncogene in the 14 cell lines. By Northern-blot analysis, c-fms transcripts were not detected in any of the cell lines (Fig. 1). Production
of M-CSF
M-CSF levels in the culture supernatants of the 14 cell lines were studied (Table 2). The 4 cell lines which expressed the M-CSF gene secreted significant amounts of M-CSF into their culture supernatants, while the 10 cell lines which did not express the MCSF gene secreted no M-CSF protein. Southern-blot
analysis
Using 2 kinds of restriction enzymes, Southernblot analysis was carried out to investigate whether or not genetic changes were involved in the mechanism of M-CSF production by these cell lines. As
FIG. 1. Northern-blot analysis of M-CSF and c-fms in the various cell lines. Six T-cell lines (a) and 8 B-cell lines (b) were analyzed. RNA was extracted from cultured cells and from monocytes stimulated with 100 ng/ml lipopolysaccharide (LPS) for 2 h. An aliquot of total RNA (20 ug) was analyzed with the cDNA probe for M-CSF and rehybridized with the probe for c-fms. Hybridization with a probe for pactin was performed as a control. Mono-LPS, normal monocytes stimulated by LPS.
821
FIG. 2. Southern-blot analysis. DNA from cells producing M-CSF was digested with the indicated restriction enzymes. An aliquot of digested DNA (15 ng) was applied to 0.8% agarose gel, subjected to electrophoresis, transferred to a nylon membrane, and then analyzed with the cDNA probe for M-CSF. PBMC, peripheral blood mononuclear cells.
822
M-CSF production by lymphoid cell lines TABLE 1. SURFACE
PHENOTYPE
823
ANDEPSTEIN-BARR VIRUSINFECTION INHUMANLYMPHOBLASTOID CELLLINES Surface markers (%)
Cell line RPM1 8226 HS-SULTAN CCRF-SB RAII BALL-l RAMOS WI-L2 NS IM-9 MT-1 CCRF-HSB2* TALL-l MOLT-4 MOLT-3 CCRF-CEM
CD2
CD3
CD4
CD8
1 0 1 0 1 0
0 0 10 2 0 2 0 0 tIl
0 0 0 0 2 0 02
:
0 10 1 97 61 89 60
18 14 2 7
87 0 DPt 0 18 DP
1 0 2 0 4 1 23 0 DP 0 2 DP
CD10
CD19
CD20
1 4 0 63 3 0 0 0 1 0 13 25
0 91 57 54 94 71 70 97 4 0 1 3 0 0
24 99 98 94 100 100 99 83 t!l 2 1 1 1
HLA-DR
99 100 100 59 100 100 5 0 7 0 3 0
PCA-1
EBV
95 1
+ + + +
+ -
Surface marker percentages indicate the percentage of cells expressing the surface antigens detected by the various monoclonal antibodies. The nresence or absence of EBV infection is indicated on the extreme right of the table. * 100% positive for CD5. 1 t DP, double-positive for both CD4 and CD8.
shown in Fig. 2, there was no evidence of amplifications or gross structural changes around the MCSF gene in the 4 cell lines expressing M-CSF mRNA when compared with normal human lymphocyte DNA.
