©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Generation and Biological Characterization of Membrane-bound, Uncleavable Murine Tumor Necrosis Factor (*)

(Received for publication, February 28, 1995; and in revised form, June 5, 1995)

Els Decoster (§) Bart Vanhaesebroeck(¶)(**) Peter Vandenabeele (**) Johan Grooten Walter Fiers (§§)

From the Laboratory of Molecular Biology, Gent University, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Tumor necrosis factor (TNF) is produced as a membrane-bound, 26-kDa proform from which the mature, 17-kDa TNF subunit is released by proteolytic cleavage. In order to compare the biological activity of membrane-bound versus soluble TNF, mutational analysis of potential cleavage sites in murine TNF was carried out. The biological activity was assessed after transfection in L929 cells. Deletion of the first nine codons of the mature part of the murine TNF gene still led to the production of secretable TNF, indicating alternative cleavage sites separate from the -1/+1 junction. However, an additional deletion of 3 amino acids, generating TNFDelta1-12, resulted in a membrane-bound form of TNF. Site-directed mutagenesis revealed Lys as the critical residue for alternative cleavage. Mutation of this residue to Glu in a TNFDelta1-9 mutant gave rise to uncleavable, membrane-bound TNF with biological activities similar to wild-type TNF. Induction of apoptosis, proliferation, or cytokine production by triggering of either 55-kDa or 75-kDa TNF receptors in appropriate cell lines occurred efficiently both with soluble and with membrane-bound TNF. The latter was, however, less active in the cytotoxic assays on U937 cells in which the 75-kDa TNF receptor is not signaling, but contributes to maximal TNF activity by ligand passing. This indicates that membrane-bound TNF cannot be passed from the 75-kDa to the 55-kDa TNF receptor.


INTRODUCTION

The cytokine tumor necrosis factor (TNF) (^1)plays a crucial role in immunological and inflammatory reactions as a result of an infection or a tumor burden (reviewed in Vassalli(1992), Fiers(1993), Tracey and Cerami(1994)). TNF interacts with two specific receptors present on the cell surface of almost every cell type: TNF-R55 and TNF-R75, corresponding to a molecular mass of 55 and 75 kDa, respectively (Loetscher et al., 1990; Smith et al., 1990; Lewis et al., 1991; Goodwin et al., 1991). Since the active form of TNF in solution is a trimer (Wingfield et al., 1987; Schoenfeld et al., 1991) and contains three TNF-R-binding sites (Van Ostade et al., 1991), signal transduction after TNF binding presumably occurs by TNF-R clustering (Espevik et al., 1990; Pennica et al., 1992; Tartaglia and Goeddel, 1992). Almost the whole spectrum of TNF activities is mediated by TNF-R55 (Engelmann et al., 1990; Espevik et al., 1990; Tartaglia et al., 1993c). So far, the role of TNF-R75 as a signal transducer seems to be restricted to T lymphocytes (Tartaglia et al., 1991, 1993a; Vandenabeele et al., 1992). Because of the higher affinity and rate of dissociation, it has also been proposed that TNF-R75 can concentrate TNF and pass it on to neighboring TNF-R55 molecules (Tartaglia et al., 1993b).

The mature, secreted 17-kDa TNF subunit is derived from a 26-kDa TNF precursor. It has been demonstrated that the proform of TNF is present on the cell surface as a type II transmembrane protein (Kriegler et al., 1988). The latter is then proteolytically cleaved by a membrane-associated Ser protease (Scuderi et al., 1989; Kim et al., 1993) or metalloprotease (Mohler et al., 1994; Gearing et al., 1994; McGeehan et al., 1994) in order to release mature 17-kDa TNF (52 kDa in the native, trimeric form).

At present, there is only indirect evidence that the membrane-bound precursor of mTNF, presumably as a trimer, is biologically active. It was found that paraformaldehyde-fixed murine macrophages still exhibit TNF-mediated cytotoxicity (Decker et al., 1987). Likewise, immunoreactive TNF, detected in the course of mouse embryogenesis, was not secreted but rather membrane-bound, suggesting that membrane-bound TNF may function during that process (Osawa and Natori, 1989). Moreover, TNF-expressing tumor cells display reduced tumorigenicity in vivo. Considering the absence of detectable, soluble TNF in the serum of such mice, membrane-bound TNF might be responsible for this effect (Vanhaesebroeck et al., 1991).

