RAW macrophages induce apoptosis selectively in transformed fibroblasts: intercellular signaling based on reactive oxygen and nitrogen species

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1 RAW macrophages induce apoptosis selectively in transformed fibroblasts: intercellular signaling based on reactive oxygen and nitrogen species Stefanie Heigold and Georg Bauer Abteilung Virologie, Institut für Medizinische Mikrobiologie und Hygiene, Universität Freiburg, Germany Abstract: The rationale for this study was to determine whether macrophages induce apoptosis selectively in transformed compared with nontransformed fibroblasts and to elucidate the underlying intercellular signaling chemistry. Murine fibroblasts transformed by oncogene expression (ras, src) or methylcholanthrene treatment were sensitive for apoptosis induction by RAW macrophages, whereas parental cells and revertants were insensitive. Moreover, RAW macrophages induced apoptosis in normal rat kidney (NRK) fibroblasts transiently transformed by epidermal growth factor/transforming growth factor-. Sensitivity for intercellular apoptosis induction was based on target cell-derived superoxide anions and effector cell-derived peroxidase and nitric oxide (NO). Superoxide anions dismutate to hydrogen peroxide, which is converted to HOCl by the peroxidase. The interaction of HOCl with superoxide anions then generates hydroxyl radicals. In parallel, NO interacts with superoxide anions and generates apoptosis-inducing peroxynitrite. Signaling by reactive oxygen and nitrogen species seems to represent a hitherto unrecognized signaling principle for the selective elimination of potential tumor cells by macrophages. J. Leukoc. Biol. 72: ; Key Words: peroxidase nitric oxide superoxide anion INTRODUCTION Macrophages represent an essential part of the natural antitumor defense system. They use several different signaling mechanisms for apoptosis induction in tumor cells. The specificity of these mechanisms with regard to the transformed state of potential target cells and their potential interdependencies and interactions is not yet completely understood. Release of tumor necrosis factor (TNF) and transforming growth factor- (TGF- ) represents one aspect of macrophage anti-tumor action [1 6]. TNF interacts with transformed and nontransformed cells, but selective apoptosis induction in transformed cells derives from a selective target cell response, which seems to be based on a decreased concentration of endogenous apoptosis inhibitors [7]. TGF- has been shown to induce apoptosis in certain tumor cell lines [8 17] and to modulate the sensitivity of certain tumor cells for apoptosis induction by the apo/fas system [18] or by DNA-damaging agents [19]. The molecular basis of TGF- -mediated anti-tumor action is not completely resolved. However, there is strong evidence for an involvement of reactive oxygen species (ROS) in this process [11, 20]. Direct apoptosis induction by TGF- seems not to be restricted to tumor cells, as nontransformed cells have also been reported to be affected under certain conditions [8, 10, 20, 21]. So far, it remains enigmatic how sensitivity to apoptosis induction by TGF- is controlled on the molecular level. Macrophages release nitric oxide (NO), which is known to be involved in anti-tumor defense [22, 23]. Recent evidence indicates that NO may interact with transformed, cell-derived superoxide anions and thereby generate the apoptosis inducer peroxynitrite [24, 25]. As extracellular superoxide anion production represents a hallmark of the transformed state [26, 27] (for review, see refs [28, 29]), peroxynitrite generation based on interaction of NO with target cell-derived superoxide anion may represent the key for selective, NO-based anti-tumor action by macrophages. Myeloperoxidase (MPO), which is known to be involved in the tumoricidal activity of granulocytes (in cooperation with their oxidative burst) [30, 31] (for review, see ref [32]), is not considered a typical macrophage-specific enzyme. However, direct measurements indicated that RAW macrophages synthesize MPO, although in much lower concentration than granulocytes [33]. Therefore, macrophages might also use the peroxidase/hocl-dependent signaling pathway for selective apoptosis induction as recently described [24, 28, 29] and discussed below. We have recently shown that TGF- -pretreated, nontransformed fibroblasts use target cell-generated ROS for selective apoptosis induction in transformed target cells [24] (for review, see refs [28, 29]). Signaling is based on superoxide anion generation by transformed target cells and uses two signaling pathways: the HOCl/hydroxyl radical and the NO/peroxynitrite pathway. The HOCl/hydroxyl radical pathway requires dismutation of superoxide anions to hydrogen peroxide. Effector cell-derived peroxidase then converts hydrogen peroxide to hypochlorous acid, which interacts with target cell-derived Correspondence: Georg Bauer, Abteilung Virologie, Hermann-Herder Str. 11, D Freiburg, Germany. tgfb@ukl.uni-freiburg.de Received July 12, 2001; revised February 26, 2002; accepted April 16, Journal of Leukocyte Biology Volume 72, September

