Induction of Myeloid Colony-Stimulating Activity in Murine Monocyte Tumor Cell Lines by Macrophage Activators and in a T-Cell Line by Concanavalin A1

[CANCER RESEARCH 38, 1414-1419, May 1978]

Induction of Myeloid Colony-stimulating Activity in Murine Monocyte Tumor Cell Lines by Macrophage Activators and in a T-Cell Line by Concanavalin A1

Peter Ralph, Hal E. Broxmeyer,2 Malcolm A. S. Moore, and Ilona Nakoinz

Sloan-Kettering Institute for Cancer Research, Rye, New York 10580

ABSTRACT activating agents and in T-lymphomas by T-lymphocyte mitogens. Certain fibrosarcoma lines in culture and the WEHI-3 myelomonocytic leukemia cell line have previously been shown to secrete myeloid colony-stimulating activity MATERIALS AND METHODS (CSA) spontaneously. We describe here other hemato- Murine Tumor Cell Lines. Monocyte and macrophage poietic tumor cell lines in which CSA is either produced tumor cell lines are described in Ref. 32, except for Abelson constitutively or inducible by immunostimulators. CSA leukemia virus-induced line RAW264 (33). T-lymphoma lines production in macrophage and monocyte tumor lines is EL4, RBL-5, BW5147, and S49; myelomas P3 and induced by lipopolysaccharide, zymosan, Mycobacterium MOPC315; mastocytoma P815; lymphoma P388; and Abel- strain Bacillus Calmette-Guerin, tuberculin purified pro son line R8 are described in Ref. 31. Rauscher leukemia tein-derivative preparation from mycobacteria, and dex- virus line RBL-3 and chemically induced leukemia L1210 tran sulfate. Myeloma, mastocytoma, and T-lymphoma (39) were obtained from K. Chang (NIH, Bethesda, Md.); lines do not produce CSA with or without these agents. In fibrosarcoma L929 (5) was obtained from B. Williams contrast, the T-lymphocyte mitogen concanavalin A (but (Sloan-Kettering Institute, Rye, N. Y.); bone marrow fibro- not phytohemagglutinin) induces CSA synthesis in one of blast JLSV9 and Rauscher leukemia virus-infected JLSV9- seven T-lymphomas tested. In most cases induction of RLV (9) were obtained from A. Demsey (Sloan-Kettering CSA is correlated with conditions of cell growth inhibition Institute); Abelson lymphoma RAW309.1 was obtained from by the immunomodulators. However, other drugs that W. Raschke (Salk Institute for Biological Studies, San cause cytostasis or cytotoxicity do not lead to CSA pro Diego, Calif.); F22.WC-2 T-lymphoma was obtained from T. duction. Leukemic cells thus may retain sensitivity to Watanabe (Osaka University Hospital, Osaka, Japan); me- normal regulatory events with resultant effects on host sothelioma BALTNMS201, T-lymphomas P1798 and BAL- hematopoietic cell functions. ENTL5, and Abelson lymphomas ABPL1 and ABPL2 were obtained from M. Potter (National Cancer Institute, Be INTRODUCTION thesda, Md.). Substances Tested for Induction of CSA. Dialyzed latex Elevated numbers of granulocytes and macrophages are beads 0.81 Â¿Â¿mindiameter, PHA P, and LPS (Salmonella associated with immunological reactions and inflammatory typhosa W0901; Difco Laboratories, Detroit, Mich.); PPD conditions. Marrow and blood monocytes have been iden (gift from Dr. A. Gray, Merck Sharp & Dohme, West Point, tified as producers of CSA,3 necessary for the development Pa.); BCG (Tice strain; University of Illinois, Chicago, III.); of granulocytes and macrophages in vitro (6,8, 25). Bacte zymosan and dextran sulfate (M.W., 500,000; Sigma Chem rial LPS stimulates macrophages to increased CSA produc ical Co., St. Louis, Mo.); and Con A (Pharmacia Fine tion (8, 10, 35). However, CSA synthesis is also induced Chemicals, Inc., Piscataway, N. J.) were dissolved or sus during T-lymphocyte-dependent mitogen or antigen stim pended in phosphate-buffered saline, pH 7.4 (8 g NaCI, 0.2 ulation of spleen or blood cells (7, 22, 26, 37, 38). g KCI, 1.15 g Na, HPO,, 0.2 g KH,PO4> 0.1 g CaCL, and 0.1 g We have described constitutive CSA production in the MgCL-6H,O per liter), at 20 to 100 times the final concentra mouse myelomonocyte tumor cell line WEHI-3 (30) and tion. induction of CSA in a macrophage tumor cell line PU5-1.8 Assay for CSA (3). Mouse bone marrow cells (7.5 x 104) by LPS, BCG, PPD, yeast zymosan, and phorbol myristate were suspended in 1 ml 0.3% Difco agar culture medium (29). This study investigates the induction of CSA in other containing enriched McCoy's medium plus 10% fetal calf monocyte-macrophage tumor cell lines by macrophage- serum. Medium conditioned by cell lines (0.1 ml) was placed on the bottom of Petri dishes prior to addition of 1 Supported by National Science Foundation Grant PCM 75-19734. Na agar cell suspensions. Cultures were incubated in 5% CO2- tional Cancer Institute Grants CA 17353 and CA 17085, and the Gar Reichman humidified air, and colonies (>40 cells/aggregate) and Foundation. clusters (3 to 40 cells/aggregate) were scored after 5 to 7 2 Special Fellow of the Leukemia Society of America 3 The abbreviations used are: CSA, myeloid colony-stimulating activity; days. Under these conditions a maximum of 100 to 200 LPS, lipopolysaccharide; BCG, Mycobacterium strain Bacillus Calmette- colonies of granulocytes and macrophages can be obtained Guerin; PPD, tuberculin purified protein derivative; PHA, phytohemaggluti with optimal concentrations of CSA. For intracellular CSA, nin; Con A, concanavalin A; T-cell, thymus-derived lymphocyte; B-cell, bone marrow-derived lymphocyte. cell pellets were suspended in phosphate-buffered saline to Received November 7. 1977; accepted February 2. 1978. the original volume and lysed by 2 cycles of freeze-thaw. No