In order to develop a system allowing us to study the biological activity of membrane-bound TNF, we investigated the proteolytic conversion of the 26-kDa mTNF proform to a 17-kDa, secreted TNF subunit by site-directed mutagenesis. The mTNF mutants generated were expressed in the murine fibrosarcoma cell line L929 and analyzed for their biochemical and biological characteristics. This approach permitted us to generate a nonsecretable, biologically active mutant of mTNF. L929 cells expressing this mutant induced proliferation of the murine CT6 cell line and GM-CSF secretion by a rat thymoma cell line; moreover, they were cytotoxic to U937 cells. These results directly demonstrate that the membrane-bound form of TNF is able to trigger biological activities mediated by TNF-R55 as well as by TNF-R75. However, depending on the receptor signal transduction system, different bioactivities of membrane-bound versus secreted mTNF could be observed.


MATERIALS AND METHODS

Cell Lines and Cell Culture

The fibrosarcoma cell lines L929sA and WEHI 164 cl 13 (Espevik and Nissen-Meyer, 1986) were cultured as described (Vanhaesebroeck et al., 1992). PC60-R55/R75 is a rat mouse T cell hybridoma transfected with both hTNF-R55 and hTNF-R75 cDNA under control of the SV40 early promoter (Vandenabeele et al., 1995). CT6 is an interleukin-2-dependent murine cytotoxic T cell line (Ranges et al., 1989). PC60-R55/R75, CT6, and the human myeloid U937 cell line were cultured in RPMI 1640 supplemented with 10% fetal calf serum, antibiotics, 2 mML-glutamine, 1 mM sodium pyruvate, and 50 µM 2-mercaptoethanol. All cell lines were repeatedly screened for mycoplasma by a DNA-fluorochrome assay and were found to be negative.

Cytokines and Antisera

Purified Escherichia coli-derived mTNF and hTNF were obtained in our laboratory and had a specific biological activity of 2 10^8 IU/mg and 8.4 10^7 IU/mg, respectively. International standards for TNF quantification (IU/ml) were obtained from the National Institute for Biological Standards and Control (Potters Bar, United Kingdom). Recombinant mGM-CSF was kindly provided by Dr. J. DeLamarter (Glaxo IMB, Geneva, Switzerland). Polyclonal rabbit antiserum against mTNF was provided by J. Van der Heyden (Roche Research, Gent, Belgium). Polyclonal rabbit antiserum directed against the tip region of mTNF (amino acids 99-115; Lucas et al., 1994) was a generous gift from Dr. R. Lucas (Brussels University, Brussels, Belgium). Anti-hTNF-R55 and anti-hTNF-R75 monoclonal antibodies were generously provided by Dr. M. Brockhaus (Hoffmann-La Roche, Basel, Switzerland; Brockhaus et al., 1990).

Determination of TNF Bioactivity in Culture Supernatant

Supernatant of transfected cell lines was concentrated 100-fold by centrifugation in Centriprep-10 and Centricon-10 microseparation devices (Amicon, Danvers, MA). TNF was quantified in an 18-h cytotoxicity assay using WEHI 164 cl 13 cells in the presence of 1 µg of actinomycin D/ml (Espevik and Nissen-Meyer, 1986). The detection limit of this assay was about 1 pg/ml.

Site-directed Mutagenesis

Site-directed mutagenesis was carried out using pMa and pMc phasmids as described (Stanssens et al., 1989). Mutations were verified by DNA sequencing.

Plasmid Constructions and Transfection

Mutant TNF or wtTNF genes were inserted in the eukaryotic expression vector pSV23S under control of the SV40 early promoter (Huylebroeck et al., 1988). The pSV2neo plasmid encoding the neo^r gene under control of the SV40 early promoter was used as a selectable marker in L929 transfections (Southern and Berg, 1982). Plasmid DNA used for transfection was purified using pZ523 columns (5 Prime 3 Prime, West Chester, PA). Stable transfection of L929 cells was carried out by an improved DNA-calcium phosphate coprecipitation method as described (Vanhaesebroeck et al., 1992).