2 superoxide anions to yield highly reactive, apoptosis-inducing hydroxyl radicals. As superoxide anions have an extremely short free-diffusion pathway [34], hydroxyl radical generation is confined to the intimate vicinity of the membrane of target cells and thus warrants selectivity of apoptosis induction. In a parallel signaling pathway, effector cell-derived NO is converted to apoptosis-inducing peroxynitrite through target cellderived superoxide anions. Extracellular superoxide anion production through membrane-associated reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase represents a general feature of many transformed and tumor cells [26, 27, 35 37] and therefore may be the key for selective apoptosis induction by natural anti-tumor systems. The aim of this study was to elaborate whether transformed cell-derived superoxide anions are the basis for efficient and selective apoptosis induction by macrophages. As the src- or ras-transformed rat fibroblasts used as target cells in this study are not sensitive to direct apoptosis induction by TNF or TGF- under the conditions of our assays (G. Bauer, unpublished observation), they should allow for studying peroxidase- and NO-based signaling pathways exclusively. MATERIALS AND METHODS 4-Hydroxy-3-methoxyacetophenone [acetovanillone, apocynin (APO)] was obtained from Calbiochem (San Diego, CA) and kept as a stock solution of 2.5 mg/ml in medium at 20 C. APO represents a specific inhibitor of NADPH oxidase [38 40]. N-omega-nitro-L-arginine methylester hydrochloride (NAME) and N6- methyl-l-arginine (NMMA) were obtained from Sigma Chemical Co. (St. Louis, MO). Stock solutions (60 mm) in medium were kept at 20 C. NAME and NMMA inhibit NO synthesis. 5-, 10-, 15-, 20-Tetrakis(4-sulfonatophenyl)porphyrinato iron(iii) chloride (FeTPPS) was obtained from Calbiochem. Stock solutions (10 mm) were kept at 20 C. FeTPPS represents a specific decomposition catalyst for peroxynitrite [41, 42]. Control experiments ensured that FeTPPS effectively decomposes peroxynitrite but that it does not affect hydrogen peroxide or superoxide anions [25]. Superoxide dismutase (SOD; from bovine erythrocytes) was obtained from Sigma Chemical Co. Stock solutions [30,000 units/ml in phosphate-buffered saline (PBS)] were kept at 20 C and only used once per aliquot. Catalase (from Aspergillus niger) was obtained from Sigma Chemical Co. and applied at a final concentration of 77 U/ml. Taurine (Sigma Chemical Co.) was kept as a stock solution of 500 mm in medium at 20 C. The solution had been passed through a sterile filter. Taurine represents a specific scavenger of HOCl [43]. 4-Aminobenzoyl hydrazide (ABH; Acros Organics, Geel, Belgium) was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 1 M. It was then diluted with medium to a concentration of 1 mm (stock solution). The stock solution was kept at 20 C. The remaining 0.1% DMSO in the stock solution was diluted to % final concentration in the assays, which was found to be without effect on intercellular induction of apoptosis or MPO activity. ABH represents a mechanism-based inhibitor of MPO [44 46]. Terephthalate was obtained from Sigma Chemical Co. It was kept as a stock solution of 40 mm in water at 20 C. Terephthalate represents a specific scavenger of hydroxyl radicals [47]. TGF- 1 has been purified from human platelets. Cell lines and cell culture Murine RAW macrophages were a gift of Dr. Bruene (Erlangen, Germany). They were kept in suspension in RPMI medium containing 10% fetal calf serum (FCS) that had been heated for 30 min at 56 C prior to use. Medium was supplemented with penicillin (40 U/ml), streptomycin (50 g/ml), neomycin (10 g/ml), moronal (10 U/ml), and glutamine (280 g/ml). Cell culture was performed in plastic tissue culture flasks. Cells were passaged once or twice weekly. Cell density was never lower than 300,000 cells/ml. Nontransformed rat fibroblasts 208 F and their src oncogene-transformed derivatives 208 F src3 and 208 F cells with an inducible H-ras oncogene (IR-1) were a generous gift of Drs. C. Sers and R. Schäfer (Charité, Berlin). Ras oncogene induction through addition of 20 mm isopropyl -D-thioglactoside (IPTG) to IR-1 cells caused the expression of the transformed phenotype within h. 208 F src3 cells and IPTG-treated IR-1 cells show crisscross morphology, form colonies in soft agar, and are sensitive to intercellular induction of apoptosis by TGF- -pretreated fibroblasts [24, 48, 49], whereas parental 208 F cells do not exhibit these features of transformed cells. Revertants derived from 208 F src3 cells have been recently described [49]. Revertants have lost the morphological characteristics of transformed cells such as criss-cross morphology and colony formation in soft agar. They have been shown to be resistant to intercellular induction of apoptosis by TGF- pretreated fibroblasts. The nontransformed mouse fibroblast line C3H 10 T1/2 and its methylcholanthrene-transformed derivative MCA 18 Cl18 have been recently described [50]. Normal rat kidney (NRK) 536 rat fibroblasts were obtained from Dr. U. Rapp (Würzburg). Treatment of NRK 536 with TGF- and epidermal growth factor (EGF) causes transient morphological transformation [51 53]. Fibroblasts were kept in Eagle s minimal essential medium containing 5% FCS that had been heated for 30 min at 56 C prior to use. Medium was supplemented with penicillin (40 U/ml), streptomycin (50 g/ml), neomycin (10 g/ml), moronal (10 U/ml), and glutamine (280 g/ml). Cell culture was performed in plastic tissue culture flasks. Cells were passaged once or twice weekly. Determination of