1414 CANCER RESEARCH VOL. 38

additional CSA was detected in producing lines following untreated cultures of a variety of macrophage, monocyte, further cycles of freeze-thaw. T-lymphoma, myeloma, or mastocytoma cell lines (Table 1); in a number of these lines tested, an inhibitor of the CSA assay was also undetected, indicating the absence of the RESULTS molecule(s). Spontaneous Production of CSA by Tumor Cell Lines. A Induction of CSA by LPS and Other Macrophage Acti number of murine cell lines adapted to culture were inves vators in Monocyte-related Lines. Since LPS will induce tigated for spontaneous production of CSA. Besides the CSA synthesis in the macrophage line PU5-1.8 (29), this previously described L-cell fibrosarcoma (2) and myelo- agent was tested in the other hematopoietic lines. We use monocytic leukemia WEHI-3 (30), 2 lymphoid lines secreted the term induction to denote the stimulation of CSA produc CSA constitutively (Table 1). Large amounts of CSA were tion in experimental cultures when control cultures do not found in supernatants of RBL-3 and, to a lesser extent, of contain detectable activity. As shown in Table 2, LPS L1210 lymphoid cell lines. A fibroblastoid cell line derived from normal mouse bone marrow (JLSV9) and its Moloney leukemia virus-infected counterpart, JLSV9-RLV, also pro Table 2 Induced CSA production and drug toxicity in hematopoietic tumor duced CSA spontaneously, as described for the similar cell lines JLSV5 virus-transformed line (44). The fibroblastoid lines CSA"Cell produced only or predominately macrophage CSA. CSA from the other constitutive lines caused bone marrow colonies of granulocyte and macrophage morphology (not lineMyelomonocyteWEHI-3MacrophagePU5-1neousLPS''1210000000000000014024000001305556786"000000000012692570000(99)Â°(90)(76)(10)(100)(10)(10)(29)(0)(0)(0)(0)(0)(0)(0)(0)(10)(26)(33)(14)(34)(0)ConA*1170000108000000000132300000ND'(13)(0)(37)(0)(31)(40)(70)(K)(8)(72)(100)(46)(14)(25)(0)(13)(24)(46)(100)(0)(51)PHA''128 shown). In the CSA-secreting lines studied by us, undiluted supernatants and 1:10 dilutions in most cases produced (20)0 greater than 100 colonies/7.5 x 104bone marrow cells, and dilutions greater than 1:10 were required before titrating out the activity. No CSA was found in supernatants of .8J774P388D1RAW264T-lymphomaEL4S49RBL-5P1798F22.WC-2BALENTL5BW5147MyelomaP3MOPC315MastocytomaP815LymphomaRBL-3L1210R8C(0)0 (39)0 (16)0 Table 1 (41)0 Spontaneous CSA production in tumor cell lines (66)0 CelllineMyelomonocyteWEHI-3MacrophagelineT-lymphomaEL4S49RBL-5P1798F22.WC-2 (K)e0 (K)0 Â±8*0000068 (71)0 (K)0 (K)0 (16)0 PU5-1.8P388D1J774RAW264MastocytomaP815MesotheliomaBALTNMS201FibrosarcomaL929MarrowBALENTL5BW5147MyelomaP3MOPC315LymphomaRBL-3L1210R8RAW309.1C1498ABPL1ABPL2CSA000000000140 (0)0 (0)0