Immunoprecipitation and Immunoblotting of TNF

TNF was immunoprecipitated from concentrated cell supernatant or from cell lysates as described (Vanhaesebroeck et al., 1992). Immunoprecipitates were separated by electrophoresis in an SDS-containing 12.5% polyacrylamide gel under reducing conditions. After electrophoresis, the proteins were blotted on a nitrocellulose membrane using a Novablot II apparatus (Pharmacia Biotech Inc.) according to the manufacturer's instructions. Blocking was performed overnight at 4 °C in 50 mM Tris-HCl, pH 8.0, 2 mM CaCl(2), 80 mM NaCl, 0.2% (w/v) Nonidet P-40, 0.02% (w/v) dry milk. The blot was then incubated for 4 h at 4 °C with polyclonal rabbit antiserum to TNF, followed by a 1-h incubation at room temperature with 0.2 µCi of I-protein A (Amersham International, Amersham, UK; 30 mCi/mg) in 20 ml of 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20. Radioactivity was detected by autoradiography.

Flow Fluorocytometry

Membrane-bound TNF was analyzed by incubation of the cells for 1 h at 4 °C with polyclonal antiserum to the tip of mTNF (amino acids 99-115; 1 µg of antibody/4 10^5 cells/200 µl), 1 h of incubation at 4 °C with biotinylated donkey anti-rabbit polyclonal antiserum (Amersham International), and 1 h at 4 °C incubation with phycoerythrin-conjugated streptavidin. Analysis was performed by flow fluorocytometry using a Coulter Epics 753 equipped with an argon-ion laser (Coulter, Hialeah, FL).

Bacterial Expression of TNF

wtTNF and mutant TNF-K11E genes (both without presequence) were cloned in the vectors pMaTrp and pMcTrp (Stanssens et al., 1989), respectively, under control of the inducible Trp promoter. After 3 days of induction at 18 °C, bacteria were sonicated, and TNF bioactivity was tested in a WEHI 164 cl 13 cytotoxicity assay. In order to determine the specific bioactivity of E. coli-derived mTNF-K11E, the recombinant mutant protein was purified as described previously for wtTNF (Tavernier et al., 1990).

Cellular Coculture

The bioactivity of TNF in transfected L929 cells was determined by a coculture between transfected L929 effector cells and TNF-responsive target cells (CT6, PC60-R55/R75, or U937). Adherent effector cells were seeded in flat bottomed 96-well microtiter plates at 20,000 cells/well 24 h before the addition of 25,000 nonadherent target cells/well. The biological effects of in situ produced TNF on the various target cells were established as detailed below.

CT6 Proliferation Assay

The proliferation of effector L929 transfectants was inhibited by 4 h of pretreatment with mitomycin C (50 µg/ml) at 37 °C followed by three washes. After 20 h of coculture with CT6 cells, proliferation was measured by [^3H]dThd incorporation for 4 h (Ranges et al., 1989).

Induction of GM-CSF Production by PC60-R55/R75 Cells

L929 transfectants were cocultured with PC60-R55/R75 cells. These transfectants were used in order to enhance the TNF-induced response. After 24 h of coculture the supernatants were harvested and assayed in an FDCp1 cell proliferation assay (DeLamarter et al., 1985; Vandenabeele et al., 1990).

Determination of Apoptosis in U937 Cells

U937 cells were pretreated for 1 h at 4 °C or for 3 h at 37 °C in the presence or absence of 2 µg/ml utr-1 antiserum, which is a monoclonal antibody against hTNF-R75 (Brockhaus et al., 1990). U937 cells were incubated with L929 effector cells in the presence of 50 µg/ml cycloheximide. After 4 h, nonadherent U937 cells were harvested, incubated with propidium iodide, frozen, and analyzed by flow fluorocytometry. The extent of apoptosis was quantified as the percentage of cells with a hypoploid DNA profile (Grooten et al., 1993). Contaminating L929 effector cells were electronically gated out on the basis of their different forward angle and 90° angle light-scattering properties.