intercellular induction of apoptosis in transformed cells cocultured with RAW macrophages without direct cell-to-cell contact For cocultivation of cells without cell-to-cell contact, cell-culture clusters with inserts were used [pore-size of inserts, 0.4 m (Falcon, obtained from Becton Dickinson, Heidelberg, Germany); distance between cell layers, approximately 2 mm]. RAW macrophages as effector cells were seeded into the inserts ( cells per insert or as indicated in the respective figure legends). The inserts were placed above 40,000 transformed target cells in six-well plates. Determination and quantitation of apoptosis were based on the classical, morphological criteria membrane blebbing, nuclear condensation, and nuclear fragmentation. These were determined using inverted-phase contrast microscopy, as recently described [49, 50]. The percentage of apoptotic cells (apoptotic cells/total number of cells inspected) was determined from at least a total of 200 cells categorized per assay. Apoptotic cells were attached or rounded and showed membrane blebbing, membrane blebbing and nuclear condensation/fragmentation, or nuclear fragmentation/condensation without blebbing. (These cells seem to represent later stages of apoptosis where the blebs have already been lost.) Care was taken to differentiate apoptotic cells from nonapoptotic rounded cells with intact nuclei, reflecting mitotic stages. All quantitative data in this paper were derived using this method. Parallel assays ensured that apoptotic cells characterized by morphological criteria as described above showed a positive TUNEL reaction, indicative of free 3 hydroxyl groups of the DNA, one of the hallmarks of apoptotic cells. DNA strand breaks (free 3 hydroxyl groups) were detected by the TUNEL reaction [54] using a commercially available detection kit (Boehringer, Mannheim, Germany). It is based on the incorporation of fluorescein-labeled deoxyuridine triphosphate by terminal deoxynucleotidyl transferase. After the TUNEL reaction, cells were stained with 1 g/ml bisbenzimide (in PBS) for 30 min for the verification of chromatin condensation and fragmentation. Differentiation of intact and depolarized mitochondria through staining with rhodamine 123 is described in the legend to Figure 3. Statistical analysis In all experiments, assays were performed in duplicate. The mean values (from duplicate assays within the same experiment) and the empirical standard deviations were calculated and are shown in the figures. Absence of standard Heigold and Bauer Apoptosis inclination by macrophages 555

3 RESULTS Fig. 1. Apoptosis induction in transformed cells by RAW macrophages depends on the number of effector cells. Transformed 208 F src3 fibroblasts (40,000) were seeded in Costar six-well tissue-culture clusters as target cells. After the cells had attached, tissue-culture inserts (pore size, 0.4 m) were placed above the target cells. Tissue-culture inserts received the indicated numbers of RAW macrophages. Controls remained without macrophages. All assays were performed in duplicate. The percentage of apoptotic cells (characterized by membrane blebbing and/or nuclear condensation/fragmentation) was determined kinetically. deviation bars for certain points indicates that the standard deviation was too small to be reported by the graphic program; i.e., results obtained in parallel were nearly identical. Empirical standard deviations were calculated merely to demonstrate how close the results were obtained in parallel assays within the same experiment and not with the intention of statistical ANOVA, which would require larger numbers of parallel assays. The Yates continuity-corrected -square test was used for the statistical determination of significances. Src-oncogene transformed target cells were seeded in six-well tissue culture clusters, and RAW macrophages were added in tissue culture inserts. These allow exchange of signaling molecules between the two populations of cells, and a distance of about 1 mm prevents direct cell-to-cell contact. RAW macrophages induced apoptosis in transformed target cells, dependent on the time and on the concentration of effector cells (Fig. 1). Apoptotic transformed cells were characterized by membrane blebbing and chromatin condensation or fragmentation. For further verification of apoptotic cell death, transformed cells in coculture with RAW macrophages were stained for chromatin structure (bisbenzimide staining) and DNA strand breaks (TUNEL) reaction. As can be seen in Figure 2, RAW cells induced chromatin condensation/fragmentation and DNA strand breaks in transformed target cells. Based on these two criteria and on the morphological appearance of challenged target cells, their death was characterized as apoptosis. Staining for mitochondrial activity revealed that apoptosis induction in transformed cells through coculture with RAW macrophages was paralleled by mitochondrial depolarization (Fig. 3). Depolarization was frequently found in cells that did not yet show the morphological signs of apoptosis, indicating that mitochondrial depolarization preceded chromatin condensation and membrane blebbing. To test for the role of target cell density during intercellular signaling, 10,000 transformed 208 F src3 cells were seeded dispersely or in four distinct clumps of fourfold higher density. In parallel, 40,000 target cells were seeded