Cell, Vol. 70, 47-56,

July IO, 1992, Copyright

0 1992 by Cell Press

The p70 Tumor Necrosis Factor Receptor Mediates Cytotoxicity Renu A. Heller, Kyung Song, Nancy Fan, and David J. Chang Institute of Cancer and Developmental Syntex Research 3401 Hillview Avenue Palo Alto, California 94303

Biology

Summary Tumor necrosis factor a (TNF) selectively kills tumor cells, but this specificity is not clearly understood. Two distinctly different cell surface receptors (TNFRs), proteins of 55 kd (~55) and 70-80 kd (p70), mediate TNF action. Mouse TAl cells are not killed by human (h) TNF, but are killed by mouse (m) TNF alone. Since the mouse p70 TNFR is recognized only by mTNF, these results implicate p70 receptor action in TAl cell killing. Human HeLa cells have mainly the p55 receptor and are not killed by hTNF alone. When transfected with the human p70 TNFR, HeLa p70 die within 24 hr. HeLa p70 cells also show reduced c-fos and manganous superoxide dismutase induction by TNF. NIH 3T3 mouse fibroblasts are sensitive to only mTNF, but overexpression of the human p70 receptor causes cell death by hTNF and increased sensitivity to mTNF. These results provide a direct function for the p70 TNFR in TNF-induced cytotoxicity.

introduction Tumor necrosis factor a (TNF) is a multifunctional cytokine secreted primarily by mitogen-activated macrophages (for reviews see Old, 1985; Beutler and Cerami, 1988). Its biological activities include TNF enhancement of cell growth (Sugarman et al., 1985; Vilcek et al., 1988) cytotoxicity toward certain cells and tumors (Urban et al., 1988; Larrick and Wright, 1990) modulation of differentiation of myelogenous leukemia cells into the monocyte macrophage pathway (Takeda et al., 1988) repression of adipocyte and myoblast differentiation (Torti et al., 1985; Miller et al., 1988) and mediation of endotoxic shock (Tracey et al., 1988). TNF also promotes inflammation, possibly through the induction of cell adhesion molecules (Dustin and Springer, 1988; Osborn et al., 1989) neutrophil and macrophage chemotactic factors (Streiter et al., 1989; Dixit et al., 1990) and the production of other cytokines such as IL-1 and IL-8 (Kaushansky et al., 1988; Kohase et al., 1988). TNF exerts these responses by binding to specific cell surface receptors present on almost all cells analyzed (Kull et al., 1985; Yoshie et al., 1986). The existence of more than a single form of the receptor was predicted from TNF cross-linking studies and monoclonal antibodies to the receptors (Hohmann et al., 1989; Brockhaus et al., 1990) and confirmed by protein purification and cDNA cloning (Heller et al., 1990a, 1990b; Loetscher et al., 1990;

Schall et al., 1990; Smith et al., 1990). The two completely different TNF receptors that bind TNFa and TNF6 with high affinity are proteins of 55 kd (~55) and 70-80 kd (p70), respectively. The genes for the two receptors map to human chromosomes 12 and 1 and in the mouse to conserved syntenic regions on chromosomes 6 and 4 (Milatovich et al., 1991; Goodwin et al., 1991). Their ligands, TNFa and TNF8, are encoded by closely linked genes (Spiess et al., 1986). Based on their amino acid sequence, the two receptors are only 29% identical to each other, and both share cysteine-rich repeats in their extracellular domains that are similar to the repeats in the NGF receptor and define a unique structural motif shared by several other cell surface receptors (Loetscher et al., 1990; Smith et al., 1990; ltoh et al., 1991; Dijrkop et al., 1992). TNF has been widely implicated in the killing of tumor cells, but not many tumor cell lines are killed by TNF alone (Sugarman et al., 1985; Shepard and Lewis, 1988). Cytotoxicity of TNF is enhanced by other cytokines such as interleukin-1 and interferon? but lowered by growth factors such as transforming growth factor a and 8 (Sugarman et al., 1987). From the many surveys conducted to analyze the responses of different cell types to TNF, a few sensitive cell lines have become favored model systems, such as the L929 mouse fibroblasts, ME180 human cervical carcinoma, and MCF-7 human breast carcinoma cells. More often TNF cytotoxicity has been studied indirectly in combination with metabolic inhibitors such as actinomycin D and cycloheximjde (CHX), compounds that make most cells vulnerable, while TNF alone has no effect on these cells (Kull and Cuatrecasas, 1981). Despite these various studies, the role of TNF per se in cell killing is not clear. Recently, with the cloning of the mouse homologs of the human ~55 and p70 receptors, it was observed that the mouse p70 receptor did not bind human TNF (Lewis et al., 1991; Goodwin et al., 1991). This result has an important implication, in that responses in mouse cells treated with hTNF are those mediated by the p55 receptor. For example, hTNF represses TAl mouse adipogenic cell differentiation into fat cells and also reverses differentiated TAl fat cells to fibroblasts (Toni et al., 1985). Cytotoxicity in TAl cells has only been tested with hTNF, but like HeLa and NIH 3T3 cells, TAl cells are not killed by hTNF alone. They are killed by the combined actions of hTNF and CHX (Reid et al., 1989) via a process in which the activity of A6-desaturase, an enzyme required for arachidonic acid biosynthesis, is involved (Reid et al., 1991). This indicates a requirement for arachidonic acid and possibly phospholipase Aa action in hTNF-CHX-induced killing process. In light of the binding of hTNF to onlythe mouse ~55 receptor, these actions in TAl cells are mediated by the mouse ~55 receptor. Other studies conducted with monoclonal antibodies specific for each of the two receptors have demonstrated common roles for the two receptors (Shalaby et al., 1990; Hohman et al., 1990). Using polyclonal antibodies as well as the specificity of murine TNF for mouse p70 receptor, cell proliferation in mouse thymocytes and CT6