(0)129

(51)26 (57)0 (20)0 Â±496 1498ABPL1ABPL2RAW309.1Sponta (60)0 (3)0 (55)ND Â±2160 Â±1624 Â±200000 " Colonies/7.5 x 104mouse bone marrow cells stimulated by 0.1 ml tumor cell line supernatant in 1-ml agar cultures. Cell line cultures were initiated at 2 x 105cells/ml and incubated with drugs fibroblastJLSV9JLSV9-RLVCSAÂ«121 Â±11170 for 3 days. Viable cells were then counted, percentage of inhibition Â±7Cell of tumor cell growth was compared to control cultures calculated, and supernatants were tested for CSA. " Colonies/7.5 x 10" mouse bone marrow cells stimulated by 0.1 * LPS, 1 Â¿ig/ml,and Con A and PHA, 10 /Â¿g/ml,were incubated ml tumor cell line supernatant in 1-ml agar cultures. Cell line with tumor cell lines for 3 days. Control solutions of these mitogens cultures were initiated at 2 x 105cells/ml, and supernatants were alone had no effect on the bone marrow colony assay. collected when cultures reached 10Vml (2 to 3 days). WEHI-3, c Numbers in parentheses, percentage of tumor cell growth L929, JLSV9, JLSV9-RLV, and RBL-3 supernatants stimulated 90 to inhibition. 150 colonies also when tested at 1:10 dilution. The other constitu '' LPS, 0.01 /Â¿g/ml. tive producers and inducible lines (described below) showed less e K, killed, fewer viable cells after 3 days than initial concentra activity when their supernatants were diluted more than 1:2. tion. 6 Mean Â±S.E. f ND, not done.

MAY 1978 1415

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1978 American Association for Cancer Research. P. Ralph et al. induced CSA to varying degrees in all of the monocyte and 0.1 and 1 us LPS per ml must be due to high-dose inhibition macrophage lines except for the constitutive producer of CSA production, as these concentrations do not affect WEHI-3. Results are shown for a 3-day incubation with LPS, the assay system. Latex beads, which are actively phago- 1 /xg/ml. This concentration of LPS inhibits the growth of cytosed by the cell lines without interfering with growth the monocyte-macrophage lines (except P388D1) more than (32), did not induce CSA. 50% but not of the other cells shown in Table 1 (32). In Induction of CSA by T-Lymphocyte Mitogens. Since some experiments, control WEHI-3 cultures had lower activ mitogen stimulation of normal T-lymphocytes induces CSA ity (40 to 80 colonies/0.1 ml supernatants) than was shown production, these agents were tested on a number of T- in Table 2, and then LPS stimulated CSA production up to lymphoma cell lines. PHA and Con A are preferentially toxic 2-fold (4). Cell lysates (at 106 cells/ml) of control cultures of to murine (27) and human (28) T tumor cell lines compared inducible tumor cell lines neither contained CSA nor in to myelomas; however, many other hematopoietic cell lines hibited the activity of preformed CSA (0.1 ml tested in are as sensitive as T lines (Ref. 27; unpublished observa assay), except for slight inhibition with J774. tions). Con A was very effective in stimulating granulocyte- Production of CSA by L1210 cells was augmented by macrophage CSA in the T-lymphoma EL4 (Table 2). Six incubation with LPS, and induction of CSA occurred in an other T-lymphomas and other types of hematopoietic cell Abelson lymphoma line, R8. These 2 lines and RBL-3 lack T- lines were not induced by Con A. Dose titration (Chart 2) lymphocyte Thy-1 antigen, surface Â¡mmunoglobulin, and shows that the threshold for CSA induction and tumor cell myeloid markers of phagocytosis and lysozyme but bear Fc growth inhibition occurred at 2 to 5 ^g Con A per ml. Con receptors and possess an alkaline phosphatase activity A, up to 50 /ng/ml, strongly induced CSA production despite characteristic of lymphoid tumors (unpublished observa the fact that no viable EL4 cells remained after 3 days of tions). LPS did not induce CSA in a number of T-lymphoma, incubation in this high toxic concentration. myeloma, mastocytoma, or other lymphoma lines (Table 2). PHA showed a toxicity curve almost identical with that of The monocyte-related tumor lines were also tested with Con A, but no CSA was induced in EL4 by this lectin. other macrophage-active agents known to induce CSA in Controls showed that PHA did not inhibit the activity of PU5-1.8 (29) and to inhibit specifically growth of these preformed CSA or the induction of CSA in EL4 cells by Con tumor types (32). As shown in Chart 1, the immunopotentia- A. PHA did not induce CSA in any cell line tested (Table 1). tors LPS, BCG, PPD, zymosan, and dextran sulfate induced Kinetics of CSA Production. Chart 3 shows that small CSA in monocyte and macrophage tumor lines at concen amounts of CSA were detected in supernatants of EL4 trations generally correlated with tumor cell cytostasis. The cultures incubated for 16 hr with Con A, 10 or 30 Â¿ig/ml. At decline in CSA activity in supernatants of several lines as this time 30% of EL4 cells were killed by the higher Con A LPS concentration is increased to 10 pig/ml may be due to concentration. There was a great increase in supernatant inhibition of the assay by LPS (32). However, the suboptimal CSA during the subsequent 2 days of incubation. At 30 Â¿ig activity in cultures of the most sensitive line, RAW264, at Con A per ml, viable cells decreased to 5% of initial numbers at Day 2 and 0% at Day 3, and total live and dead PU5-I.8