dispersely. Target cells were then challenged with RAW macrophages. The rationale for this experiment was based on recent work from our group that had shown that 10,000 dispersely seeded transformed target cells did not produce optimal hydrogen peroxide for efficient intercellular signaling (with TGF- -pretreated, nontransformed fibroblasts as effector cells), whereas 10,000 cells seeded at high local density or 40,000 cells seeded Fig. 2. Apoptosis induction in transformed fibroblasts by RAW macrophages: verification by the TUNEL reaction. Assays were performed as described in Figure 1 using transformed 208 F src3 fibroblasts as target and RAW macrophages as effector cells. Transformed target cells were stained for DNA strand breaks following the TUNEL reaction described by Gorcyca et al. [54] after 3 days of coculture between effector and target cells. After performance of the TUNEL reaction, cells were stained with bisbenzimide as recently described [49]. (A, B) Transformed target cells challenged by RAW macrophages; (C, D) transformed cells cultivated without macrophages. (A, C) Bisbenzimide staining (for detection of condensed/fragmented nuclei); (B, D) TUNEL staining of the same area (for detection of DNA strand breaks). Note the condensed nuclei (A), which correspond to a positive TUNEL reaction (B). (C, D) Only a minor background of apoptotic cells. 556 Journal of Leukocyte Biology Volume 72, September

4 Fig. 3. Intercellular induction of apoptosis in srctransformed fibroblasts by RAW macrophages aims at target-cell mitochondria. Assays were performed as described in Figure 1, using transformed 208 F src3 fibroblasts as target and RAW macrophages as effector cells. After 3 days of coculture, rhodamine 123 was added to target cells at a concentration of 5 g/ml, and the assays were incubated for 30 min at 37 C. Medium was removed, and the cells were washed twice with PBS before the assays were inspected by fluorescence microscopy. Rhodamine 123 stains functional but not depolarized mitochondria. Mitochondrial depolarization and cytochrome C release are found during many signaling pathways involved in apoptosis induction and precede activation of caspase 3 [55 57]. (A, B) Transformed target cells challenged by RAW macrophages; (C, D) transformed cells cultivated without macrophages. (A, C) Phase-contrast microscopy of cells using visible light; (B, D) rhodamine staining for functional mitochondria. Note that practically all cells in the control show functional mitochondria, stained by rhodamine 123, whereas transformed target cells challenged by macrophages show a substantial number of cells with depolarized mitochondria, which are not stained by rhodamine 123. During the staining procedure, most of the cells with morphological signs of apoptosis (like blebbing and chromatin condensation/fragmentation) detach from the plate. These cells show no staining with rhodamine 123 (data not shown) and are absent in this photograph. The unstained cells (B) represent cells that already have depolarized mitochondria but do not exhibit the final signs of apoptosis such as membrane blebbing or chromatin condensation (therefore, these cells are still attached to the plate). This finding indicates that mitochondrial depolarization precedes the final state of apoptosis in transformed cells challenged by macrophages. dispersely did [24, 58]. Hydrogen peroxide derives from spontaneous dismutation of superoxide anions generated by target cells and therefore its generation depends on the cell density. Figure 4 demonstrates that RAW macrophages achieved higher apoptosis induction in 40,000 transformed target cells or in 10,000 target cells seeded at high local density than in 10,000 target cells seeded at lower density. Therefore, the efficiency of apoptosis induction seems to depend on target-cell density rather than target-cell number. This finding also shows that transformed target cells significantly contribute to signaling, which leads to their own apoptosis. To test whether the apoptosis-inducing effect of RAW macrophages was specific for target cells exhibiting the transformed phenotype, several types of target cells were challenged with RAW macrophages, and sensitivity for apoptosis induction was measured (Figs. 5 7). Whereas rat fibroblasts with constitutive expression of the src oncogene were sensitive for apoptosis induction by RAW cells, nontransformed, parental 208 F cells remained insensitive (Fig. 5). 208 F cells with an inducible ras oncogene were only sensitive when ras expression had been induced through the addition of IPTG. Similarly, mouse fibroblasts transformed by the chemical carcinogen methylcholanthrene (MCA) [50] were sensitive for apoptosis induction by RAW cells, whereas their nontransformed parental cells (C3H 10 T1/2) were not. Revertants derived from 208 F src3 cells that had lost their transformed phenotype had also lost their sensitivity to apoptosis induction by RAW macrophages (Fig. 6). NRK 536 cells that were transiently induced to express the transformed phenotype through the combined action of TGF- and EGF showed sensitivity to apoptosis induction (Fig. 7). Treatment with either cytokine alone (which did not result in establishment of the transformed state) was not sufficient to induce sensitivity. These data demonstrate that RAW macrophages induce apoptosis selectively in cells that express the