RESULTS

Biochemical Characterization of TNF Mutants with Deletions at the N Terminus of Mature TNF

In order to determine which amino acids are crucial for the proteolytic processing of the membrane-bound proform of mTNF into a secreted mature form, mutants were constructed in which coding sequences for the N-terminal part of mature mTNF were deleted. The mutants were stably expressed in L929 fibroblast cells, which have been shown to become resistant to autocrine TNF production (Vanhaesebroeck et al., 1992).

In order to remove the authentic cleavage site between presequence and mature mTNF, a mutant was generated in which the first 9 amino acids of mature TNF were deleted (referred to as TNFDelta1-9) (Fig. 1). It is indeed known that deletion of up to 9 amino acids does not change the biological activity of hTNF (Creasey et al., 1987). Immunoprecipitation and immunoblotting of mTNFDelta1-9 transfectants showed a truncated 25-kDa TNF precursor in cell lysates (Fig. 2A) and different TNF species in the supernatant (Fig. 2B). Furthermore, flow fluorocytometric analysis of L929 transfected with wtTNF or TNFDelta1-9 revealed a membrane-bound TNF form (Fig. 3). Acidic treatment, applied to dissociate soluble TNF from the TNF-R, did not alter the level of immunofluorescence (data not shown). These results demonstrate that TNFDelta1-9 is present not only as a membrane-bound form but also as a cleaved, soluble form. Thus removal of the cleavage site between presequence and mature mTNF (-1/+1) (Fig. 1) (Sherry et al., 1990) did not abolish mTNF processing and still permitted secretion and membrane expression of TNFDelta1-9.


Figure 1: Overview of TNF mutants generated. An invertedfilledtriangle shows the known cleavage site between the TNF presequence and mature TNF (-1/+1). The potential N-glycosylation site Asn at position 7 is indicated by an asterisk. Openbars indicate deleted amino acid sequences. mTNFDelta1-9,K(11)E, mTNFDelta1-9,K11E.




Figure 2: Expression of TNF by L929 cells transfected with various TNF secretion mutants as detected in cell lysates and culture supernatants. Open and closedarrowheads point to 26- and 17-kDa TNF species, respectively. A, immunoprecipitation followed by immunoblot analysis with antiserum to 17-kDa mTNF on cell lysates of L929 cells transfected either with the neo^r gene alone (lane1), or combined with plasmid coding for wt-mTNF (lane2), mTNFDelta1-9 (lane3; a, 25 kDa), mTNFDelta1-12 (lane4; b, 24.6 kDa), or mTNFDelta1-9,K11E (lane5; c, 25 kDa). B, immunoprecipitation followed by immunoblot analysis with anti-mTNF on the supernatant from clonal L929 cells transfected with either the neo^r gene alone (lane1) or combined with plasmid coding for wt-mTNF (lane2), mTNFDelta1-9 (lane3), mTNFDelta1-12 (lane4), or mTNFDelta1-9,K11E (lane5).




Figure 3: Flow-cytometric analysis of expression of cell-associated TNF by L929 cells transfected with TNF secretion mutants. Cells were transfected either with the neo^r gene alone (A) or with the neo^r gene combined with plasmid coding for wtTNF (B), mTNFDelta1-9 (C), mTNFDelta1-12 (D), or mTNFDelta1-9,K11E (E). Cells were treated with secondary antibody alone (curve1) or with antiserum to the tip of TNF (amino acids 99-115) and secondary antibody (curve2). No signal other than staining with secondary antibody alone was found with an irrelevant rabbit IgG (not shown).



In order to identify the cleavage site responsible for this alternative processing, the deletion was extended to the first 12 amino acids of mature mTNF, generating the mutant TNFDelta1-12. Although the corresponding cell lysate contained the truncated TNF proform with a predicted molecular mass of 24.6 kDa (Fig. 2A), protein bands reactive with antiserum against TNF were not detectable in the supernatant of TNFDelta1-12 transfectants, as assayed by immunoprecipitation and immunoblotting (Fig. 2B). Moreover, flow fluorocytometric analysis of L929 transfectants with antiserum to the tip of the TNF molecule (amino acids 99-115) revealed the presence of membrane-bound TNF at expression levels similar to the ones observed with wtTNF or TNFDelta1-9 (Fig. 3). These data show that TNFDelta1-12 was expressed at the cell surface but, unlike TNFDelta1-9, could not be secreted. Consequently, the additional cleavage site responsible for processing of TNFDelta1-9 is presumably located between amino acids 9 and 13.