50 IOO -IOO Ã³

25 50 I

J774

50 IOO

25 50 I80 S RAW264 iÂ«

50 IOO

20 30 50 25 50 on A or PHA Chart 2. Induction of CSA in T-lymphoma EL4 by Con A. EL4 cultures were initiated at 10s cells/ml and incubated with Con A or PHA, 0 to 50 Â¿ig/ I05 IO6 I 10 100 10 roo i ra loo ml. After 3 days viable cells were counted, and the supernatant was assayed LPS BCG PPD Z DS for CSA. Increase in viable cells in experimental cultures was compared to Chart 1. Induction of CSA and inhibition of growth in macrophage cell the increase in controls to determine the percentage of control growth. lines by macrophage activators. Cultures were initiated at 2 x 105 cells/ml Control cultures contained 28.6 x 105 EL4 cells/ml at 3 days. *, high plus final concentrations of LPS, PPD, zymosan (Z), and dextran sulfate (OS) concentrations of Con A and PHA were toxic to EL4. No viable cells were in ng/m\ or BCG in bacteria/ml as shown. After 3 days of incubation, viable found after 3 days with 30-^g/ml doses of either lectin. Supernatants of tumor cells were counted (percentage of growth inhibition equals 100 times control EL4 cultures to which Con A was added at the end of 3 days of the increase in experimental-increase in control culture), and supernatants incubation contained no detectable CSA. Con A and PHA added to pre were tested for CSA (colonies stimulated per 7.5 x 1Q.4bone marrow cells formed WEHI-3 CSA did not inhibit the activity of the latter in the assay plated in agar with 0.1 ml cell line supernatant). For several lines tested the system. PHA, 5 ng/ml, did not affect the induction of CSA in EL4 cells by Con product induced was granulocyte-macrophage CSA. A, 5 pglm\.

