Stimulation of Tumor Necrosis Factor Release from Monocytic Cells by the A375 Human Melanoma Via Granulocyte-Macrophage Colony-Stimulating Factor1

[CANCER RESEARCH 50, 2673-2678. May 1, 1990] Stimulation of Tumor Necrosis Factor Release from Monocytic Cells by the A375 Human Melanoma via Granulocyte-Macrophage Colony-stimulating Factor1

Massimo Sabatini,2 Jeffery Chavez, Gregory R. Mundy, and Lynda F. Bonewald

Division of Endocrinology and Metabolism, Department of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284- 7877

ABSTRACT cell line A375, the target cell line used to show that GM-CSF induced monocyte-mediated cytoxicity (3), we noted that con It has long been known that complex interactions occur between tumors ditioned medium harvested from A375 tumor cell cultures and normal host immune cells. The human melanoma cell line A375 has been used previously as an indicator cell for tumor cell cytotoxicity induced TNF production in human blood monocytes and the mediated by monocytes. During other studies on this tumor cell line, we human monocytoid cell line U937 by the secretion of a soluble noted that the conditioned media harvested from A375 cultures induced factor. By multiple criteria, we have identified this soluble factor both the human monocytoid cell line U937 and human blood monocytes which causes TNF production as GM-CSF. These results sug to release the cytokine tumor necrosis factor (TNF). We characterized gest that in this human tumor, production of GM-CSF by the this tumor factor which induced TNF release by monocytic cells. Purifi tumor may retard tumor growth by causing release of cytotoxic cation was performed using ammonium sulfate precipitation, ion exchange cytokines of host cell origin. (DEAE) chromaiography, gel filtration, and reversed-phase high per formance liquid chromatography. The factor copurified with granulocyte- macrophage colony-stimulating factor (GM-CSF). The purified material MATERIALS AND METHODS caused the release of TNF by U937 cells and stimulated formation of Culture Media and Reagents. RPMI 1640, Eagle's minimal essential granulocyte-macrophage colonies in methyl cellulose. TNF release by medium, and PBS were from Hazleton (Lenexa, KS), trypsin-EDTA U937 cells in response to A375-conditioned medium was inhibited by was from GIBCO (Grand Island, NY), and PCS was from Hyclone neutralizing antibodies to GM-CSF. The TNF-inducing activity in A375- (Logan, UT). BSA, PMA, HEPES, and Tris buffer were from Sigma conditioned medium was completely removed by an anti-GM-CSF affinity (St. Louis, MO). column. Western blotting using antibodies to GM-CSF confirmed a single The reagents and apparatus for electrophoresis and Western blotting M, 27,000 band in A375-conditioned medium. We found that recombinant were from Bio-Rad (Richmond, CA) unless specified. All the reagents human GM-CSF stimulated TNF production by the same cells as the for purification of GM-CSF from A375-conditioned medium were from tumor-conditioned medium. Sigma, unless specified. These data show that A375 human melanoma cells produce GM-CSF, Cytokines and Antibodies. rhTNF, rhTGFa, and rh 7-interferon were which in turn causes TNF production by cells in the monocyte lineage. kindly provided by Dr. H. Michael Shepard (Genentech, Inc., South The combination of GM-CSF production by the tumor and TNF produc San Francisco, CA), and rhGM-CSF by Dr. Steven C. Clark (Genetics tion by immune cells may influence not only tumor growth but also some Institute, Boston, MA). Monoclonal mouse anti-rhGM-CSF antibody of the paraneoplastic syndromes associated with malignancy such as was purchased from Genzyme (Boston, MA), rhEGF was from Collab hypercalcemia, cachexia and leukocytosis. orative Research (Lexington, MA), and polyclonal goat anti-rhTNF was from R & D Systems (Minneapolis, MN). INTRODUCTION