DISCUSSION Flow cytometric analysis showed that the 14 cell lines in this study comprised 6 T-lineage and 8 Blineage lines. All the B-cell lines expressed CD19, CD20, or PCA-1. Four of the 6 T-cell lines expressed CD2, while MT-l and CCRF-HSB2 did not do so. As MT-l expressed CD4 and CCRF-HSB2 expressed CD5, both of which are T-lineage markers [34], these two cell lines were also determined to be of the T lineage. Among these 14 cell lines, M-CSF gene expression was detected in lines of both B- and T-cell origin by Northern-blot analysis including CCRF-SB, BALL1, IM-9, and CCRF-CEM. The mRNA detected in all 4 cell lines was 4.0 kb in size, indicating that it was the full-length M-CSF transcript [3]. M-CSF is produced by any kinds of normal cells including activated monocytes [l], endothelial cells [4], and fibroblasts [5,6], as well as by various tumor cells [791. However, its constitutive expression by lymphoid cell lines is comparatively rare. A considerable amount of M-CSF protein was detected in the culture supematants of all 4 cell lines which expressed M-CSF mRNA, indicating that the translational mechanisms were functioning normally
in these cell lines and allowing M-CSF transcripts to be translated to the secretory form of M-CSF protein. Since neither monocytosis nor granulocytosis were observed in the patients from whom these 4 cell lines were established [16,18,21,22], it seems that MCSF production by the original leukemic cells had no effect on the clinical features. However, longestablished cell lines are known to change their characteristics [35,36], so the investigation of fresh leukemic cells will be necessary to determine the exact relation between the clinical features of leukemia and CSF production by the tumor cells. We also examined the expression of c-fms oncogene mRNA to determine whether the cells producing M-CSF also expressed the M-CSF receptor, since the biological effect of M-CSF is mediated through specific binding to its receptor, which is a product of the c-fms oncogene [30]. Northern-blot analysis failed to detect c-fms mRNA in any of the 4 M-CSF-producing cell lines, indicating that M-CSF did not act as an autocrine growth factor for any of these cell lines. Since various CSFs are reported to act as autocrine growth factors [37,38], we also investigated the production of several other CSFs using ELISA or Northern-blot analysis. Analysis of culture supernatants showed that none of the 14 cell lines produced either granulocyte CSF (G-CSF) or granulocyte-macrophage CSF (GM-CSF) , but WI-L2 NS and CCRF-HSB2 produced trace amounts of GM-CSF when stimulated by phorbol myristateacetate (data not shown). In addition, Northern-blot analysis failed to detect interleukin 3 (IL-3) gene transcripts in any of these cell lines (data not shown).
C. KAWASAKI et al.
824
TABLE 2. M-CSF LEVEL IN THE CULTURE SUPERNATANT OF EACH OF THE 14 CELL LINES Cell line RPM1 8226 HS-SULTAN CCRF-SB RAJI BALL-l RAMOS WI-L2 NS IM-9 MT-l CCRF-HSB2 TALL-l MOLT-4 MOLT-3 CCRF-CEM
M-CSF (ng/ml)*
0145-2124191 33.00 + .oo Pergamon Press plc
PRODUCTION OF MACROPHAGE COLONY-STIMULATING FACTOR BY HUMAN LYMPHOBLASTOID CELL LINES* CHIYUKI KAwAsAk&t$ SEIICHIOKAMURA,$SEIJIKONDO,t MUNEO YAMADA§
and
YOSHIYUKI NIHOt tl’he First Department of Internal Medicine; *Cancer Center, Faculty of Medicine, Kyushu University, Fukuoka; and BMorinaga Milk Industry Company Ltd, Kanagawa, Japan (Received 15 January 1991. Revision accepted 25 March 1991)
Abstract-Thein vitro production of macrophage colony-stimulating factor (M-CSF) was studied in 14 human lymphoblastoid cell lines and their lineages were ascertained by surface phenotype analysis. M-CSF gene transcripts were detected in a T-lymphocyte-derived cell line (CCRF-CEM) and 3 Blymphoblastoid cell lines (IM-9, BALL-l, and CCRF-SB) by Northern-blot analysis. The secretion of M-CSF protein into the culture supematant by each cell line was also studied using an enzymelinked immunosorbent assay for M-CSF. The 4 cell lines which expressed the M-CSF gene secreted considerable amounts of M-CSF into their culture supematants, while the 10 cell lines without MCSF gene expression did not do so. The cell lines which constitutively produced M-CSF were then subjected to Southern-blot analysis of the M-CSF gene structure, and all 14 cell lines were examined for infection by the Epstein-Barr virus. Neither structural changes nor amplification of the M-CSF gene were detected, and Epstein-Barr virus infection was found to be not directly related to M-CSF production. Key words: M-CSF,
nosorbent
lymphoblastoid assay, gene expression.
cell lines, Epstein-Barr
immu-
culture supernatants [l&12]. However, the production of M-CSF by lymphoblastoid cell lines without exogenous stimulation has not yet been clearly demonstrated [3,13]. In this article, we report that various human lymphoblastoid cell lines of either Tor B-cell origin can constitutively produce M-CSF.