1416 CANCER RESEARCH VOL. 38

Stimulation of CSA by dextran sulfate in bone marrow cells, probably macrophages, has been described by Gron- owiczef al. (14). In addition to these agents and BCG, PPD, and zymosan used in the present paper, we previously showed that phorbol myristate induces CSA in the mono cyte cell line PU5-1.8 (29) and in normal adherent bone marrow cells (unpublished observations). Phorbol myristate is a potent stimulator of enzyme release and other activities of granulocytes (11, 34), induces plasminogen activator production in macrophages (43), and is a T-lymphocyte mitogen (42). Other macrophage properties stimulated by activators in the macrophage tumor cell lines include anti body-dependent cellular immunity against erythrocyte (33) and tumor targets," production of T-lymphocyte-activating factor (21, 24), prostaglandin release (Ref. 20; E. Rietschel, personal communication), /3-glucuronidase, elastase, plas 0 8 16 20 30 4O 48 Hours minogen activator, collagenase, endogenous pyrogen, and Chart 3. Kinetics of CSA induction. EL4 cultures with Con A, 10 factors required for induction of murine T killer cells (16), ( ) or 30 ng/m\ ( ), and PU5-1.8 cultures with LPS, 1 ^g/ml, were murine antibody-producing cells,5 and human B-lympho- initiated at 2 x 10scells/ml. At the times shown viable cells in duplicate 2-ml cytes (17) not replaceable by mercaptoethanol. A summary cultures were counted, and the supernatant was assayed for CSA (colonies/ 7.5 x 10* bone marrow cells/0.1 ml supernatant). Con A, 10 /Â¿g/ml,inhibited of macrophage properties retained by murine and human EL4 growth 58%, and 30 pg/ml was toxic, while LPS inhibited PU5-1.8 monocyte-related cell lines is shown in Table 3. growth more than 95% when measured at 48 hr. The constitutive production of CSA by the least differen tiated myelomonocytic line, WEHI-3, but not by other lines cell numbers never exceeded initial concentrations by more with more properties of mature macrophages (33) deserves than 10%. Thus, CSA induction can occur under conditions comment. Normal, immature myeloid cells or their progen in which the inducing agent almost completely inhibits cell itors are dependent on CSA for growth. There must be growth. The induction of CSA in the monocyte line PU5-1.8 stringent controls to prevent self-stimulation of the progen by LPS was also a slow process, taking at least 3 days for itor by premature synthesis of CSA, which is a function of maximal expression (Chart 3). In contrast to lectin induction the mature cells of the monocyte branch of the myeloid of EL4, LPS stimulation of CSA in PU5-1.8 cells was accom series. Production of CSA associated with murine (24) and panied by reversible cytostasis of the tumor cells rather human (13) myelomonocytic leukemias suggests that 1 path than by cytotoxicity (32). DNA synthesis is not required for to leukemogenesis in this early cell type is the inappropriate induction of CSA in normal lymphocytes by Con A (36) nor activation of genes for the molecule that stimulates its own growth. There is evidence that the murine WEHI-3 tumor in PU5-1.8by LPS (29). (24) and WEHI-3 culture line cells (4) are still dependent on CSA for growth. A granulocyte inhibitor of CSA production DISCUSSION blocks WEHI-3 colony formation in agar but does not block Several facts are apparent from this study of CSA induc colony formation of other tumor lines, and this growth tion in different types of hematopoietic tumor cell lines, (a) inhibition is overcome by adding an external source of CSA Only cell type-specific agents (macrophage activators for or by stimulating endogenous synthesis of CSA (4). monocyte-related lines; Con A for the T line) can induce Induction of CSA in spleen and peripheral blood cells by CSA production, (b) All inductions of CSA are accompanied Con A and PHA (and perhaps by pokeweed mitogen) is a property of T-lymphocytes (26, 27). However, there is evi by drug effects strong enough to block tumor cell prolifer ation. Other inhibitors of cell growth [dibutyryl cyclic aden- dence for separate subsets of T-cells responding to PHA osine 5'-phosphate, high levels of thymidine, dimethyl sulf- and Con A, in terms of induction of DNA synthesis (41) and oxide (29), PHA, and Con A in most cell lines] do not induce immune functions of helper, suppressor, and killer cells. The induction of CSA in the T-cell line EL4 by Con A but not CSA. (c) The macrophage lines differ in sensitivity to induc by PHA suggests that these cells belong to a Con A-respon- ing agents, presumably because of differences in quantity sive, PHA-unresponsive subset and that other T lines may or affinity of specific receptors for each kind of activating molecule, (d) The inducible T-cell line EL4 must share with be found to be activated by the other T mitogens. Pokeweed the other T lines binding sites for PHA and Con A leading to mitogen did not induce CSA in EL4 cells. However, adher toxicity but has a unique Con A-binding site for functional ent cells are required for activation of some normal T-cell activation of CSA production. The LPS induction of CSA in the monocyte-related tumor 1 P. Ralph, M. Ito, H. E. Broxmeyer, and I. Nakoinz. Corticosteroids Block Newly-induced but Not Constitutive Functions of Macrophage Cell Lines: lines described here is similar to the results of experiments CSA Production, Latex Phagocytosis, Antibody-dependent Lysis of RBC and of Eaves and Bruce (10) with normal mouse macrophages Tumor Targets, submitted for publication. and of Ruscelli and Chervenick (35) wilh human monocytes * M. K. Hoffmann, C. Galanos, S. Koenig, R. S. Mittler, P. Ralph, H. F. Oettgen, and U. HÃ¤mmerling. Differentiation of B Lymphocytes: Factor in the dose tilralion and kinelics requiring several days for Released by LPS-stimulated Macrophages Induces Functional and Pheno- maximal stimulation. typic Differentiation, submitted for publication.

MAY 1978 1417

Table 3 General properties of monocyte-related tumor cell lines in culture immunity''RBCLyso-

genouspyro-gen'++++NT'"NTNT+--NTProsta-glandin"+++++NTNT---- Tumorzyme" Phago- phago- RBC lysis+ cytosis* CSA'' cytosis lysis teases''+++++NTNTNT---Endo LineWEHI-3P388D1J774PU5-1.8RAW264RAW309CrWR19MU937T-lymphomasMyelomasMastocytomaP815StrainC"DBA/2CCBAB/14CXC.BCHumanDBA/2EtiologyOilS'?OilSVirusVirusVirusDHLCell-mediated (-)+ + + (-) (-)j++ + + + (-)â€¢*++ + + + +k++ +k + + (-) +++ + + + +' NT++ + NT + + NT++ + NT + + + (-)_ n-----(-)Neutralpro