Cell 48

murine T cells has been recognized as a mouse p70 receptor-mediated response, but TNF-CHX-mediated cytotoxicity of murine LM cells was associated with the p55 receptor (Tartaglia et al., 1991). Therefore, while many functions can be assigned to the p55 receptor (Torti et al., 1985; Reid et al., 1989, 1991; Shalaby et al., 1990; Hohman et al., 1990; Tartaglia et al., 1991) information on the function of p70 is sparse. In an attempt to identify a role for the p70 receptor, the well described TAl system was used to compare human and mouse TNF activities. Our results show that while TAl cells are not killed by hTNF, they are killed by mouse TNF alone, a dramatic and unexpected response that implies a role of p70 TNFR in cell killing. We have extended this observation to other cell lines such as HeLa and NIH 3T3. These cells are not killed by hTNF alone, but transfectants overexpressing the human p70 receptor become extremelysensitive and are killed. Theseobservations, while they do not address the role of the p55 TNFR, have led us to the conclusion that the p70 TNFR has a critical role in TNF-induced cell killing. Results This paper focuses on three cell lines: murine TAl and NIH 3T3 and human HeLa cells. These cells were characterized for the presence and relative abundance of the two TNFRs and for their cellular responses to human and mouse TNF. Determination of the Total Number and Presence of Both ~55 and p70 TNFRs The numberof receptors in the threecell linesduring exponential growth was measured by ‘251-labeled TNF binding experiments and derived from Scatchard plots. Analyses

showed that both HeLa and NIH 3T3 cells possess more than 4000 receptors per cell, and TAl cells contain about 2400 receptors per cell (Figure 1A). In view of the observation that hTNF binds only to murine ~55 TNFR (Lewis et al., 1991) we performed binding assays with TAl and NIH 3T3 cells in which bound ‘251-labeled mTNF was displaced by lOO-fold excess of either human or mouse unlabeled TNF, and the percent of total binding displaced by each of these ligands was determined. In TAl cells, hTNF could displace approximately 80%-85% of the total specific binding, whereas in NIH 3T3 cells this value was between 50%-60%. These observations were taken to indicate that both receptors are present in these cells. Support for these results was obtained from Northern blot analysis in which the expression of p55 and p70 mRNAs was examined with species-specific cDNA probes (Figure 16). A sirjgle mRNA transcript of 2.5 kb for p55 was observed in all three cell lines. For p70 two mRNA transcripts of 4.5 kb and 3.8 kb were present in the two murine NIH3T3 and TAl cell lines. Previously reported sizes for the single mRNA transcript for the murine p55 receptor are 2.3 kb or 2.6 kb, while the two transcripts for the murine p70 receptor were estimated as 4.1 and 3.2 kb or 4.5 and 3.6 kb (Goodwin et al., 1991; Lewis et al., 1991, respectively). No p70 mRNA was observed in HeLa cells. Nonetheless, the presence of p70 mRNA could be demonstrated in HeLa cells when its cDNA was amplified in the polymerase chain reaction with hp70 receptor sequen:e-specific oligonucleotide primers (data not shown). Taken together we can conclude from the above set of experiments that both receptors occur in all three cell lines, but their relative levels are different. Induction of c-fos and NF-~6 and the Repression of Fat-Specific Clone 27 (FSP27) in TAl Cells Three separate cellular responses were analyzed to test

Figure 1. Characterization and HeLa Cells for TNFR TAl

1 4ooI NIH3T3

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of TAl, expression

NIH 3T3,

(A) Specific binding of ‘%labeled TNF to TAI , NIH 3T3, and HeLa cells was performed. Each assay used 2 x 1O8 cells from exponentially growing cultures incubated with increasing amounts of ‘251-labeled TNF, with or without IOO-fold excess unlabeled TNF. Cells were washed, and the bound ligand was determined by counting cell-associated radioactivity. With TAl and NIH 3T3 cells binding of ‘%labeled mTNF (open squares) was displaced by a 1OOfold excess of human TNF (solid diamonds) or mouse TNF (solid squares), to determine presence of both ~55 and ~70 receptors. For HeLa cells ‘r”l-labeled hTNF (open squares) was displaced by lOO-fold excess of human TNF (solid diamonds). Estimated receptor numbers from Scatchard analysis for TAl are -2400 per cell; for NIH 3T3 -5100 per cell; and for HeLa 4400 P70 per cell. (6) RNA blot analyses for TNFR mRNAs were conducted with polyadenylated RNA (10 pg per lane) isolated from exponentially growing TAl, NIH 3T3, and HeLa cells and hybridized with mouse or human p55 cDNA probes (left panel) or ~70 cDNA probes (right panel). Positions of 2% and l&S ribosomal RNA bands are indicated. Estimated size for the ~55 receptor mRNA is 2.5 kb and for the two mouse ~70 receptor mRNA bands is 4.5 and 3.6 kb. mRNA for p70 in HeLa cells was not observed under these conditions.

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of mTNF versus hTNF. FSP27 gene is abundantly expressed in adipocytes and rapidly declines in expression in the presence of TNF. Therefore, our results shown here imply that at least in this cellular system these p55mediated responses are not significantly altered by p70 receptor activation.