Cell Lines. The A375 human melanoma, the U937 human histiocytic lymphoma, the SaOS-2 human osteosarcoma, and the NCTC-L929 It has long been appreciated that the immune system is murine fibrosarcoma were obtained from the American Type Culture activated by the presence of a tumor and that tumor growth Collection (Rockville, MD). The HOSO human maxillary carcinoma may depend not just on the characteristics of the tumor itself was kindly provided by Dr. Toshiyuki Yoneda, of our group. The but also on the defense mechanisms which host cells mount (1, UMR-106rat osteosarcoma was kindly provided by Dr. T. John Martin 2). Although all of the signals which are responsible for tumor (University of Melbourne, Australia). The A375, U937, SaOS-2, and cell-host cell interactions are not known, recently it has become UMR-106 cell lines were cultured in 10% FCS-RPMI 1640 medium. The NCTC-L929 cells were cultured in 10% FCS-Eagle's minimal apparent that several of the cytokines which may be involved include GM-CSF3 and TNF. The human melanoma cell line essential medium. For preparation of CM, tumor cells were grown until confluent; then the medium was discarded, the flasks washed three A375 has often been used as an indicator target cell line for times with PBS, and either 0.1 % BSA-RPMI or 50 miviHEPES-RPMI cytotoxic effects of immune cells. Grabstein et al. (3) found medium was added. After 2 days the conditioned medium was collected, that recombinant human GM-CSF induced normal peripheral centrifuged to remove debris, and stored at â€”20Â°Cuntiluse. blood monocytes to become tumoricidal for A375 cells. Can- Human Blood Monocytes. Peripheral blood was drawn from healthy nistra et al. showed that human GM-CSF induced expression volunteers and the mononuclear cell fraction was separated by gradient of TNF by peripheral blood monocytes and by the human centrifugation on Ficoll-Paque (Pharmacia, Piscataway, NJ). Aliquots of 1.2 x 10" cells in 1 ml of 5% FCS-RPMI were seeded in 24-well monocytoid cell line U937 (4) and that antibodies to TNF plates and, after l h of incubation at 37Â°Cin 5% CO2, washed three completely inhibited the cytotoxic activity released by mono cytes in response to GM-CSF (5). times with PBS to remove the nonadherent cells. The adherent cells (monocytes) were then cultured in 0.5 ml of 5% FCS-RPMI with the During the course of other studies with the human melanoma indicated treatments for 24 h, at the end of which the media were Received 7/17/89; revised 2/1/90. collected for TNF assay. The costs of publication of this article were defrayed in part by the payment Treatment of U937 Cells. U937 cells, 1 x 10" cells/ml, were incubated of page charges. This article must therefore be hereby marked advertisement in for 24 h, unless indicated, with various dilutions of cytokines or tumor- accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported by Grant CA-40035 from the NIH. conditioned media in 5% FCS-RPMI in the absence or presence of 10 2To whom requests for reprints should be addressed. ng/mlof PMA. 3The abbreviations used are: BSA, bovine serum albumin; CM, conditioned TNF Cytotoxicity Assay. TNF was assayed biologically as cytocidal medium; EGF, epidermal growth factor; PCS, fetal calf serum; GM-CSF, gran activity against NCTC-L929 cells. Each sample was assayed in dupli ulocyte-macrophage colony-stimulating factor; PBS, phosphate-buffered saline; cate in serial dilutions in 96-well plates as described (6). TNF activity PMA, phorbol 12-myristate 13-acetate; rh, recombinant human; TGFÂ«,trans forming growth factor a; TNF, tumor necrosis factor; HEPES, 4-(2-hydroxy- was quantitated as the reciprocal of the dilution that results in 50% ethyl)-l-piperazineethanesulfonicacid; II. I. interleukin 1. survival. One cytotoxic unit is the amount of TNF that results in 50% 2673