0 Lysozyme synthesis and predominate secretion (30). h Phagocytosis of zymosan and latex beads; P815 has slight ingestive activity against latex beads (32). '' Granulocyte-monocyte CSA, constitutive in WEHI-3 and inducible in the other macrophage lines by LPS, BCG, PPD, zymosan, or dextran sulfate (Chart 1). '' Antibody-dependent phagocytosis or lysis of sheep RBC and tumor targets (33). *' Plasminogen activator, collagenase, elastase (J. Hamilton and Z. Werb, personal communication). fP. Bodel. Endogenous Pyrogen Production by Murine Macrophage Cell Lines, submitted for publication. " Prostaglandin E production, especially in response to LPS or CSA (20). '' Mouse strain: C, BALB/c. ' S, spontaneous; virus, Abelson leukemia virus (W. C. Raschke, S. Baird, I. Nakoinz, and P. Ralph. Functional Macrophage Cell Lines Transformed by Abelson Leukemia Virus, submitted for publication); DHL, patient with diffuse histiocytic lymphoma (30). 1 Ascites preparations are very active in lysis of RBC targets (Ref. 33; unpublished observations). * Latex bead phagocytosis and antibody-dependent tumor lysis by PU5-1.8 line is stimulated by preincubation with LPS or PPD.4 ' Stimulated further by preincubation with LPS or PPD (33). '" NT, not tested. " One (EL4) of 7 T-lymphomas produces CSA in response to Con A (Table 2).

functions, and this is being tested with the cell line. Other antiimmunoglobulin activation of rabbit B-lymphocytes functions of T-cell lines have been described, such as (Ref. 18 and Footnote 6). Study of macrophage lines differ inhibition (12, 40) and stimulation (T. Watanabe, personal ing in expression of receptors for microbial immunostimu- communication) of in vitro antibody production, but the lating agents (Chart 1) and differing in functional properties present experiments are the first examples of an induced of antibody-dependent cellular cytotoxicity to RBC and function in a lymphoid cell line. tumor targets will aid investigations of similar diversity in In addition to macrophage and T-cell sources of CSA, normal macrophage subsets. Defining tumor cell retention there are suggestions that B-lymphocytes can produce of sensitivity to physiological controls, as in this paper or in myeloid CSA. Since LPS-induced B-cell mitogenesis is the inhibition of growth and cancer accompanying stimula correlated with CSA production by mouse spleen cultures tion of differentiated properties in myeloblastic leukemia in kinetics, genetics, and dose response, Apte ef al. (1) cell lines by CSA (15, 19), is a continuing goal in these have implied that B-cells are the source of CSA. However, studies. spleen macrophages produce CSA in response to LPS, and no attempt was made to exclude this possibility. We find that spleen cell production of CSA in response to LPS is ACKNOWLEDGMENT macrophage dependent, whereas LPS-induced prolifera The authors thank Sa Schrader for excellent technical assistance. tion of B-lymphocytes is independent of macrophages, implying that B-cells are not a major contributor to LPS- REFERENCES induced CSA (unpublished observations). What uses can be made of these hematopoietic tumor 1. Apte. R. N., Hertogs, C. F., and Pluznik, D. H. Regulation of Lipopolysac- charide-lnduced Granulopoiesis and Macrophage Formation by Spleen line systems? Due to their homogeneity, they are ideally Cells. I. Relationship between Colony-Stimulating Factor Release and suited for biochemical analysis of induction and regulation, Lymphocyte Activation in Vitro. J. Immunol., 118: 1435-1440, 1977. from surface membrane receptors for external stimuli to 2. Austin, P. E., McCulloch, E. A., and Till, J. E. Characterization of the Factor in L-Cell Conditioned Medium Capable of Stimulating Colony cytoplasmic transduction of activating signals to nuclear Formation by Mouse Marrow Cells in Culture. J. Cellular Physiol., 77: events. For example, LPS at CSA-inducing concentrations 121-133, 1971. stimulates intracellular cyclic adenosine 5'-phosphate lev els and phosphorylation of nonhistone nuclear protein in â€¢¿Y.Nishizawa, T. Kishimoto, H. Kikutani, Y. Yamamura, and P. Ralph, the J774 macrophage line, similar to that described for manuscript in preparation.