Lys is frequently present in protease recognition sites (Harris, 1989) and is present at position 11 of mature mTNF (Fig. 1). Since this Lys is a possible candidate for the alternative cleavage site of mTNF, we replaced in TNFDelta1-9 the positively charged Lys with the negatively charged Glu by site-directed mutagenesis (Fig. 1). Cell lysate from the resulting TNFDelta1-9,K11E transfectants clearly showed the truncated 25-kDa TNF proform (Fig. 2A), while the supernatant did not reveal any TNF-specific band (Fig. 2B). Further analysis by flow fluorocytometry demonstrated the presence of membrane-bound TNF at a density similar to that observed for L929-TNFDelta1-12 or L929-wtTNF transfectants (Fig. 3). These findings confirm that Lys constitutes the alternative cleavage site by which mTNF is processed in the absence of the proper cleavage site at -1/+1.

Functional Characterization of Secretable and Nonsecretable TNF Deletion Mutants

The biological activity of secreted TNF was assessed on the highly sensitive WEHI 164 cl 13 cells (Espevik and Nissen-Meyer, 1986). As expected, 100-fold concentrated culture supernatant of L929 cells transfected with wtTNF or secretable TNFDelta1-9 had a cytotoxic activity of 16.4 IU/ml and 70 IU/ml TNF, respectively, in originally unconcentrated supernatant (Fig. 4). This cytotoxicity was mediated by TNF, since it was abolished by the addition of neutralizing anti-TNF antiserum (data not shown). But no cytotoxicity could be detected in the supernatant of TNFDelta1-12 or TNFDelta1-9,K11E transfectants (Fig. 4). This is in agreement with the absence of immunoprecipitable TNF in these culture supernatants and further supports the absence of production of mature mTNF by such transfectants.


Figure 4: Cytotoxic activity present in the supernatant of L929 cells transfected with various TNF mutants. L929 cells were transfected either with the neo^r gene alone (▴) or with the neo^r gene combined with plasmid coding for wtTNF (bullet), mTNFDelta1-9 (), mTNFDelta1-12 (▪), or mTNFDelta1-9,K11E (). The cytotoxic activity was tested on WEHI 164 cl 13 cells.



The biological activity of uncleavable TNFDelta1-12 and TNFDelta1-9,K11E was investigated by coculture of L929 transfectants with various TNF-responsive target cell lines. A first assay was based on the TNF-R75-mediated proliferation of CT6 cells, in which TNF-R75 is signal-transducing (Tartaglia et al., 1993a). As shown in Fig. 5A, L929 cells producing wtTNF or TNFDelta1-9,K11E exerted a similar growth-inducing activity, while TNFDelta1-12 exerted this activity to a lower extent. These results show that both uncleavable TNF mutants trigger mTNF-R75-mediated signaling.


Figure 5: Determination of the bioactivity of membrane-bound TNF mutants by cellular coculture experiments. A, TNF-induced proliferation of CT6 cells incubated for 24 h with L929 transfectants pretreated with mitomycin C. Control experiments showed that mTNF clearly induced CT6 to proliferate; CT6 alone or cocultured with L929-neo^r cells hardly showed CT6 proliferation. Experiments were done in triplicate, and S.D. is indicated. B, induction of GM-CSF production by PC60-R55/R75 cells incubated for 24 h with L929 transfectants. GM-CSF released in the supernatant was assessed in an FDCp1 cell proliferation assay. 500 pg/ml mTNF induced GM-CSF levels similar to those recorded after coculture of PC60-R55/R75 cells with L929 wt-mTNF transfectants; L929 transfectants did not produce more than 10 pg/ml GM-CSF. C, TNF-dependent induction of apoptosis of U937 cells treated with cycloheximide after cocultivation with TNF-expressing L929 transfectants. Unpretreated U937 cells (filledbars) or U937 cells pretreated with 2 µg/ml utr-1 (openbars) (utr-1 binds to hTNF-R75 and prevents ligand passing) were incubated with mTNF-expressing L929 cells for 4 h in the presence of 50 µg/ml cycloheximide. The inset shows the induction of apoptosis in U937 cells, either untreated or after treatment with htr-1 (10 ng/ml) or TNF (0.1 ng/ml); filled and openbars refer to control and utr-1 pretreatment. Apoptosis in U937 cells was quantified by the percentage of hypoploid particles. Experiments were done twice in triplicate; bars indicate S.D. TNFDelta1-9,K(11)E, TNFDelta1-9,K11E.