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1990 American Association for Cancer Research. A375 MELANOMA CELLS INDUCE TNF RELEASE VIA GM-CSF PRODUCTION survival of the indicator cells. TNF specificity was shown by using anti- (11). Mononuclear cells were isolated using Ficoll-Paque (Pharmacia) human TNF antibody. and the nonadherent fraction was separated by adherence overnight to TGFa Assay. TGFa was measured as EOF equivalents by radiore- plastic. The nonadherent fraction was enriched for colony-forming cells ceptor assay on UMR-106 as described (7). by positive indirect immunoadherence using human progenitor cell Immunoaffinity Column. Monoclonal anti-rhGM-CSF antibody, 500 antibody (Becton Dickinson, Mountain View, CA), which is the com /ig, was covalently bound to 2 ml of Amino Link, cross-linked, 6% mercial preparation of monoclonal anti-My-10 antibody (12), and the beaded agarose gel, using Immuno Pure Antibody/Antigen Immunob- "panning" technique of Engleman et al. (13). The cells were cultured ilization Kit I (Pierce, Rockford, IL). The antibody-linked gel, loaded as described previously (11) in duplicate, and the total number of in a 1- x 0.5-cm column, had the capacity to retain 500 ng of rhGM- colonies per dish was counted on day 14. CSF. A375 CM, 500 ml in 50 mM HEPES-RPMI, were applied to the column at 4Â°Cby a peristaltic pump at the rate of 0.5 ml/min. The column was washed of the unbound protein with 16 ml of RPMI, and RESULTS the retained material was eluted using 12 ml of 0.1 M glycine. pH 2.8. During the course of studies on the mechanism of hypercal- Fractions of 1 ml were collected and immediately neutralized with 50 n\ of l M Tris-HCl, pH 9.5. cemia which occurs in the A375 tumor, we found that condi Electrophoresis and Western Blotting Procedure. Fractions contain tioned medium harvested from cultured A375 cells consistently ing TNF-inducing activity as determined by their capacity to stimulate stimulated the human monocytoid cell line U937 and human TNF production by U937 cells were pooled, dialyzed extensively against monocytes to release TNF activity (Tables 1 and 2). This 50 mM NH4HCO.,, pH 8.3, at 4Â°Cina M,3500 molecular weight cutoff activity could clearly be ascribed to TNF, inasmuch as it was membrane (Spectrum, Los Angeles, CA), lyophilized, and resuspended abolished by anti-rhTNF antibody (data not shown). A time in 20 fi\ of H2O. Fifteen /Â¿Iofthis sample were subjected to sodium course study of TNF production by U937 cells in response to dodecyl sulfate/polyacrylamide gel electrophoresis according to the A375 tumor cell-conditioned medium is shown in Fig. 1. In all method of Laemmli and Favre (8) on a Bio-Rad Mini-Protean appa these experiments, the U937 cells were costimulated with the ratus. The gel was 1 mm thick 13.5% polyacrylamide and was stained with a silver nitrate staining kit from Bio-Rad. phorbol ester PMA, known to cause differentiation of U937 cells to a mature phenotype capable of differentiated cell func The samples were transferred from the gel to an Immobilen transfer membrane (Millipore, Bedford, MA) according to the method of Tsang tion (14). No TNF activity could be detected in the media of et al. (9) and immunoblotted according to the method of Thomas et al. U937 cells cultured in absence of PMA (data not shown). (10). The primary antibody was monoclonal anti-rhGM-CSF diluted Normal human macrophages were also tested for TNF produc 1:750, the secondary antibody was alkaline phosphatase-conjugated tion in response to A375-conditioned media. We saw that the rabbit anti-mouse IgG 1:2000 (Zymed, San Bruno, CA). Antigen- A375-conditioned medium specifically stimulated TNF produc