1418 CANCER RESEARCH VOL. 38

3. Broxmeyer, H. E., Moore, M. A. S., and Ralph, P. Cell-Free Granulocyte 1001,1969. Colony Inhibiting Activity Derived from Human Polymorphonuclear Neu- 25. Moore, M. A. S., Williams, N., and Metcalf, D. In Vitro Colony Formation trophils. Exptl. Hematology, 5: 87-102, 1977. by Normal and Leukemic Human Hematopoietic Cells: Interaction be 4. Broxmeyer, H. E., and Ralph, P. Regulation of a Mouse Myelomonocytic tween Colony Forming and Colony-Stimulating Cells. J. Nati. Cancer Leukemia Cell Line in Culture. Cancer Res., 37: 3578-3584, 1977. Inst.,50. 591-602, 1973. 5. Carswell, E. A., Old, L. J., Kassel, R. L., Green, S.. Fiore, N., and 26. Parker, J. W., and Metcalf, D. Production of Colony-Stimulating Factor Williamson, B. An Endotoxin-lnduced Serum Factor That Causes Necro in Mitogen-Stimulated Lymphocyte Cultures. J. Immunol.. 112: 502-510, sis of Tumors. Proc. Nati. Acad. Sei. U. S., 72: 3666-3670, 1975. 1974. 6. Chervenick, P. A., and LoBuglio, A. F. Human Blood Monocytes: 27. Ralph, P. Retention of Lymphocyte Characteristics by Myelomas and V Stimulators of Granulocyte and Mononuclear Colony Formation in Vitro. Lymphomas: Sensitivity to Cortisol and Phytohemagglutinin. J. Immu Science, 178: 164-166, 1972. nol., 110: 1470-1475, 1973. 7. Cline, M. J., and Golde, D. W. Production of Colony-Stimulating Activity 28. Ralph, P. Differential Toxicity of Con A and PHA on Murine and Human by Human Lymphocytes. Nature, 248: 703-704, 1974. Hematopoietic Cell Lines. In: H. Bittiger and H. P. Schnebli (eds.), 8. Cline, M. J., Rothman, B., and Golde, D. W. Effect of Endotoxin on the Concanavalin A as a Tool, pp. 613-621. New York: John Wiley and Sons, Production of Colony-Stimulating Factor by Human Monocytes and Ltd., 1976. Macrophages. J. Cellular Physiol., 84: 193-196, 1974. 29. Ralph, R., Broxmeyer, H. E., and Nakoinz. I. Immunostimulators Induce 9. Demsey, A., Kawka. D., and Stackpole, C. W. Application of Freeze- Granulocyte/Macrophage Colony-Stimulating Activity and Block Prolif Drying Intact Cells to Studies of Murine Oncornavirus Morphogenesis. eration in a Monocyte Tumor Cell Line. J. Exptl. Med.. 746. 611-616, J. Virol., 21: 358-365, 1977. 1977. 10. Eaves, A. C., and Bruce, W. R. In Vitro Production of Colony-Stimulating 30. Ralph, P., Moore. M. A. S., and Nilsson. K. Lysozyme Synthesis by Activity. I. Exposure of Mouse Peritoneal Cells to Endotoxin. Cell Tissue Established Human and Murine Histiocytic Lymphoma Cell Lines. J. Kinet., 7: 19-30, 1974. Exptl. Med., 743: 1528-1533, 1976. 11. Estensen, R. D., White, J. G., and Holmes, B. Specific Degranulation of 31. Ralph, P., and Nakoinz, I. lipopolysaccharides Inhibit Lymphosarcomas Human Polymorphonuclear Leukocytes. Nature, 248: 347-348, 1974. of Bone Marrow Origin. Nature, 249: 49-51, 1974. 12. Feldman, M., Boylston, A., and Hogg, N. M. Immunological Effects of 32. Ralph, P., and Nakoinz. I. Direct Toxic Effects of Immunopotentiators on IgT Synthesized by ThÃªta-Positive Cell Lines. European J. Immunol., 5. Monocytic, Myelomonocytic, and Histiocytic or Macrophage Tumor 426-431, 1975. Cells in Culture. Cancer Res., 37: 546-550, 1977. 13. Goldman, J. M.. Th'ng, K. H., Catovsky, D., and Galton, D. A. G. 33. Ralph, P.. and Nakoinz. I. Antibody-Dependent Killing of Erythrocyte Production of Colony-Stimulating Factor by Leukemic Leukocytes. and Tumor Targets by Monocyte-Related Cell Lines: Enhancement by Blood, 47: 381-388, 1976. LPS and PPD. J. Immunol., 779: 950-954, 1977. 14. Gronowicz, E., Biberfeld, P., Wahren, B., and Coutinho, A. Characteri 34. Repine, J. E., White, J. G., Clawson, C. C., and Holmes. B. M. Effects of zation of Dextran Sulfate-Sensitive Cells. Scand. J. Immunol., 5: 573- Phorbol Myristate Acetate on the Metabolism and Ultrastructure of 582, 1976. Neutrophils in Chronic Granulomatous Disease. J. Clin. Invest., 54: 83- 15. Ichikawa, Y., Maeda, M., and Horiuchi, M. Induction of Differentiated 90, 1974. Functions Which Are Reversibly Suppressed by Cytochalasin B. Exptl. 