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(A) Induction of c-fos and NF-KB and repression of FSP27 mRNA. c-fos expression was analyzed in confluent TAI preadipocytes treated with increasing concentrations of mTNF or hTNF for 30 min. Total RNA prepared from cells was analyzed for expression of the 2.2 kb c-fos mRNA. For NF-xB and FSP27, cells were treated with 50 nglml mTNF or hTNF for the times indicated. Total RNA was analyzed for the induction of the 3.8 kb NF-KB and 1.8 kb FSP27 mRNA. (B) Cytotoxic action of mTNF in TAl preadipocytes. Left panel is a time course of TNF treatment of TAl cells with 50 nglml human (open circles) or mouse (closed squares) TNF. The right panel is a dose response of TAI cells treated with 0, 2, 10,50, and 200 nglml human (open circles) or mouse (closed squares) TNF for 4 days. Results are expressed as percentage of surviving cells determined by crystal violet staining. The absorbance of the eluted dye in untreated cells is expressed as 100% and the data represent mean absorbance of eight treatment wells. Standard deviations ranged from 0.5% to 5%.

the differential effects of human and mouse TNF in TAl cells (Figure 2A). Two of these are the induction of the transcription factors c-fos and NF-KB by hTNF (Haliday et al., 1991; Meyer et al., 1991). The time course for c-fos mRNA induction in these cells is rapid and transient; it peaks in 30 min and declines to basal levels by45 min with an E& of lo-20 nglml (Haliday et al., 1991). In a doseresponse experiment, confluent TAl cells were treated with a range of mouse or human TNF concentrations for 30 min and then harvested for RNA preparation. Our data confirmed earlier observations of c-fos induction by a wide concentration range but did not provide any obvious difference between the relative effectiveness of mTNF versus hTNF in triggering this response. Likewise, in a time course of the induction of NF-KB, increased mRNA level was observed within 4 hr with both mTNF and hTNF, but no differential effects were noted. Using the repression of the fat-specific gene FSP27 (Danesch et al., 1992) as a measure of the ability of hTNF to reverse differentiation of TAl adipocytes, we detected no differences in the actions

Human TNF Is Not Toxic to TAl Cells, but Mouse TNF Causes Cell Death All of the cellular responses used in the comparative studies described above are relatively short term effects of TNF that occur within hours of treatment. The ability of TNF to inhibit adipocyte differentiation or mediate cytotoxicity are responses that require more time, in the order of days, and offer alternative systems for comparative studies of mouse and human TNF. TAl cells are resistant to the cytotoxic effects of hTNF alone, but their conversion from preadipocytes to adipocytes is blocked by hTNF (Torti et al., 1985). When the differentiation process in TAl preadipocytes was analyzed with mTNF, we observed a dramatic difference. Cells treated with mTNF alone could not survive this treatment and were completely killed by mTNF in a 3-4 day period. This killing process was both time and dose dependent (Figure 28) and suggested an involvement of the p70 receptor in initiating cytotoxicity. To examine further the involvement of the p70 receptor in cytotoxic action, we have performed experiments with other cell lines and overexpressed the human p70 TNFR in them to analyze any changes in their response to TNF. Studies with HeLa Cells and HeLa p70 Cells Overexpressing the Human p70 Receptor From ‘251-labeled hTNF binding studies, we had determined that HeLa cells contain approximately 4400 receptors per cell (Figure 1 A), and Northern analysis for transcript abundance had shown little p70 TNFR mRNA expression in these cells(Figure 1 B). Cross-linking experiments were also conducted to determine levels of receptor proteins. In HeLa cells, ~55 cross-linked products with ‘%labeled TNF of 75 kd and 95 kd have been detected, and in U937 and HL60 cells, p70 cross-linked complexes of primarily 98-100 kd have also been reported (Hohman et al., 1989). In our studies with HeLa cells, specific crosslinked products of p55 were recognized as complexes of 85-100 kd with a faint presence of additional higher sized products in the 105-l 25 kd range, most likely due to p70 (Figure 48, lanes 1 and 3). We have also characterized HeLa p70 cells for overexpression of the p70 receptor by using several methods. Flow cytometric analysis with fluoresceinated hTNF (FITCTNF) has shown a significant increase in binding to these cells (Figure 3A). 1251-labeled hTNF binding measurements have provided a value of 190,000 receptors per cell (Figure 38). Northern analysis has demonstrated the presence of the expected 4 kb mRNA transcript from the introduced plasmid encoding the p70 receptor (Figure 4A). The 2.5 kb mRNA for ~55 was also observed in these cells, and its level, of expression is unaltered compared with the parental HeLa cells (Figure 4A). Cross-linking experiments have provided evidence for an abundant increase in the

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of cell density and thereby survival in different concentrations of TNF. mTNF or hTNF did not interfere with the growth rate of HeLa cells over the wide concentration range tested. These cells doubled in approximately 24 hr, beginning at day 1 with a density of approximately 1 x 104, reaching a density at day 2 of about 2 x lo4 and at day 3 of around 3.6 x lo4 as calculated from the optical density measurements. Actually, TNF between 2 and 10 nglml slightly stimulated proliferation of these cells (Figure 5C). Similarly, HeLa cells cotransfected with the pCDM8 vector and pSM-hygro’ plasmids behaved like parental HeLacells in their growth response to TNF, with increases in cell density per well from 0.8 x lo4 to 1.7 x lo4 to 3.2 x lo4 cells in 24,48, and 72 hr, respectively (Figures 5A, 58, 5C). When these experiments were performed with HeLa p70 cells overexpressing the human p7g receptor, they were killed by either hTNF or mTNF within 24 hr. The effect was not a decreased growth rate, but complete lethality. Since HeLa p70 cells are killed within a day at all TNF concentrations shown here, we further tested their cytotoxic response to lower doses of TNF beginning with 0.1 nglml and have obtained an E& for cell killing of 0.2 to 0.3 nglml for both mouse and human TNF (data not shown). Since TNF cytotoxicity in HeLa p70 cells occurs within 24 hr, we tested shorter term actions of TNF such as the induction of mRNA transcripts for c-fos, c-jun, manganous superoxide dismutase (MnSOD; Wong and Goeddel, 1988), and cytosolic phtbspholipase % (cPLA2; Clark et al., 1991). Except for cPLA2, induction of these genes was observed within a 2 hr period (Figure 6A) and over a wide TNF concentration range (Figure 6B). It appears from the quantitative analyses and comparison of these responses in both cell types that in HeLa p70 the expression of these genes is reduced relative to parental HeLa cells. Both in the time course and with TNF concentrations of up to 200 nglml, the induction of c-fos mRNA in HeLa p70 cells was about half themaximum responseobserved in the parental HeLa cells. Since these are all p55-mediated responses (Wong and Goeddel, 1988; Haliday et al., 1991) it is possible that a reduced level of this receptor in HeLa p70 cells could account for these results. But as mentioned above, examination by Northern and cross-linking analysis had shown that p55 mRNA and protein levels are not lower in these p70overexpressing cells (Figures 4A, 48). Alsd, these reduced responses do not appear to be due to competition for ligand, since even at 200 nglml of ligand-a concentration at least 4-fold above that required to saturate receptor binding in the overexpressing cell lines-the induction of these genes did not approach levels seen in parental HeLa cells.