antibody complexes were detected using the alkaline phosphatase im- tion by monocytes. This stimulation was more evident in pres munostain kit from Bio-Rad. ence of a secondary stimulus such as 7-interferon or PMA Purification of TNF-inducing Activity from A375 CM. One liter of (Table 1). A375 CM in 0.1% BSA-RPMI was subjected to precipitation in 80% These studies show that A375 tumor-conditioned medium (NH4)2SO4 while stirring at 4Â°Cfor 4 h. After centrifugation at 15,000 rpm for 45 min at 4Â°C,the pellet was resuspended in H2O, dialyzed at Table 1 Effects of tumor-conditioned media on TNF production by cultured a molecular weight cutoff of 3500 against 50 volumes of 0.01 Msodium monocytes in absence or presence oflFN-y" or PMA phosphate, pH 8.3, and then applied to a DEAE-cellulose aniÃ³nex Human peripheral blood monocytes were cultured with the indicated treat change column (8 x 2.5 cm). The bound protein was eluted by a stepwise ments for 24 h, at the end of which time the TNF activity in the culture media gradient of increasing concentrations of sodium phosphate, pH 8.3, was assayed. from 0.01 to 0.1 M PÂ¡.The protein elution profile was monitored (units/ml)Treatment TNF spectrophotometrically at 280 nm. The fractions collected at 0.04 and 0.06 M sodium phosphate, containing most of the TNF-inducing activ DilutionControl (100ng/ml)1545 ng/ml)84543 ity, were pooled, dialyzed against 50 mM NH4HCG\,, pH 8.3, lyophi mediumA375 lized, resuspended in 50 m\i NH4HCO.,, pH 8.3, and applied to a Sephadex G-200 gel filtration column (100 x 2.5 cm). The fractions CMSaOS corresponding to the peak of TNF-inducing activity were pooled, di :4 3 32 :8:2 <2<2<2IFN-i 95 2117 alyzed against 50 mM NH4HCO3, pH 8.3, and resuspended in pyridine acetate buffer before application to a Nova Pak C,Â«reversephase HPLC CM:2 column, 15 x 0.39 cm (Waters, Milford, MA). The buffer system :4None<25 6PMA(1 10 consisted of: A, 0.2 M pyridine-0.9 M acetic acid, pH 4.0; B, 50% A " IFN-v, -x-interferon. and 50% 1-propanol (all from Burdick & Jackson, Muskegon, MI). The gradient was linear from 100% A to 30% A and 70% B in 60 min. Table 2 Effects of tumor-conditioned media on TNF production by U937 cells Thymidine Incorporation. The effect of TNF or U937 tumor-condi U937 cells were treated with tumor conditioned media in the presence of PMA (10 ng/ml) for 24 h, at the end of which time the TNF activity in the culture tioned medium on the growth of A375 cells was determined as follows. media was assayed. TNF activity was measured by the L929 cytotoxicity assay. A375 cells (1000/well/80 ^1) were seeded in 96-well plates. After 24 h, TNF was not detectable in A375 CM (TNF units/ml < 2). 80 p\ of control or treatment medium were added; after 72 h, the cells were labeled with 0.25 ^Ci/well of ['HJthymidine (ICN, Irvine, CA). TNF production by U937 cells (units/ml) After 6 h, the medium was aspirated from the plate and 100 /Â¿I/wellof trypsin-EDTA were added. After 10 min the cells were collected by a Treatment Dilution Experiment 1 Experiment 2 PHD cell harvester (Cambridge Technology, Watertown, MA) onto Control medium 32 15 filter paper discs. The radioactivity retained by the filters was measured A375CMSaOS by a liquid scintillation counter (Beckman, Fullerton, CA) and the effects of each treatment were calculated as the percentage of change compared with the control. Colony Formation Assay in Methylcellulose Using My-10* Cells. CMHOSO A375-conditioned medium and partially purified TNF-inducing activity from A375-conditioned medium were tested for colony-stimulating CM:2:4:8;2:4:2:4175952216831315231245584 activity using panned My-10* bone marrow cells as described previously 2674