transformed phenotype. Fig. 4. Apoptosis induction in transformed target cells depends on their density. Transformed 208 F src3 fibroblasts were seeded in Costar six-well tissue-culture clusters in three different configurations: 10,000 cells disperse (10 T), 10,000 cells at high local density (10 T HD), and 40,000 cells disperse (40 T). High local density was achieved by seeding four clumps of 2500 cells in 7 l medium. After the cells had attached (1 2 h), medium was added to 3 ml. Target cells were then cocultured with tissue-culture inserts containing 120,000 RAW macrophages. Control assays remained free of effector cells. Assays were performed in duplicate The percentage of apoptotic cells (characterized by membrane blebbing and/or nuclear condensation/fragmentation) was determined at day 3 of coculture. Heigold and Bauer Apoptosis inclination by macrophages 557

5 Fig. 6. Revertants from src-transformed cells have lost the transformed phenotype as well as sensitivity for apoptosis induction by RAW macrophages. Transformed 208 F src3 fibroblasts (40,000) or their revertants R2, R4, or R5 [49] were seeded in Costar six-well tissue-culture clusters. Assays received 120,000 RAW macrophages in tissue-culture inserts or remained free of effector cells (control). All assays were performed in duplicate. The percentage of apoptotic cells (characterized by membrane blebbing and/or nuclear condensation/fragmentation) was determined kinetically. Fig. 5. Selective apoptosis induction in src-, ras-, or chemically transformed fibroblasts. The following target cells were seeded in Costar six-well tissue clusters (40,000 cells per assay; target cell line indicated in the left corner of each graph): 208 F src3 cells (constitutive src expression); nontransformed 208 F parental cells; IR-1 cells pretreated with IPTG for 2 days for induction of RAS expression (and IPTG treatment continued during the experiment); IR-1 control cells without RAS induction; MCA 18 Cl18 (C3H 10 T1/2 cells transformed by methylcholanthrene); and parental C3H 10 T1/2 cells. Tissueculture inserts received 120,000 RAW macrophages. Control assays of target cells remained free of effector cells (control). Assays were performed in duplicate. The percentage of apoptotic cells (characterized by membrane blebbing and/or nuclear condensation/fragmentation) was determined kinetically. Oncogenic transformation has been shown to induce extracellular superoxide anion production through a membraneassociated NADPH oxidase [26]. Published as well as ongoing work in our laboratory have confirmed that 208 F cells transformed by an inducible ras oncogene or a constitutively expressed src oncogene, C3H 10 T1/2 cells transformed by the chemical carcinogen MCA, and NRK 536 cells transiently transformed by the addition of EGF plus TGF- -generated extracellular superoxide anions. Extracellular superoxide anion generation (followed by spontaneous dismutation to hydrogen peroxide) rendered the cells sensitive to peroxidase-based apoptosis induction through HOCl synthesis and hydroxyl radical generation [24, 53, 58]. In contrast, nontransformed 208 F, C3H 10 T1/2, or NRK cells showed no or only marginal extracellular superoxide anion production. To test whether Fig. 7. Transient induction of the transformed phenotype in NRK 536 cells causes transient sensitivity to apoptosis induction by RAW macrophages. NRK 536 fibroblasts (30,000) were seeded in Costar six-well tissue-culture clusters. They were pretreated for 2 days with EGF (1 ng/ml), TGF- (1 ng/ml), EGF plus TGF- (1 ng/ml each), or without addition. Then 120,000 RAW macrophages in tissue-culture inserts were placed above the cultures. Control assays (control, EGF, TGF, and TGF plus EGF) remained without effector cells. All assays were performed in duplicate. The percentage of apoptotic cells (characterized by membrane blebbing and/or nuclear condensation/fragmentation) was determined kinetically. 558 Journal of Leukocyte Biology Volume 72, September

6 apoptosis induction in transformed fibroblasts by RAW macrophages was dependent on extracellular superoxide anion generation, transformed fibroblasts were challenged with RAW macrophages in the absence and presence of APO. APO represents a selective and efficient inhibitor of NADPH oxidase [38 40]. As can be seen in Figure 8, APO markedly blocked apoptosis induction in transformed fibroblasts by RAW macrophages, thus demonstrating the central role of superoxide anions for this process. To test whether the interaction of RAW macrophages with transformed target cells is based on the same ROS and reactive nitrogen species (RNS)-mediated, intercellular signaling chemistry as recently described for intercellular induction of apoptosis by nontransformed fibroblasts, src-transformed fibroblast were challenged by RAW macrophages in tissue-culture inserts in the presence of inhibitors that interfere with the HOCl/hydroxyl radical signaling pathway and the NO/peroxynitrite signaling pathway. The following inhibitors were used: SOD (to delineate the role of extracellular superoxide anions), catalase (to test for an involvement of hydrogen peroxide), the mechanism-based peroxidase inhibitor ABH (to test for an involvement of peroxidase), taurine (to test for the potential role of HOCl), terephthalate (to test for an involvement of hydroxyl radicals), and NAME (to delineate the potential role of