35. Ruscetti, F. A., and Chervenick, P. A. Release of Colony-Stimulating Cell Res., 90: 20-30, 1975. Activity from Monocytes by Endotoxin and Potyinosinic-Polycytidylic 16. Igarashi, T., Okada, M., Kishimoto, T., and Yamamura, Y. In Vitro Acid. J. Lab. Clin. Med.,83: 64-72, 1974 Induction of Polyclonal Killer T Cells with 2-Mercaptoethanol and the 36. Ruscetti, F. W., and Chervenick, P. A. Regulation of the Release of Essential Role of Macrophages in This Process. J. Immunol., 118, 1697- Colony-Stimulating Activity from Mitogen-Stimulated Lymphocytes. J. 1703, 1977. Immunol., 774: 1513-1517. 1975. 17. Kishimoto, T., Hirano, T., Kuritani, T., Yamamura, Y., Ralph, P.. and 37. Ruscetti, F. W.. and Chervenick, P. A. Release of Colony-Stimulating Good, R. A. Induction of IgG Production in Human B Lymphoblastoid Activity from Thymus-Derived Lymphocytes. J. Clin. Invest., 55: 520-526, Cell Lines with Normal Human T-Cells. Nature, 277. 756-758, 1978. 1975. 18. Kishimoto, T., Nishizawa, Y., Kikutani, H.. and Yamamura, Y. Biophasic 38. Ruscetti, F. W., Cypress, R. H., and Chervenick, P. A. Specific Release Effect of Cyclic AMP on IgG Production and on the Changes of Non- of Neutrophilic- and Eosinophilic-Stimulating Factors from Sensitized Histone Nuclear Proteins Induced with Anti-lmmunoglobulin and En Lymphocytes. Blood. 47: 757-762. 1976. hancing Soluble Factor. J. Immunol., 118: 2027-2033, 1977. 39. Shevach, E. M., Stobo, J. D., and Green, I. Immunoglobulin and H- 19. Krystosek, A., and Sachs, L. Control of Lysozyme Induction in the Bearing Murine Leukemias and Lymphomas. J. Immunol . 708: 1146- Differentiation of Myeloid Leukemic Cells. Cell, 9: 675-684, 1976. 1151,1972. 20. Kurland, J. I., and Bookman, R. Prostaglandin E Production by Human 40. Stocker, J. W., Marchalonis, J. J., and Harris, A. W. Inhibition of a T Blood Monocytes and Mouse Peritoneal Macrophages. J. Exptl. Med., Cell-Dependent Immune Response in Vitro by Thymoma Cell Immuno in press, 1978. globulin. J. Exptl. Med., 739: 785-790, 1974. 21. Lachman, L. B., Hacker, M. P., Blyden, G. T., and Handschumacher, R. 41. Strobo, J. D., Paul, W. E., and Henney, C. S. Allogeneic Mixed Lympho E. Preparation of Lymphocyte-Activating Factorfrom Continuous Murine cyte Reactivity and Cytolytic Activity as Functions of Distinct T Cell Macrophage Cell Lines. Cellular Immunol., 34: 416-419, 1977. Subsets. J. Immunol.. 770: 652-660, 1973. 22. McNeill, T. A. Release of Bone Marrow Colony Stimulating Activity 42. Touraine, J.-L., Hadden, J. W.. Touraine. F., Hadden. E. M , Estensen. during Immunological Reactions in Vitro. Nature New Biol., 244: 175- R., and Good, R. A. Phorbol Myristate Acetate: A Mitogen Selective for 176,1973. a T-Lymphocyte Subpopulation. J. Exptl. Med , 745 460-465. 1977. 23. Meltzer, M. S., and Oppenheim, J. J. Bidirectional Amplification of 43. Vassalli, J.-D., Hamilton, J.. and Reich, E. Macrophage Plasminogen Macrophage-Lymphocyte Interactions: Enhanced Lymphocyte Activa Activator: Induction by Concanavalin A and Phorbol Myristate Acetate. tion Factor Production by Activated Adherent Mouse Peritoneal Cells. J. Cell, 77: 695-705, 1977. Immunol., 118: 77-82, 1977. 44. Watson, J., and Prichard, J. Characterization of a Factor Required for 24. Metcalf, D., Moore, M. A. S., and Warner, N. L. Colony Formation in the Differentiation of Myeloid and Lymphoid Cells in Vitro. J. Immunol.. Vitro by Myelomonocytic Leukemic Cells. J. Nati. Cancer Inst.. 43: 983- 708: 1209-1217, 1972.

MAY 1978 1419

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1978 American Association for Cancer Research. Induction of Myeloid Colony-stimulating Activity in Murine Monocyte Tumor Cell Lines by Macrophage Activators and in a T-Cell Line by Concanavalin A

Peter Ralph, Hal E. Broxmeyer, Malcolm A. S. Moore, et al.

Cancer Res 1978;38:1414-1419.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/38/5/1414

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/38/5/1414. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.