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(A) Binding of FITC-hTNF to HeLa and HeLa p70 cells. Cells incubated with FITC-hTNF were washed and their fluorescence intensity measured in a flow cytometer. HeLa/a, unstained HeLa cells; HeLa/b, stained with FITC-hTNF at 0.8 f@rnl and HeLa p70 stained with FITChTNF at 0.9 pg/ml. Binding specificity of HeLa p70 cells with FITChTNF was seen by displacement of its fluorescent intensity curve with 1 O&fold excess of hTNF to overlap that of HeLa cells (data not shown). (B) Specific binding of ‘251-IabeIed hTNF to HeLa p70 cells. Cells (2 x IO8 per assay) were incubated with increasing concentrations of ‘Wabeled hTNF alone (open squares) or with 100-fold excess of unlabeled hTNF(closed squares)for2 hr at 4%, andtheamountof radioactivity associated with washed cells was determined. Scatchard analysis provided a value of ~190,000 receptors per cell. (C) Binding of FITC-hTNF to NIH 3T3 cells and NIH 3T3 p70 cells conducted as described for HeLa p70 cells in (A). An increased amount of FITC-TNF was bound to NIH 3T3 p7tI cells compared with parental NIH 3T3 cells. (D) Specific binding of ‘Wabeled mTNF to NIH 3T3 p70 cells. Experiments conducted were as described in (8) for HeLa p70 cells, except that ‘*Wabeled mTNF was used and competed with lOOfold excess of mTNF. A similar displacement curve was obtained with lOO-fold excess of hTNF. Scatchard analysis provided a value of ~110,000 receptors per cell.

p70 receptor protein with major TNF cross-linked bands of 105-l 25 kd, as well as additional, larger sized complexes (Figure 48, lanes 5 and 7). The ~55 protein products of 85-100 kd are also visible in the p70overexpressing ceils but show little or no increase compared with parental HeLa cells (Figure 48, lanes 3 and 5). These estimated sizes for TNF-p55 and TNF-p70 receptor complexes are in the expected range produced by the binding of dimer or trimer TNF molecules to the receptors. For viability studies, HeLa cells were seeded in 96-well microtiter plates at approximately 1 x lo4 cells per well and were grown overnight before the addition of mouse or human TNF at concentrations ranging from 2 to 100 ngl ml. Although hTNF bindsonlyto the mp55 receptor, mTNF recognizes both hp55 and hp70 and displaces bound 1251-labeled hTNF with efficiency equal to that of hTNF. After 24, 48, and 72 hr (Figures 5A, 58, 5C), cells were fixed and stained with crystal violet to provide a measure

Studies with Murine NIH 3T3 and NIH 3T3 p70 Cells Overexpreeelng the Human p70 Receptor If indeed the p70 TNFR confers increased susceptibility in cells to TNF, its overexpression in murine cell lines already sensitive to mTNF should increase their sensitivity to TNF. An example of this increased cytotoxic response was seen in murine NIH 3T3 cells, which contain mu4900 receptors per cell of both the p55 and p70 types (Figure 1A and see

TNFR-Specific 51

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Figure 4. Comparisons of TNFRs mRNA and Protein Levels

p55 and p70

(A) RNA blot analysis of ~55 and ~7’0 mRNAs in HeLa and HeLa p70 cells. The two left lanes were hybridized with human p55 cDNA and the astwo right lanes with human p70 cDNA probes. P70 - p70 A single p55 mRNA of 2.5 kb is seen in both cell types. No p70 transcript is observed in I p55HeLa cells. In HeLa p70 a single 4 kb p70 ISmRNA transcribed from the introduced p70 cDNA is present. The p55 transcript levels were quantitated in HeLa and HeLa p70 cells by densitometric scans and normalized with values obtained from probing blots with 8-actin. No B D difference in expression was obsenred (data HeLa p70 NIH3T3 NIH3T3 p70 HeLa not shown). 1 rN C N C “N C N C’ ‘N CmCh”N CmCh (B)Cross-linked products of radiolabeled hTNF with TNFRs in HeLa and HeLap cells. Lanes 1 and 3 show protein complexes (CP) of 85100 kd and faint bands of 105-125 kd in HeLa cells from two separate experiments. Lane 5 shows major CP of 105 and 125 kd in HeLa pi’0 and the 85-100 kd complex similar to that in HeLa cells. All are 5 day exposures, except lanes 7 and 8 are an 18 hr exposure of lanes 5andEincluded todemonstratethetwodistinct c bands of 105 and 125 kd (N, cross-linking in absence or C, presence of excess unlabeled 1 2 345678 123456 hTNF). (C) RNA blot analysis of p55 and p70 mRNA in NIH 3T3 and NIH 3T3 p70 cells. The two left lanes are probed with mouse p55 cDNA and the two right lanes with mouse p70 cDNA probes. A single ~55 mRNA of 2.5 kb is present in both cell types. Two endogenous murine p70 transcripts of 4.5 and 3.8 kb are seen in NIH 3T3 cells, and in NIH 3T3 p70 cells two additional bands of approximately 4.0 kb and 2.5 kb derived from the introduced cDNA are also observed. The 3.8 kb and the expected 4.0 kb bands are superimposed as judged by the high intensity of this band. Quantitation of endogenous transcript levels using p-actin to normalize expression showed no significant differences in p55 mRNA in these cells. (D) Cross-linked products of radiolabeled TNF with TNFRs in NIH 3T3 and NIH 3T3 p70 cells. Lanes 1 and 4 show CP 85-100 kd and 105-125 kd in the absence(N) of excess unlabeled mTNF. Lanes 2 and 5 show cross-linking in presence of excess mTNF (Cm) and lanes 3 and 8 in presence of excess hTNF (Ch). Only mTNF competes for binding to both complexes; hTNF only displaces radiolabeled mTNF from the 85-100 kd complex.