160

36 48 60 72 Tima (hours) Fig. 1. Time course of the production of TNF by U937 cells in response to (O) control medium, (â€¢)A375 CM. and (A) SAOS CM. Each point represents the mean of duplicate wells. L'937 cells were treated with tumor-conditioned media in the presence of PMA (10 ng/ml) for 24 h. at the end of which time TNF activity in the culture media was assayed. induces cells in the monocyte lineage to produce TNF by the release of a soluble mediator. We then attempted to identify the soluble mediator responsible for TNF production by human U937 cells. Because the A375 melanoma is a producer of TGFÂ« (15) (we could detect in the conditioned medium a concentra tion of approximately 1 ng/ml), we tested TGFÂ«(50 ng/ml) as well as the related peptide EGF (50 ng/ml), for their effects on TNF production by U937 cells. No stimulatory effect was seen (data not shown). Conditioned medium was collected from serum-free A375 cultures, concentrated by ammonium sulfate precipitation, and fractionated by ion exchange chromatography, gel filtration, and reverse phase HPLC. A purification profile similar to that described for GM-CSF (16) was obtained, with the TNF-inducing activity eluting from a ds reverse phase HPLC column at 31% 1-propanol (Fig. 1C). 0.0 Since conditioned medium harvested from A375 tumors was 10 15 20 25 SO 35 40 behaving like GM-CSF and previous studies had shown that Fraction No. GM-CSF induced-TNF mediated monocyte cytotoxicity for Fig. 2. Purification profile of TNF-inducing activity in A.175 CM. Flution profile of (A) DEAE ion-exchange chromatography. (IS) gel filtration using A375 cells (3-5), we then attempted to determine if GM-CSF Sephadcx G-200. and (C) reverse phase HPLC'. (â€¢)absorbance profile. Columns, was the soluble factor produced by A375 cells which stimulated TNF-inducing activity. 1'937 cells were incubated with aliquots of each fraction in the presence of PMA (10 ng/ml) for 24 h. at the end of which time TNF TNF production. Firstly, we tested A375 crude conditioned activity in the culture media was assayed. ,\aPl. sodium phosphate. medium and purified fractions for GM-CSF activity and found it to be present (Table 3). However, in the samples treated with column after washing with low pH glycine buffer (Fig. 3). crude conditioned medium the colonies were smaller in size These data show that neutralizing antibodies to GM-CSF than those seen with the purified fractions or with rhGM-CSF block the activity in A375 tumor cell conditioned medium and (data not shown). The partially purified TNF-inducing activity that all of the TNF-inducing activity in tumor cell-conditioned from A375-conditioned medium was converted to GM-CSF media can be absorbed to a GM-CSF antibody affinity column. equivalents in molarity on the basis of the TNF-inducing activ To confirm that the activity was in fact due to GM-CSF, we ity of rhGM-CSF. Recombinant human GM-CSF and purified A375-conditioned medium caused similar effects on colony size examined purified fractions using Western analysis. We frac tionated a purified sample obtained from immunoaffinity col and number. umns by sodium dodecyl sulfate-polyacrylamide gel electropho- Next, we examined whether neutralizing antibodies raised resis and then transferred the proteins to nitrocellulose. Im- against recombinant human GM-CSF abrogated the TNF- inducing activity of the A375-conditioned medium. We saw munoblot analysis was performed using specific monoclonal that monoclonal anti-rhGM-CSF substantially decreased the antibody to GM-CSF as primary antibody, and alkaline phos- TNF-inducing activity of the A375-conditioned medium and phatase-conjugated secondary antibodies in order to visualize rhGM-CSF (Table 4). The inhibition by the antibody of the the corresponding protein by enzymatic reaction. Recombinant activity in the A375-conditioned medium was incomplete. How human GM-CSF was used as a positive control. As shown in Fig. 4, a distinct band was present in the A375 affinity-purified ever, at the dilution used, the antibody completely inhibited only concentrations of rhGM-CSF lower than 0.5 ng/ml and conditioned medium (M, 27,000) within the range assigned to partially inhibited concentrations of GM-CSF capable of giving native hGM-CSF( 17). the same effect as the A375-conditioned medium. We then used TNF has been shown recently to be directly tumoricidal for the monoclonal antibody to construct an affinity column for the A375 melanoma (18). We found similar results by testing GM-CSF. We found that all of the TNF-inducing activity the effects of TNF on thymidine uptake by A375 cells (Fig. contained in an aliquot of A375-conditioned medium was 5A). In addition, the TNF-containing culture media from U937 bound to the column. No activity was detected in the flow- cells treated with either A375-conditioned medium or GM-CSF through. The TNF-inducing activity was eluted from the affinity inhibited, as expected, the uptake of thymidine by A375 cells. 2675