NO). As can be seen in Figure 9, catalase, ABH, taurine, SOD, and terephthalate showed a strong, inhibitory effect, and NAME showed a weaker yet distinct, inhibitory effect on apoptosis induction in src-transformed cells by RAW macrophages. These findings point to the involvement of the HOCl/hydroxyl radical and the NO/peroxynitrite signaling pathway for apoptosis induction. Fig. 9. Intercellular signaling during the interaction of macrophages and transformed fibroblasts. Transformed 208 F src3 cells (40,000) were seeded in Costar six-well tissue-culture clusters. Assays received tissue-culture inserts with 120,000 RAW macrophages (TCI) or no effector cells (CONTROL, etc.). Assays received the following inhibitors: none (CONTROL and TCI); catalase, 77 U/ml (CAT); ABH, 50 g/ml: taurine, 25 mm (TAU); SOD, 150 U/ml; terephthalate, 200 M (TER), and NAME, 1.2 mm. All assays were performed in duplicate. The percentages of apoptotic cells (characterized by membrane blebbing and/or nuclear condensation/fragmentation) were determined after 2 days of coculture. Statistical analysis showed that inhibition of RAW macrophage-mediated apoptosis induction by catalase, ABH, SOD, taurine, and terephtalate was highly significant (P 0.001), whereas the weak inhibition by NAME was border-line significant (P 0.05) at the time point shown. One day later, inhibition by NAME was still much weaker than inhibition by the other scavengers but was significant (P 0.001). A follow-up experiment is shown in Figure 10. Fig. 8. Apoptosis induction in transformed fibroblasts by RAW macrophages is inhibited by APO, a specific inhibitor of NADPH oxidase. Transformed 208 F src3 cells (40,000) were seeded in Costar six-well tissue-culture clusters. Assays received tissue-culture inserts with 120,000 RAW macrophages ( RAW) or no effector cells (CONTROL). Assays received 50 /ml NADPH oxidase inhibitor APO or no addition. All assays were performed in duplicate. The percentage of apoptotic cells (characterized by membrane blebbing and/or nuclear condensation/fragmentation) was determined kinetically. As NAME had only shown a weak, inhibitory effect, the significance of the NO/peroxynitrite signaling pathway for intercellular induction of apoptosis in transformed fibroblasts by RAW macrophages was tested in a repeat experiment. In this experiment, a second inhibitor of NO synthesis, NMMA, was applied in parallel to NAME. To test for the potential role of peroxynitrite (potentially generated by the interaction of NO with target cell-derived superoxide anions) as an ultimate apoptosis inducer, FeTPPS, a selective peroxynitrite-decomposition catalyst [41, 42], was also applied. As shown in Figure 10, both inhibitors of NO synthesis as well as FeTPPS showed a significant inhibition of RAW macrophage-dependent apoptosis induction in transformed fibroblasts. Both inhibitors of NO synthesis as well as FeTPPS showed a similar degree of inhibition, indicating that NO synthesis as well as peroxynitrite formation were involved in apoptosis induction. As the effect of these inhibitors of the NO/peroxynitrite signaling pathway was weaker than the effect of taurine (an inhibitor of the HOCl/ hydroxyl radical signaling pathway), it may be concluded that the NO/peroxynitrite signaling pathway plays a minor yet distinct role during intercellular signaling. Heigold and Bauer Apoptosis inclination by macrophages 559

7 Fig. 10. Involvement of the NO/peroxynitrite signaling pathway during intercellular signaling. Transformed 208 F src3 cells (40,000) were seeded in Costar six-well tissue-culture clusters. Assays received tissue-culture inserts with 120,000 RAW macrophages or no effector cells (CONTROL). Where indicated, assays received the following additions: NAME (1.2 mm), NMMA (1.2 mm), FeTPPS (20 M), or taurine (TAU; 25 mm). All assays were performed in duplicate. The percentage of apoptotic cells (characterized by membrane blebbing, nuclear condensation/fragmentation) was determined after 2 days of coculture. NAME and NMMA represent inhibitors of NO synthesis, whereas FeTPPS is a specific decomposition catalyst for peroxynitrite. Taurine scavenges HOCl. Therefore, inhibition by taurine indicates the degree of involvement of the HOCl/hydroxyl radical signaling pathway in addition to the NO/signaling pathway, which seems to be less prominent. Statistical analysis showed that NAME, NMMA, or FeTTPS inhibited RAW macrophagemediated apoptosis significantly (P 0.001). There was no significant difference among the inhibitory effects of NAME, NMMA, or FeTPPS. This result was expected, as they seem to act at the same signaling pathway. Taurine also caused significant inhibition (P 0.001) of RAW macrophage-mediated apoptosis induction. Inhibition of apoptosis induction by taurine (an inhibitor of the HOCl/hydroxyl radical pathway) was significantly different (P 0.001) from inhibition by NAME, NMMA, or FeTTPS, which represents inhibitors of the NO/peroxynitrite pathway. DISCUSSION Our data show that RAW macrophages cause apoptosis induction selectively in transformed fibroblasts. Selectivity was assured, as cells exhibiting the transformed phenotype through constitutive src expression, induced ras expression, transformation by methylcholanthrene, or TGF- /EGF treatment were sensitive, whereas nontransformed parental cells were insensitive to apoptosis induction. Furthermore, src-transformed