below). NIH 3T3 p70 cells cotransfected with the human p70 receptor expression plasmid and a plasmid for hygromycin resistance were characterized by the same criteria as used for HeLa p70 cells. Increased surface expression of receptors was observed with FITC-TNF binding (Figure 3C), and 1Z51-labeled mTNF binding measurements provided a value of ~110,000 receptors per cell (Figure 3D). RNA blot analysis demonstrated the existence of a single 2.5 kb mRNA transcript for p55 in both NIH 3T3 and the p70 overexpressors, and the relative levels of this transcript were not different (Figure 4C). For ~70, thetwo different size endogenous transcripts of 4.5 kb and 3.8 kb were seen in the NIH 3T3 cells. In NIH 3T3 p70 cells two additional transcripts of 4 kb and 2.5 kb were prominent. The 4 kb mRNA is the size expected from the transfected cDNA, and the 2.5 kb mRNA is also p70 receptor specific and most likely derived from the introduced cDNA (Figure 4C). Cross-linking data again revealed both ~55 and p70 protein products as bands of 85-l 00 kd and 105-l 25 kd, respectively (Figure 4D). In these experiments excess unlabeled hTNF displaced radiolabeled mTNF from the 85100 kd complex (Figure 4D, lanes 1 and 3) and from the introduced hp70 protein (Figure 4D, lanes 4 and 8), but not from the endogenous murine p70 receptor. mTNF competed for binding to all, human and murine, receptor pro-

teins in these cells (Figure 4D, lanes 2 and 5). The results also demonstrate that the level of the p70 protein was amplified, while the p55 protein levels remained relatively equivalent (Figure 40, lanes 1 and 4). NIH 3T3 cells were tested for cytotoxicity with mTNF or hTNF (Figure 7A) and similar to the results obtained with mouse TAl cells, NIH 3T3 cells were not killed by hTNF alone over a 2-100 ngl ml concentration range in a 24 to 72 hr period (data from only 24 hr and 48 hr treatments is presented here). However, cell killing with mTNF was observed within 2 days, with a 50% cell death occurring in 3 days at 50 nglml mTNF. In contrast, NIH 3T3 p70 cells were sensitive to hTNF, with an ECmof m50-54 nglml required for cell killing in 48 hr. Their sensitivity to mouse TNF, which binds both mouse and human p70 receptors, showed an ECso of 1720 nglml and reflects an increase in sensitivity. Cells transfected only with the pCDM8 plus pSVP-hygro plasmid8 exhibited TNF killing patterns like those of parental NIH 3T3 cells (data not shown). As with HeLa and HeLa p70 cells, the induction by TNF of MnSOD (Figure 7B) and c-fos (data not shown) was measured, primarily to test whether the p70overexpressing cells could still exhibit these short term responses. Again, although NIH 3T3 p70 cells showed the expected increase in MnSOD mRNA levels, the magnitude of this response was reduced.

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Figure 5. Cytotoxic Response of Human HeLa Cells, HeLa p70 Cells, and HeLa pCDM8 Cells to Treatment with hTNF and mTNF 98-well microtiter plates seeded with 1 x”lO’cells per well were treated with 2, 10, 25, 50, and 100 rig/ml of hTNF (open circles) or mTNF (closed squares) for 24 hr (A), 46 hr (B), or 72 hr (C) and their growth rate compared with untreated controls. At the end of each experimental period viable cells were stained with crystal violet, and their density was determined as described in Experimental Procedures.

Discussion Two distinct receptors for TNFa, referred to here as ~55 and ~70, have been identified. Both receptors bind TNF with high affinity, although the p70 receptor has been reported to have a higher affinity for TNF (Hohmann et al., 1990). Both receptors are present on almost all cells analyzed, but their relative abundance in different cells and tissues varies, implying that separate regulatory mechanisms control their expression. Both receptors are functional in transducing signals (Shalaby et al., 1990; Hohmann et al., 1990), but a unique function for the p70 receptor has not been identified. A recent paper, however, has reported cell proliferation, demonstrated by an increase in [3H]thymidine incorporation, to be a p70 receptor-mediated response in two selected murine cell types (Tartaglia et al., 1991). Murine homologs of these two receptors have been cloned (Lewis et al., 1991; Goodwin et al., 1991), and using species-specific TNF, studies have demonstrated that mTNF bound to the mouse p70 recep-

Figure 6. Induction of c-fos, HeLa and HeLa p70 Cells

MnSOD,

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mRNA

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in

(A) Time course of induction with confluent cells treated with 50 nglml hTNF. Total RNA (10 ugllana) was analyzed for expression of the 2.2 kb c-fos, 1 and 4 kb MnSOD, and 3.4 kb cPLA* mRNAs. c-fos mRNA expression was quantified bydensitometric scanning, and values were normalized with signals obtained for 8-actin. Relative induction was obtained by dividing the value for each experimental sample with the expression in the corresponding untreated controls. (B) Dose response of c-jun and c-fos mRNA expression by hTNF in HeLaand HeLap70cells. Confluentcells treated with increasing doses of hTNF ranging from 0.1 nglml to 200 @ml for 30 min were used, and their total RNA was analyzed for c-fos and c-jun mRNA transcripts and then probed with 8-actin. c-fos mRNA expression was quantified as explained above.