Table 3 Colony-stimulating activity ofA375 conditioned media and partially purified T.\'F-inducing activity compared with rhGM-CSF AB My-10* hemopoietic progenitor cells were cultured with the indicated treat kd ments for 2 weeks, at the end of which time the number of colonies per each well was counted. The assay was performed in duplicate. -67 TreatmentControl mediumA375

CMUndiluted1:51:10Immunoaffinity-purified -25

-13

fractionIO'' Fig. 4. Western blot showing (Lane A) an approximate molecular weight of MÂ°IO"10 27.000 for (he immunoaffinity-purified A375 TNF-inducing material and (Lane B) recombinant human GM-CSF. kd, molecular weight in thousands. MIO'"

MrhGM-CSFIO'' 20

10 M10-'Â° O

MIO'" -10

MColonies/dish32232014201716201714181052830IS181210 -20 o 0 -30 " The immunoaffÃ¬nity-purifiedfraction was quantified in molar equivalents of * -40 rhGM-CSF on the basis of its TNF-inducing activity compared to that of rhGM- o CSF. 1 -50 o 31 125 500 O Table 4 Effects of monoclonal anti-rhGM-CSF on TNF production by U9ÃŒ7cells TNF(ng/ml) stimulated with either rhGM-CSF or A375 CM 20 j

(units/ml)- 10- B antibodyantibody3 + o TreatmentControl -10 mediumrhGM-CSF1.5 3119 -20 ng/ml1.0 3793 ng/ml0.5 3362 -30 ng/ml0.1 1121 ng/mlA375 5120 -40 CM1/21/4J 20115 -50 2325.0pH l/B 1/4 1/2 1/DII. Fig. 5. Cytotoxic effects of U937 CM and TNF on A375 cells. (A) Dose response of TNF (8-500 ng/ml). (A) O. CM from untreated U937 cells; â€¢.CM 250-)^^ from U937 cells treated with A375 CM; A. CM from U937 cells treated with rhGM-CSF; A. CM from U937 cells treated with A375 CM plus anti-rhGM- buffar1('>â€¢-â€¢â€¢*2.8 Gly CSF. U937 cells were cultured in the presence of PMA (10 ng/ml). Data are 200fc i means Â±SE (bars) of 3 replicates. j-15.0 DISCUSSION 150Â£â€¢=2 Â«I

A\i\5 S-1L.O The monocytic cell line U937 is a well-established model for 100-oo*CÃ¶ Â¿-5.0 the study of maturation and activation of cells of the monocyte- macrophage lineage (19, 20). GM-CSF has been shown to 50-TJ_CU. ,. induce differentiation of these cells (21) and to inhibit their Bflâ€¢-*\'&Hiflgx&flJiâ€¢ttUk20.0 growth via TNF expression (4). Several investigators have used QRPMI|.4\â€¢ nU n cultured A375 cells as an indicator target cell for investigating 0^8 g g 2 6 1 38Fraction1B 22 26 30 34 macrophage tumoricidal activity (3, 18). In our study, we found Â¡C -DTNF No. "3 that A375-conditioned medium stimulates monocytes and U937 cells to produce TNF and that the factor produced by A375 cells which is responsible for this effect is GM-CSF. Fig. 3. Protein and activity profile of A375 CM processed over an anti-GM- CSF affinity column. (â€¢)absorbance profile. Columns. TNF-inducing activity. GM-CSF is a glycoprotein originally isolated from a T- The A375 CM was diluted 1/5 in control media (RPMI 1640). Fractions were lymphoblast cell line (22) on the basis of its capacity to stimulate assayed as described in Fig. 2. the formation of granulocyte-macrophage colonies in cultures of bone marrow progenitor cells. Infusion of recombinant Antibody to hGM-CSF prevented the production of inhibitory hGM-CSF into healthy and pancytopenic monkeys led to a activity by U937 cells treated with A375-conditioned medium dramatic leukocytosis and reticulocytosis (23). An improve (Fig. 5B). ment of granulocyte, monocyte, and reticulocyte counts was 2676