Whether cell surface-expressed TNF could induce the production of GM-CSF in PC60-R55/R75 cells (Vandenabeele et al., 1995), was also investigated. Induction of GM-CSF secretion by PC60-R55/R75 cells involves signaling by both types of TNF-R in a cooperative effect between TNF-R55 and TNF-R75. (^2)TNFDelta1-9,K11E was found to induce a GM-CSF response similar to that induced by wtTNF, whereas TNFDelta1-12 induced a 3-fold lower response (Fig. 5B).

Finally, TNF-dependent induction of apoptosis in U937 cells was studied. These cells die from apoptosis after an incubation of 4 h in the presence of TNF and cycloheximide. Selective triggering of hTNF-R55 in U937 cells with the agonistic htr-1 monoclonal antibody induced apoptosis (Fig. 5C, inset). But apoptosis could not be induced via selective triggering of hTNF-R75 using utr-1 monoclonal antibodies (Fig. 5C, inset). Moreover, this utr-1 monoclonal antibody, which is agonistic on transfected PC60 cells (Vandenabeele et al., 1992, 1995), did not enhance the cytotoxicity on U937 cells exerted by htr-1. These findings indicate that only hTNF-R55 is signal-transducing in U937 cells, which is in agreement with previously reported data (Shalaby et al., 1990; Barbara et al., 1994). But hTNF-R75 might play an important role by facilitating binding of TNF to hTNF-R55, presumably through a ligand-passing mechanism (Tartaglia et al., 1993b). This phenomenon is supported by our observation that blocking of hTNF-R75 by pretreatment with a monoclonal antibody (utr-1) reduced the induction of apoptosis in U937 cells by TNF, but not by htr-1 (which only interacts directly with hTNF-R55) (Fig. 5C, inset). After coculture with U937 cells, L929-TNFDelta1-9,K11E induced an appreciable level of apoptosis, though this activity was significantly lower than that induced by L929-wtTNF transfectants (Fig. 5C), which express similar amounts of membrane-bound TNF. This is in contrast with CT6 and PC60-R55/R75 cells, in which the activity patterns induced by L929-TNFDelta1-9,K11E and L929-wtTNF were identical (TNF-R75 is signal-transducing in these two target cells). The different behavior of these two L929 transfectants may be explained by ligand passing of soluble TNF produced by L929-wtTNF, since utr-1 pretreatment of U937 cells (which prevented ligand passing) resulted in a wtTNF activity similar to that of membrane-bound TNFDelta1-9,K11E. These results suggest that the membrane-bound mutant, and by extension the membrane-bound TNF, cannot be passed. The cellular cytotoxicity observed was TNF-dependent, as evidenced by abrogation of the apoptotic response by addition of TNF-neutralizing antiserum, while inclusion of preimmune serum or irrelevant antiserum did not alter the biological response (data not shown). Furthermore, TNFDelta1-12 was completely ineffective in inducing cell death (Fig. 5C). Fig. 5, A-C, shows that membrane-bound TNFDelta1-9,K11E is capable of inducing signal transduction by TNF-R75 and TNF-R55; in the latter case, however, the efficiency is less than with soluble TNF, which has a higher efficacy because of ligand passing.