INTRODUCTION
MACROPHAGE colony-stimulating CSF-1) is one of the hematopoietic
virus, c-fms, enzyme-linked
factor (M-CSF or regulators and its
action is necessary for macrophage progenitor cells to become mature macrophages [l]. Cloning of the cDNA of M-CSF has been performed by both Kawasaki et al. [2] and Wong et al. [3]. M-CSF is known to be produced by activated monocytes [ 11, endothelial cells [4], fibroblasts [5,6], and various tumor cell lines [7-91. Recently, Epstein-Barr virus (EBV)transformed B lymphocytes and a human T-leukemic cell line were reported to release M-CSF into their
MATERIALS
AND METHODS
Preparation of cells and cell culture supernatants
The following 14 lymphoblastoid cell lines were studied; RPM1 8226 [14], HS-Sultan [15], CCRF-SB [16], RAJI [17], BALL-l [18], RAMOS [19], WI-L2 NS [20], IM9 [21], CCRF-CEM [22], MT-1 [23], CCRF-HSB2 [24], TALL-l [ 181, MOLT-3 [25], and MOLT-4 [25]. All cell lines were provided by the Foundation for Promotion of Cancer Research (Tokyo, Japan). Cells were cultured at 37°C in RPM1 1640 medium supplemented with 10% fetal bovine serum (FBS), under a humidified 5% COr atmosphere. The cells were harvested in the logarithmic growth phase and analyzed by flow cytometry using a FACScan (Becton Dickinson, Mountain View, CA). The monoclonal antibodies used to determine the surface antigen profiles included Leu-Sb/CDZ, Leu-4/CD3, Leu-3a/CD4, Leu-l/ CD5, Leu-2a/CD8, Leu-12/CD19, Leu-16/CD20, HLADR, and PCA-1. They were purchased either from Becton Dickinson or from Coulter Immunology (Hialeah, FL). For the measurement of M-CSF levels in culture supernatants, the cells were cultured at a concentration of
* This work was partly supported by Grants-in-Aid for Cancer Research and Scientific Research (Nos 60770949, 02256106, 03670325 and 63015063) from the Ministry of Education, Science and Culture of Japan and from the Fukuoka Anti-Cancer Society. Abbreviations: M, macrophage; G, granulocyte; GM, granulocyte-macrophage; CSF, colony-stimulating factor; EBV, Epstein-Barr virus; FBS, fetal bovine serum; LPS, lipopolysaccharide; ELZSA, enzyme-linked immunosorbent assay; IL, interleukin. Correspondence to: Dr Seiichi Okamura, Cancer Center, Kyushu University Hospital, 3-l-l Maidashi, Higashi-ku, Fukuoka 812, Japan. 819
C. KAWASAKI et al.
820
5 x lo* cells/ml for 3 days and cell-free supernatants were obtained by centrifugation. Human monocytes were separated from the peripheral mononuclear cells obtained from a healthy donor who gave informed consent by adherence to the plastic culture dish. The monocytes were resuspended in RPM1 1640 medium with 10% FBS at 5 X lo5 cells/ml and cultured with 100 ng/ ml of bacterial lipopolysaccharide (LPS; Difco, Detroit, MI) for 2 h. Then cells were harvested by centrifugation to obtain the total RNA for use as a positive control in the Northern-blot analysis. Non-adherent and unstimulated human mononuclear cells, including T and B cells, were used as the negative control for the Northern-blot analysis. Northern-blot analysis
The procedure used for the Northern-blot analysis of human CSF genes has been reported previously [26,27]. Total cellular RNA was isolated from cultured cells by the acid-guanidinium thiocyanate-phenol method [28]. After glyoxalation, 20-ug samples of total RNA were size-fractionated by agarose gel electrophoresis and transferred to nylon membranes (Hybound-N+, Amersham, Arlington Heights, IL). The membranes were then hybridized with the cDNA probes according to the method of Meinkoth et al. [29]. The probes used for the analysis were the cDNA encoding M-CSF [3], kindly provided by the Genetics Institute (Cambridge, MA) and c-fms [30], which was purchased from Oncogene Science Inc. (North Mineola, NY). Radiolabeling of the cDNA probes was performed according to the method of Feinberg et al. [31] and the membranes were washed and autoradiographed with an intensifying screen. Measurement of M-CSF nosorbent assay (ELISA)
levels by enzyme-linked
immu-
Quantitation of M-CSF was performed by ELISA as previously reported [32]. In brief, the assay was based on the dual antibody immunometric sandwich principle, using horse and rabbit polyclonal antibodies against human urinary CSF. The minimum detectable level of M-CSF was 50 pg/ml . Southern-blot analysis
Chromosomal DNA was isolated from cultured lymphoblastoid cell lines and peripheral mononuclear cells by phenol extraction [33] and then digested with the restriction enzymes Hind III or Barn HI (Toyobo, Osaka, Japan). Samples of digested DNA (15 ug) were then subjected to electrophoresis on 0.8% agarose gel and transferred to Hybond-N+ membranes. The membranes were subsequently hybridized with the cDNA probe for M-CSF and autoradiographed as described above. Detection of Epstein-Barr virus infections
Infection of the cell lines with Epstein-Barr virus (EBV) was assessed using an EBV detection kit purchased from Enzo Diagnostics Inc. (New York, NY). In brief, cells fixed on a slide glass were incubated with the biotinylated DNA probe for the EBV genome and heated to 92°C for 2 min. The slide was then slowly cooled to room temperature, allowing the DNA probe to anneal with the viral DNA. Avidin-biotinylated horseradish peroxidase was then added to the slide and incubation was performed for 15 min. After being washed, the slide was soaked for 10 min at room temperature in a chromogenic substrate solution consisting of aminoethylcarbazole and hydrogen peroxide
in acetate buffer, and then counterstained with fast green. EBV-infected cells were then detected by microscopy as those containing a red-stained substance in their nuclei.