tor cannot be displaced by excess human TNF (Lewis et al., 1991). We have confirmed these results with murine TAl and NIH 3T3 cells, which contain mRNA transcripts for both receptors (Figure 16). In competitive ligand binding assays, receptor-bound 1251-labeled mTNF was displaced completely by excess unlabeled mTNF but only partially by hTNF (Figure 1A). In NIH 3T3 ceils, when the bound ‘251-labeled mTNF was competed with excess mouse or human TNF before cross-linking ligand to the receptors, hTNF displaced %labeled mTNF from the ~55 receptor but not the p70 receptor, while mTNF displaced radiolabel from both receptors (Figure 4D). Also, while mTNF binds to both human ~55 and human p70 receptors (Figures 5A, 58, 5C), hTNF does not bind to mouse p70 receptor. The significantly lower homology between the extracellular domains of the human and mouse p70 receptors than between the human and mouse p55 receptors has been suggested as a possible reason for this species specificity of the murine p70 receptor (Lewis et al., 1991). The unique recognition of the mouse p70 receptor by

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mTNF alone is a valuable observation. It indicated to us that in the mouse TAl cell line, all measured responses to TNF were mediated by the p55 receptor, since only hTNF had been used in these studies. Comprehensive work done with this cell line has led to the characterization of many TNF-induced responses, most notable of which has been the dedifferentiation of TAl adipocytes by TNF into fibroblasts and the suppression of fat-specific genes (Torti et al., 1985; Haliday et al., 1991; Reid et al., 1989, 1991). We have recently shown that arachidonic acid-derived products of the lipoxygenase pathway, which are important mediators of c-fos induction by TNF in these cells (Haliday et al., 1991), also mediate TNF-CHX killing (D. J. C., G. Ringold, and R. A. H., unpublished data). Therefore, in light of the activation of only the ~55 receptor by hTNF, these recorded responses are all p55 receptor-mediated events, and consequently in this system the functional role of the p70 receptor was unclear. We began by using mTNF as the ligand to trigger both the p55 and p70 receptors to test the known short term TNF-associated responses. No obvious differences were immediately apparent either in the inducibility of genes such as c-fos, NF-KB, or in the repression of fat-specific genes, e.g., FSP27. Surprisingly, when effects of long term exposure of these cells to mTNF were examined with treatments longer than 24 hr, cells were observed to undergo marked disruption, fragmentation, and ultimately cell death. These observations clearly implicated the p70 TNFR action in mTNF-mediated TAl cell killing. For further proof, we examined two other cell lines, HeLa of human epithelial origin and NIH 3T3 mouse fibroblasts. The human p70 receptor was overexpressed in these cell lines, and the consequences of p70 receptor abundance were examined. HeLa cells contain predominantly the p55 receptor and respond to hTNF by the induction of c-fos, c-jun, and MnSOD, and HeLa cells are not killed by hTNF alone. The

2

4 8h

Figure 7. Responses in Mouse NIH 3T3 NIH 3T3 p70 Cells to mTNF and hTNF

and

(A) The cytotoxic response was studied as described in Figure 4 with HeLa cells. Both NIH 3T3 and NIH 3T3 p70 cells plated in 96-well microtiter plates at 2 x IO’ cells per well were treated with 2,10,25,50, or 100 nglml of hTNF or mTNF. After 24 hr or 46 hr cell density was measured by staining attached cells with crystal violet. Upper panel is a 24 hr treatment of NIH 3T3 cells with hTNF (open circles separated by solid lines) or mTNF (closed squares separated by solid lines) and NIH 3T3 p70 cells with hTNF (open circles separated by dotted lines) and mTNF (closed squares separated by dotted lines) Lower panel is a 46 hr treatment. Wells without cells stain with dye to give an absorbance value of 0.15-0.2. (B) Time course of induction of MnSOD mRNA by hTNF in NIH 3T3 and NIH 3T3 p70 cells. Cells were treated with 50 nglml hTNF for the times indicated and used for total RNA preparation. Northern blots were hybridized sequentially with MnSOD and 6-actin probes. The MnSOD 1 kb mRNA transcript was quantified by densitometric scanning, and the values were normalized with those obtained for 6-actin.

overexpressing HeLa p70 cells contained an estimated 190,000 receptors per cell, an increase of 30- to 40-fold over wild-type parental cells. Examined for TNF cytotoxicity, these cells were found to be highly sensitive. They were killed by doses of TNF smaller than 1 nglml, with a 50% cell death occurring in 24 hr by doses in the range of 0.2-0.3 nglml. HeLa cell transfectants carrying only vector sequences, i.e., the pCDM8 vector and pSVP hygro’ plasmids, were unaffected and like the parental cells, remained totally viable following hTNF addition. Since HeLa p70 cells were killed by hTNF, their response to ligand-induced gene expression was also examined. Genes examined were c-fos, c-jut-r, MnSOD, and cPLA2 (Clark et al., 1991). It became clear that these cells were still responsive to TNF and that they had not been grossly altered by tranfection of the expression plasmid or overexpression of the p70 receptor, but the magnitude of their response was lowered. The repressive effect was not due to a depletion of TNF, since increasing ligand concentrations could not restore function to levels observed in parental HeLa cells; nor was it due to altered levels of p55 expression in p70-overexpressing cells, since p55 levels judged by both receptor protein and mRNA transcript content were comparable in both HeLa and HeLa p70 cells. We believe that these results indicate an interaction of the p70 receptor with p55-mediated functions. It is not known whether the p55 receptor selfassociates for functional activation and/or whether excess p70 receptor can alter function by causing interference in p55-mediated events. It is possible that the higher affinity of the p70 receptor for its ligand (Hohmann et al., 1990; Lewis et al., 1991; Goodwin et al., 1991) may influence the behavioral interactions of these two receptors. This repressive effect of p70 overexpression nevertheless recalls to mind the dominant negative consequences observed with other signal transducing proteins such as the

Cell 54

.