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1990 American Association for Cancer Research. A375 MELANOMA CELLS INDUCE TNF RELEASE VIA GM-CSF PRODUCTION observed after administration of rhGM-CSF to patients with cytolysin and leukoregulin (35). Recently, Zarling et al. (36) aplastic anemia and myelodysplastic syndrome (24). However, identified a growth-inhibitory factor produced by activated T- GM-CSF is not only a hematopoietic growth-regulatory factor. cells which inhibited the growth of certain tumor cell lines It controls not only the formation but also the activity of mature including the A375 melanoma. This polypeptide growth regu phagocytes. GM-CSF induces monocytes to become tumorici- lator has a molecular weight of 30,000 and is released by U937 dal for the A375 melanoma (3); stimulates TNF production by cells as well as by activated T-lymphocytes. The NH^-terminal human mononuclear cells (25), especially upon co-addition of amino acid sequence reveals that it is distinct from other lipopolysaccharide or 7-interferon (26); inhibits the migration growth-inhibitory factors. Receptors for this factor, called On- of neutrophils (22); and enhances their phagocytic activity, costatin M, have been identified on target cells. The binding antibody-dependent cytotoxicity, and IL-1 production (27-29). interactions are complex and probably represent several differ Mice transfected with the murine GM-CSF gene showed accu ent classes of binding sites (37). mulation of activated macrophages in the peritoneal and pleural Recent papers have shown that both TNF and IL-1 stimulate cavities and developed tissue damage associated with infiltra GM-CSF production by fibroblasts, endothelial cells, and tu tion of macrophages. Most of these mice died at an early age, mor cell lines (38-41), raising the likelihood of complex loops underweight and with muscle wasting, suggesting excessive between tumor factors, immune cells, and other host cells. production of the cachectic cytokines TNF and IL-1 by GM- Although the overall importance of these interactions will be CSF-activated macrophages (30). We show in the present study- difficult to unravel, they may be responsible not just for regu that the A375 melanoma cells cause, through the release of lating tumor growth but also for some paraneoplastic syn GM-CSF, the release of TNF by monocytes and the monocytic dromes associated with malignancy such as leukocytosis, cach cell line U937. Together with earlier reported studies, these exia, and hypercalcemia. Clearly, the production of GM-CSF data suggest that production of GM-CSF by tumor cells may by A375 cells may modulate specific functions in the host with limit the growth of the tumor itself by the production of TNF profound consequences on the balance between tumor growth by monocyte-macrophages. and host defense. As noted above, GM-CSF induces monocyte tumoricidal activity. This effect is mediated by TNF (5), which may be either soluble or membrane-bound. In these monocyte-cytotox- ACKNOWLEDGMENTS icity experiments coculture was performed with target tumor We would like to thank Dr. Makio Ogawa and Anne Leary for their cells; cell-cell contact may have been required. However, it is help with the CSF assays. We thank Thelma Barrios for her excellent known that TNF inhibits the growth of several tumor cell lines secretarial assistance. (31) including, as shown in this study, the A375 melanoma suggesting that cell-cell contact is not necessary in this system. 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Massimo Sabatini, Jeffery Chavez, Gregory R. Mundy, et al.

Cancer Res 1990;50:2673-2678.

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