cells that had lost their transformed state after reversion had also lost sensitivity to apoptosis induction. These data demonstrate a strict correlation between the expression of the transformed phenotype and sensitivity for apoptosis induction by macrophages. Fibroblasts that express the transformed state differ from parental cells through constitutive, extracellular generation of superoxide anions. This had been first demonstrated by Irani et al. [26] for ras-transformed fibroblasts. Src- or ras-transformed fibroblasts as well as MCA-transformed fibroblasts used in our study have also been shown to generate extracellular superoxide anions, as addition of NO caused apoptosis, which was blocked by extracellular SOD [24, 25]. In addition, src- and ras-transformed cells were subject to apoptosis induction by extracellular MPO [58]. This reaction requires superoxide anion at two distinct steps: hydrogen peroxide production through superoxide anion dismutation [59, 60] and superoxide anion interaction with HOCl synthesized by MPO [32, 58], resulting in the generation of apoptosis inducing hydroxyl radicals [61, 62]. Similarly, NRK 536 cells transiently transformed by TGF- plus EGF were sensitive to apoptosis induction by MPO in a reaction that was blocked by SOD, pointing to the role of cell-derived superoxide anions generated by EGF/TGF- treated NRK cells [53]. Therefore, all transformed cell lines that were sensitive to apoptosis induction by macrophages also exhibited extracellular superoxide anion generation, whereas insensitive cells did not. The central functional role of superoxide anion generation by transformed cells for apoptosis induction mediated by RAW macrophages is demonstrated here, as either inhibition of superoxide anion generation through the NADPH oxidase inhibitor APO or scavenging superoxide anions through SOD blocked apoptosis induction by macrophages. Apoptosis induction in transformed cells by macrophages seems to depend on the hypochlorous acid/hydroxyl radical and the NO/peroxynitrite-signaling pathway, similar to the interaction of nontransformed and transformed fibroblasts [24] (Fig. 11). The evidence for the HOCl/hydroxyl radical signaling pathway for RAW macrophage-mediated apoptosis induction is based on the inhibition by APO and SOD (which show the functional role of superoxide anions), the inhibition by catalase (pointing to the role of hydrogen peroxide), the inhibition by the mechanism-based peroxidase inhibitor ABH (which shows that a peroxidase is involved), the inhibition by taurine (which demonstrates the functional role of HOCl), and the inhibition by terephthalate (pointing to the role of hydroxyl radicals). As superoxide anions and HOCl in the molar concentration range have no apoptosis-inducing potential by themselves (for review, see ref [28]), the most likely scenario derived from the inhibitor experiments and based on the known redox potentials of the molecules involved is the following: Transformed cellderived superoxide anions foster hydrogen peroxide generation, which then allows the effector cell-derived peroxidase to synthesize HOCl (therefore, apoptosis induction by RAW macrophages is inhibited by catalase, ABH, and taurine). HOCl is converted by superoxide anions to yield hydroxyl radicals, which are efficient apoptosis inducers (therefore, apoptosis induction is inhibited by APO, SOD, and terephthalate). Transformed target cells seem to be a sufficient source for ROS required for selective apoptosis induction, as addition of purified MPO (in the absence of effector cells) caused apoptosis selectively in transformed target cells compared with nontransformed cells [58]. Apoptosis induction in these reconstitution experiments was inhibited by scavenging superoxide anions, 560 Journal of Leukocyte Biology Volume 72, September

8 Fig. 11. Proposed model for selective apoptosis induction in transformed fibroblasts by macrophages. Macrophages release TNF, TGF-, NO, and peroxidase. TNF does not discriminate between transformed and nontransformed cells per se, but transformed cells are more sensitive to the action of TNF [7]. TGF- has been shown to sensitize transformed fibroblasts for apoptosis induction [18, 19, 63] and may thus possibly enhance the effects of the NO and peroxidase-based signaling pathways. Transformed fibroblasts generate extracellular superoxide anions through a membrane-associated NADPH oxidase [24 26, 58], which is inhibited by APO. Transformed cell-derived superoxide anions interact with NO and form the ultimate apoptosis inducer peroxynitrite ( NO O 2 3ONOO k M 1 s 1 ) [64 72]. Superoxide anions dismutate and form hydrogen peroxide (2 O 2 2H 3H 2 O 2 O 2 k M 1 s 1 ) [59, 60, 73], which is used by peroxidase to synthesize HOCl (H 2 O 2 Cl H 3H 2 O HOCl) [32]. HOCl interacts with target cell-derived superoxide anions and generates highly reactive, apoptosis-inducing hydroxyl radicals (HOCl O 2 3 OH Cl O 2 k 10 7 M 1 s 1 ) [71, 74, 75]. The NO/peroxynitrite and the HOCl/hydroxyl radical signaling pathways mediated by macrophages are identical to the signaling pathways exerted by TGF- -treated, nontransformed fibroblasts [24] and by promyelocytes [76]. In each of these systems, target cell-derived superoxide anions drive the efficiency and selectivity of signaling. The limited, free-diffusion path length of superoxide anions [34] restricts generation