PDGF and EGF receptors (Ueno et al., 1991; Kashles et al., 1991) when their mutant forms are overexpressed. The results obtained with HeLa cells were confirmed with NIH 3T3 cells. These cells are killed only by mTNF, but their susceptibility to hTNF and mTNF increases with hp70 abundance, as seen with the NIH 3T3 p70 transfectants. Their increased sensitivity to mTNF may be due to the combined action of transfected and endogenous p70 receptors, since mTNF binds to both. Again, responses of TNF-inducible genes are observed, but they are reduced. Compared with HeLa p70 cells with ~190,000 receptors per cell, these NIH 3T3 p70 transfectants contain about 110,000 receptors. While HeLa p70 cells are killed by TNF within a day, the NIH 3T3 p70 cells like TAl cells show a slower cytotoxic response beginning after about 24 hr. One explanation might be the variable growth rate of these HeLa, NIH 3T3, and TAl cells that might determine a rapid versus slower response to TNF cytotoxicity, but it appears that the abundance of p70 is a critical feature in determining this response. At present we do not know the mechanism of p70-mediated cell death, and the cellular events associated with this cytotoxic process are being examined. It is important here to recall the similarity between the actions of p70 TNFR and the Fas antigen. The cDNA of this cell surface antigen has been recently cloned and shown to encode a receptor with structural similarities to the TNFR-NGFR family of proteins (Itoh et al., 1991). Fas cDNA transfected cells show anti-Fas antibody-mediated cytolytic activity, which occurs via apoptotic processes that are marked by cell surface blebs, chromatin condensation, and fragmentation of the nucleus and DNA. The cytotoxicity assays used in our studies measure the decrease in number of adheren’t cells and the inability of detached cells to proliferate upon transfer to fresh growth medium lacking TNF. A detailed analysis such as time lapse photomicrography would be useful in examining events in the progression of the p70 TNFR-mediated cell death for a valuable comparison of actions initiated by these two receptors of the same family. Recently, in two selected systems, C3H/HeJ mouse thymocytes and CT-6 cytotoxic T cells, the p70 TNFR was reported to promote DNA synthesis, and this effect was interpreted as cell proliferation (Lewis et al., 1991). While these results are difficult to reconcile with a cytotoxic function of the p70 receptor reported here, differences in responses of different cell types are always possible, and one can speculate that such proliferative responses might prove lethal. It has been suggested by recent analyses of mitotic control characteristics of mammalian cells (Kung et al., 1990) that a drive into a proliferative state can disrupt the ordered progression of cell cycle events, causing mitotic aberrations and cell death. Indeed, there is evidence to suggest that cells growth arrested in Gl phase of the cell cycle, if pretreated with IL-l, IL-6, or TNF, are less vulnerable to subsequent TNF cytotoxicity than if cells are driven through the S phase into G2 and M by reagents such as PGE2 or cholera toxin (Belizario and Dinarello, 1991). Cell cycle progression of the p70-overexpressing

cell lines might provide an insight into such unknown cellular mechanisms. It is clear that our results do not exclusively involve the p70 TNFR action in cell killing, because under the conditions examined here, both ~55 and p70 receptors are activated. However, we do not observe this killing action when only ~55 is activated. Moreover, it is important to emphasize here that overexpression of the p70 receptor is not required to cause cytotoxicity. In both TAl and NIH 3T3 cells, the endogenous levels of p70 are sufficient to induce cell death when both ~55 and p70 receptors are stimulated with mouse TNF; but if only ~55 is activated with human TNF cell death does not occur. These conclusions were drawn from the parental cell lines and were supported by the p70-overproducing HeLa p70 and NIH 3T3 p70 transfectants, which provided additional evidence for a role of the p70 receptor in pre$iisposing the cells to lethality. The down-regulation of $55 receptortransduced functions in these p7S)-overexpressing cell lines might also suggest an interaction between the two receptors. Future studies will be designed to address the biochemical contributions made by each receptor. Experimental

Procedures

Cell Lines and Transfections TAl cella are a stable mouse adipogenic cell line derived from 5-azacytidine-treated’ 1OTlh mouse embryo fibroblasts (Chapman et al., 1984). They were maintained in Eagle’s basal medium (GIBCO, Grand Island, NY) containing 10% supplemented, defined calf serum (Hyclone Laboratories, Lagan, UT) heat inactivated at 55“ for 30 min. Human HeLa cells and mouse NIH 3T3 cells were cultured in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum. All transfected cell lines were grown in medium containing 200 vglml hygromycin B containing 10% fetal bovine serum, 100 U/ml penicillin and 100 &ml streptomycin and maintained at 37OC in a 5% COz incubator. Cells were transfected by electroporation and for 1 x 10’ cells, 50 pg of the pCDM8-p70 or pCDM8 plasmid and 5 pg of pSV2 plasmid carrying the hygromycin phosphotransferase gene (pSV2hygro’) were mixed with 350 pg of yeast tRNA as carrier in 20 mM HEPES (pH 7.4) buffer and electroporated once at 250 V and 980 pFD capacitance by using a Bio-Rad Gene Pulsar. Cells were grown in hygromycin-containing medium for 2 weeks before selection of individual colonies. Construction and Use of Plasmids Full-length human TNFRp70 cDNA containing the entire coding sequence plus 2.7 kb of the 3”untranslated sequence was cloned in the pCDM8 (Aruffo and Seed, 1987) mammalian expression vector and used in these studies. A plasmid carrying the hygromycin phosphotransferase gene (pSV2-hygro? was used as a selection marker and was cotransfected with either the pCDM8 plasmid or its recombinant carrying the 4 kb p70 receptor cDNA. RNA Blot Analysis The method used has been previously described in detail (Heller et al., 199Ob). Cellular RNA was prepared by using guanidinium isothiocyanate, and poly(A)+ RNA was selected by chromatography on oligo(dT)cellulose, denatured with glyoxal and dimethyl sulfoxide, and electrophoresed in 0.8% agarose gels in IO mM sodium phosphate buffer (pH 7.0). RNA was transferred to Nytran membrane (Schleicher and Schuell), hybridized, and washed. The blots were probed with fulllength coding region cDNA inserts for both murine and human TNFR ~55 and ~70, and labeled with [“PJdCTP by using a random primer kit (Amersham) to a specific activity of lo’-109 cpmlpg. After a 24 hr hybridization at 42OC, filters were washed in 2 x SSPE, 0.1% SDS for 15 min each at room temperature and then washed two times in 0.1 x SSPE,O.l%SDSfor 15mineachat50W f 2X Filterswereexposed