RESULTS Surface phenotypes
of the cell lines
The lineages of the cell lines were ascertained by surface phenotype analysis (Table 1). Eight out of the 14 cell lines (RPM1 8226, HS-Sultan, CCRF-SB, RAJI, BALL-l, RAMOS, WI-L2 NS, and IM-9) were determined to be of B-cell lineage, because the expression of both CD19 and CD20 was detected, except in the case of RPM1 8226, which showed PCA-1 expression. The other 6 cell line cells were determined to be of T-cell origin. TALL-l, MOLT-4, MOLT-3, and CCRF-CEM showed CD2-positivity. MT-l showed the surface phenotype for helper T cells and CCRF-HSB2 expressed CD5, a marker for pan-T lymphocyte. EBV infection of the cell lines The presence of EBV infection in the 14 lymphoblastoid cell lines is also shown in Table 1. Viral genomes were detected as red dots in the nuclei of 5 cell lines, including RPM18226, HS-Sultan, CCRFSB, RAJI, and IM-9. Northern-blot
analysis
Transcripts of the M-CSF gene were detected in 4 cell lines (CCRF-SB, BALL-l, IM-9, and CCRFCEM) out of the 14 cell lines examined (Fig. 1). The size of the transcripts was 4.0 kb, which was the same as the transcript detected in LPS-stimulated and other transcripts were not macrophages, detected. No M-CSF transcripts were detected in unstimulated normal human lymphocytes. In addition to M-CSF gene expression, we investigated the expression of the c-fms oncogene in the 14 cell lines. By Northern-blot analysis, c-fms transcripts were not detected in any of the cell lines (Fig. 1). Production
of M-CSF
M-CSF levels in the culture supernatants of the 14 cell lines were studied (Table 2). The 4 cell lines which expressed the M-CSF gene secreted significant amounts of M-CSF into their culture supernatants, while the 10 cell lines which did not express the MCSF gene secreted no M-CSF protein. Southern-blot
analysis
Using 2 kinds of restriction enzymes, Southernblot analysis was carried out to investigate whether or not genetic changes were involved in the mechanism of M-CSF production by these cell lines. As
FIG. 1. Northern-blot analysis of M-CSF and c-fms in the various cell lines. Six T-cell lines (a) and 8 B-cell lines (b) were analyzed. RNA was extracted from cultured cells and from monocytes stimulated with 100 ng/ml lipopolysaccharide (LPS) for 2 h. An aliquot of total RNA (20 ug) was analyzed with the cDNA probe for M-CSF and rehybridized with the probe for c-fms. Hybridization with a probe for pactin was performed as a control. Mono-LPS, normal monocytes stimulated by LPS.
821
FIG. 2. Southern-blot analysis. DNA from cells producing M-CSF was digested with the indicated restriction enzymes. An aliquot of digested DNA (15 ng) was applied to 0.8% agarose gel, subjected to electrophoresis, transferred to a nylon membrane, and then analyzed with the cDNA probe for M-CSF. PBMC, peripheral blood mononuclear cells.