of the ultimate apoptosis inducers to the direct vicinity of the transformed target cells. These signaling pathways have been delineated from the effect of specific inhibitors and the known interdependencies and redox potentials of the molecules involved. The site of action of the inhibitors is shown in this Figure (APO; Tau taurine; TER terephthalate; SOD; CAT catalase; and ABH). In contrast to many other transformed cells, 208 F src3 cells that have been used for these studies are not sensitive to apoptosis induction by TGF- or TNF under the conditions of our experiments. Therefore, they are allowed to delineate the ROS and RNS-based signaling chemistry exclusively. Mitochondria become depolarized during apoptosis induction in transformed fibroblasts by RAW macrophages (see Fig. 3). Therefore, mitochondrial ROS and cytochrome C may be involved in triggering the execution phase of apoptosis. hydrogen peroxide, hypochlorous acid, or hydroxyl radicals, pointing to the central role of target cell-derived ROS. Taken together, these findings allow the conclusion that apoptosis induction becomes effective when target cell-derived superoxide anions interact with effector cell-derived peroxidase. The evidence for the NO/peroxynitrite signaling pathway during RAW macrophage-mediated apoptosis induction is based on the partial inhibition by the NO synthase inhibitors NAME and NMMA. This finding indicates that NO generation is involved in apoptosis induction. As NO may form peroxynitrite through interaction with target cell-derived superoxide anions in a diffusion-driven reaction, peroxynitrite may represent the ultimate apoptosis inducer. This conclusion is in line with the inhibitory effect of the peroxynitrite decomposition catalyst FeTPPS [41, 42]. This model was also verified in reconstitution experiments [25], in which NO donors induced apoptosis selectively in transformed fibroblasts compared with nontransformed cells. As apoptosis induction mediated by NO donors was blocked by scavengers for superoxide anions and by FeTPPS, peroxynitrite is shown to represent the ultimate apoptosis inducer in this system, whereas NO in the absence of superoxide anions shows no apoptosis-inducing potential. In this signaling pathway, it therefore seems sufficient for selective apoptosis induction that effector cells release NO, and target cells generate superoxide anions. Intercellular signaling mediated by macrophages therefore seems to be identical to the model recently shown for apoptosis induction in transformed cells by TGF- -treated, nontrans- Heigold and Bauer Apoptosis inclination by macrophages 561

9 formed fibroblasts [24] or by promyelocytes [77]. In these natural anti-tumor systems, transformed cell-derived superoxide anions seem to be the central elements that control efficiency and selectivity of apoptosis induction in transformed cells. In line with this model, apoptosis induction in transformed fibroblasts by RAW macrophages depended on the number of effector cells (as a result of their release of peroxidase and NO) and was modulated by the density of the target cells. High density of target cells increases spontaneous dismutation of superoxide anions to hydrogen peroxide and therefore leads to optimal efficiency of the peroxidase reaction. RAW cells have been shown to release small amounts of MPO [33]. It is not clear whether this enzyme is sufficient for the reaction observed. As our study in the fibroblast system has demonstrated that fibroblasts release a novel peroxidase, different from classical MPO [24], the occurrence of a similar enzyme in macrophages is also possible. The resolution of this question awaits further experiments. Taken together, macrophages seem to possess several tools for their attack against transformed cells (Fig. 11). The selectively acting ROS- and RNS-based mechanisms described here are paralleled by TNF and TGF- secretion. [The latter two mechanisms have been fated out in our study, as src-transformed cells are not directly induced to apoptosis by TNF or TGF- under the conditions of our study (G. Bauer, unpublished observation).] In contrast to the ROS- and RNS-based mechanisms, TNF does not act selectively on transformed cells, but transformed cells show a differential response as they are more susceptible for apoptosis induction [7]. TGF- may have several functions in the concert of macrophage tumor cell interaction. It induces apoptosis in certain tumor cells directly, but it has also been shown to sensitize cells for TNF action and for intercellular induction of apoptosis [77]. In parallel, it may turn on intercellular induction of apoptosis in other effector cells such as fibroblasts. In total, a sophisticated and selective control system is effective, which may have to be overcome by transformed cells during tumor formation. The detailed knowledge of the individual branches of this control system will possibly be helpful to understand resistance mechanisms of tumor cells and to find ways to interfere with them. ACKNOWLEDGMENTS This work was supported by the Dr. Mildred-Scheel-Stiftung für Krebsforschung (Grant Ba3) and the Deutsche Forschungsgemeinschaft (Grant BA 626/4-1). REFERENCES 1. Beutler, B., Cerami, A. (1987) Cachectin: more than a tumor necrosis factor (Review). New Engl. J. Med. 316, Fernandez, A., Ananthaswamy, H. N. 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