T$FR-Specific

to Kodak indicated.

Cell Killing

X-OMAT

film with intensifying

screens

at -7OOC

for times

Cross-Linking of ‘%Labeled TNF to Receptors These experiments were conducted essentially as described previously (Heller et al., 1990a), except that 2 x 106cells were incubated with 100 nglml of ‘251-Iabeled hTNF or mTNF with or without 50-fold excess of unlabeled hTNF or mTNF. For cross-linking 10 nl of 50 mM bis-sulfosuccinimidyl suberate was added in phosphate-buffered saline to cells at room temperature for 30 min. The cells were washed three times with phosphate-buffered saline containing 0.2 M glycine and lysed with 100 ul of 0.5% Triion X-100 in 56 mM Tris-HCI (pH 7.5) containing 50 wglml soybean trypsin inhibitor and leupeptin and 175 ng/ ml benzamidine plus 5 pM EDTA. After low speed spin at 10,000 x g for 5 min, 1 pl aliquots of the extracts were used to determine radiolabeled ligand binding, and the rest of the sample was mixed with SDScontaining sample buffer and analyzed by SDS-polyacrylamide gel electrophoresis in 7.5% to 15% gradient gels. Analysis of TNF-Mediated Functions Recombinant human and mouse TNF were purchased from Genzyme (Cambridge, MA). The probes for c-fos, 6-actin, and fat-specific clone FSP27 were obtained from Dr. Gordon Ringold (Affymax, Palo Alto); c-jun was obtained from Dr. Michael Karin (University of California at San Diego, LaJolla, CA): mouse NF-KB clone encoding the 51 kd DNA-binding subunit of NF-KB protein was a gift of Dr. Sankar Ghosh (Whitehead Institute, Cambridge, MA). A human probe for MnSOD was generated by the polymerase chain reaction using HeLacell cDNA as template, and oligonucleotide primers were prepared from the published sequence (Beck et al., 1987). The human probe for cPLA2 was obtained from Genetics Institute Inc. (Cambridge, MA). RNA isolations and Northern blot hybridizations were performed as described earlier (Heller et al., 199Ob). Radloiodinations of TNF and ‘*%Labeled TNF Receptor Blnding Assay Human ‘Z51-labeled TNF was purchased from New England Nuclear, and mouse ‘251-labeled TNF was prepared according to the previously described procedure (Heller et al., 199Oa). Methods used for the 1251-labeled TNF receptor binding assay, fluorescein conjugation of TNF, and flow cytometric analysis of TNF-receptor-expressing cell lines have also been described previously (Heller et al., 199Oa). Cytotoxiclty Assay Changes in cell viability were measured with cells plated in a 96-well microtiter plate at 1 x l(r cells per well or as indicated in 100 pl of growth medium. Twelve to 16 hr later when cells had attached to the plates, mouse or human TNF additions were made ranging in concentrations from 2 to 100 nglml, or as specified. Each treatment consisted of eight replicate wells. Cells were incubated for 24, 46, or 72 hr as specified, washed three times with phosphate-buffered saline, fixed with 5% formalin-buffered saline for 5 min, washed again with phosphate-buffered saline, and stained with crystal violet (16% ethanol, 0.1% crystal violet) for 2 hr. The wells were washed with tap water and air dried. The dye was eluted with 100 ul of 0.1 M sodium citrate (pH 4.2) in 50% ethanol for 30 min, and optical density at 570 nm was measured with a Molecular Devices kinetic microplate reader. Measurements of optical density at 570 nm as a function of cell density showed a linear relationship from 1 x 103 up to 4 x 10’ cells per well and were used to estimate cell number. Acknowledgments We would like to thank Dr. Robert Lewis for his support and encouragement, Dr. Gordon Ringold for introducing us to the TAI adipogenic cell system and for his advice during the early stages of this work, Dr. Giorgio Rovelli for generating the HeLa-pCDM6 cell line, Dr. John Dunne and Laura Chiu for flow cytometric analyses, Dr. Paul Cannon for stimulating discussions, Drs. Howard Schulman, Kurt Jarnagin, and Wolfgang Hoeck for careful reading and helpful criticism of this manuscript, and Linda Miencier and Kaylou Glenn for preparation of this manuscript.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 16 USC Section 1734 solely to indicate this fact. Received

January

6, 1992; revised

May 13, 1992.

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The p70 tumor necrosis factor receptor mediates cytotoxicity.

Tumor necrosis factor alpha (TNF) selectively kills tumor cells, but this specificity is not clearly understood. Two distinctly different cell surface...
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