822
M-CSF production by lymphoid cell lines TABLE 1. SURFACE
PHENOTYPE
823
ANDEPSTEIN-BARR VIRUSINFECTION INHUMANLYMPHOBLASTOID CELLLINES Surface markers (%)
Cell line RPM1 8226 HS-SULTAN CCRF-SB RAII BALL-l RAMOS WI-L2 NS IM-9 MT-1 CCRF-HSB2* TALL-l MOLT-4 MOLT-3 CCRF-CEM
CD2
CD3
CD4
CD8
1 0 1 0 1 0
0 0 10 2 0 2 0 0 tIl
0 0 0 0 2 0 02
:
0 10 1 97 61 89 60
18 14 2 7
87 0 DPt 0 18 DP
1 0 2 0 4 1 23 0 DP 0 2 DP
CD10
CD19
CD20
1 4 0 63 3 0 0 0 1 0 13 25
0 91 57 54 94 71 70 97 4 0 1 3 0 0
24 99 98 94 100 100 99 83 t!l 2 1 1 1
HLA-DR
99 100 100 59 100 100 5 0 7 0 3 0
PCA-1
EBV
95 1
+ + + +
+ -
Surface marker percentages indicate the percentage of cells expressing the surface antigens detected by the various monoclonal antibodies. The nresence or absence of EBV infection is indicated on the extreme right of the table. * 100% positive for CD5. 1 t DP, double-positive for both CD4 and CD8.
shown in Fig. 2, there was no evidence of amplifications or gross structural changes around the MCSF gene in the 4 cell lines expressing M-CSF mRNA when compared with normal human lymphocyte DNA.
DISCUSSION Flow cytometric analysis showed that the 14 cell lines in this study comprised 6 T-lineage and 8 Blineage lines. All the B-cell lines expressed CD19, CD20, or PCA-1. Four of the 6 T-cell lines expressed CD2, while MT-l and CCRF-HSB2 did not do so. As MT-l expressed CD4 and CCRF-HSB2 expressed CD5, both of which are T-lineage markers [34], these two cell lines were also determined to be of the T lineage. Among these 14 cell lines, M-CSF gene expression was detected in lines of both B- and T-cell origin by Northern-blot analysis including CCRF-SB, BALL1, IM-9, and CCRF-CEM. The mRNA detected in all 4 cell lines was 4.0 kb in size, indicating that it was the full-length M-CSF transcript [3]. M-CSF is produced by any kinds of normal cells including activated monocytes [l], endothelial cells [4], and fibroblasts [5,6], as well as by various tumor cells [791. However, its constitutive expression by lymphoid cell lines is comparatively rare. A considerable amount of M-CSF protein was detected in the culture supematants of all 4 cell lines which expressed M-CSF mRNA, indicating that the translational mechanisms were functioning normally
in these cell lines and allowing M-CSF transcripts to be translated to the secretory form of M-CSF protein. Since neither monocytosis nor granulocytosis were observed in the patients from whom these 4 cell lines were established [16,18,21,22], it seems that MCSF production by the original leukemic cells had no effect on the clinical features. However, longestablished cell lines are known to change their characteristics [35,36], so the investigation of fresh leukemic cells will be necessary to determine the exact relation between the clinical features of leukemia and CSF production by the tumor cells. We also examined the expression of c-fms oncogene mRNA to determine whether the cells producing M-CSF also expressed the M-CSF receptor, since the biological effect of M-CSF is mediated through specific binding to its receptor, which is a product of the c-fms oncogene [30]. Northern-blot analysis failed to detect c-fms mRNA in any of the 4 M-CSF-producing cell lines, indicating that M-CSF did not act as an autocrine growth factor for any of these cell lines. Since various CSFs are reported to act as autocrine growth factors [37,38], we also investigated the production of several other CSFs using ELISA or Northern-blot analysis. Analysis of culture supernatants showed that none of the 14 cell lines produced either granulocyte CSF (G-CSF) or granulocyte-macrophage CSF (GM-CSF) , but WI-L2 NS and CCRF-HSB2 produced trace amounts of GM-CSF when stimulated by phorbol myristateacetate (data not shown). In addition, Northern-blot analysis failed to detect interleukin 3 (IL-3) gene transcripts in any of these cell lines (data not shown).
C. KAWASAKI et al.
824
TABLE 2. M-CSF LEVEL IN THE CULTURE SUPERNATANT OF EACH OF THE 14 CELL LINES Cell line RPM1 8226 HS-SULTAN CCRF-SB RAJI BALL-l RAMOS WI-L2 NS IM-9 MT-l CCRF-HSB2 TALL-l MOLT-4 MOLT-3 CCRF-CEM
M-CSF (ng/ml)*