A Postulated Salt Bridge between Lysand C-terminal LeuIs Not Required for Bioactivity of Soluble mTNF

As shown above, the bioactivity of membrane-bound, uncleavable TNFDelta1-12 and TNFDelta1-9,K11E was lower in certain assays than the bioactivity of wtTNF. A possible explanation for these differences is that in these TNF mutants a postulated salt bridge between Lys and the C-terminal Leu of an adjacent subunit in mTNF could no longer be formed after substitution or deletion of Lys. It has indeed been suggested that this salt bridge in the soluble hTNF trimer may be required for bioactivity (Eck and Sprang, 1989). Therefore, Lys was substituted by Glu in full-length, soluble, mature mTNF. The specific bioactivity of TNF-K11E expressed in and purified from bacteria was, however, very similar to that of wtTNF, viz. 6.6 10^7 IU/mg and 2 10^8 IU/mg, respectively. This finding is in agreement with reported data that substitution of Lys (mutant K11Q, K11M, K11T, or K11N) in mature hTNF, expressed in E. coli, had virtually no effect on its bioactivity (Zhang et al., 1992). As a consequence, the almost normal biological activity of TNF-K11E indicates that removal of the hypothetical salt bridge on itself cannot explain the differences in bioactivity observed between wtTNF and uncleavable, membrane-bound TNF mutants in some of the assays. It is likely that differences in TNF-induced signaling pathways, inherent to the assays applied, account for the distinct responses observed.


DISCUSSION

The biosynthesis of TNF is a complex phenomenon. The 26-kDa proform of TNF, which presumably forms a trimer, is present as a type II transmembrane protein on the cell surface. The 17-kDa TNF subunit is then proteolytically cleaved off and released still as a trimer in the extracellular milieu (Kriegler et al., 1988). The proteolytic cleavage of mTNF normally takes place between the presequence and mature mTNF (-1/+1; Fig. 1) as shown by amino acid sequencing of the NH(2) terminus of TNF secreted by stimulated murine macrophages (Sherry et al., 1990).

In this study we eliminated the standard TNF cleavage site by mutating the N-terminal moiety of mature mTNF. We found that removal of the first 9 amino acids of mature TNF was not sufficient to prevent TNF processing and still led to the release of biologically active TNF in the supernatant. However, by deleting 3 additional amino acids, creating TNFDelta1-12, a membrane-bound, uncleavable TNF form was generated. This result is in agreement with the finding that deletion of the first 12 amino acids of mature hTNF also leads to an uncleavable mutant (Perez et al., 1990). The residual cleavage of TNFDelta1-9 versus the absence of cleavage with TNFDelta1-12 is indicative of the existence of an additional cleavage site in mature mTNF. This alternative cleavage site seems to be nonfunctional in wtTNF. When the proper site (-1/+1) is removed, the second site might become more exposed, facilitating extracellular cleavage at this noncanonical site. Lys is crucial for this cleavage, since cellular expression of TNFDelta1-9,K11E resulted in uncleavable, membrane-bound mTNF.

The biological activity of uncleavable TNF mutants was evaluated by coculturing TNF-expressing L929 transfectants with various TNF-responsive target cell lines. These included murine CT6, rat/mouse PC60-R55/R75 and human myeloid U937 cells, which respond to TNF by proliferation, GM-CSF production, and apoptosis, respectively. The proliferation of CT6 cells is exclusively mediated by mTNF-R75 signaling (Tartaglia et al., 1993a), presumably through interaction with TNF-R-associated factors 1 and 2 (Rothe et al., 1994), whereas the induction of GM-CSF production by PC60-R55/R75 involves the two TNF-Rs.^2 In both types of assay, cells expressing membrane-bound TNFDelta1-9,K11E were found to have a bioactivity roughly similar to that of wtTNF transfectants and to be more potent than TNFDelta1-12 transfectants. These results demonstrate that both TNF-R55 and TNF-R75 can be triggered by membrane-bound TNF to the same extent as by soluble TNF. But different results were obtained for the induction of apoptosis in U937 cells, where cells expressing uncleavable TNF mutants were less effective. The lesser activity of the membrane-bound TNF mutant was not due to removal of the hypothetical salt bridge linking Lys to Leu. Rather, it can be explained by the fact that induction of apoptosis in U937 cells is a hTNF-R55-mediated effect (Barbara et al., 1994); at low concentrations, hTNF-R75 might facilitate triggering of TNF-R55 by ligand passing (Tartaglia et al., 1993b). This mechanism is presumably not possible with the uncleavable, membrane-bound TNF mutant. The reduced bioactivity of TNFDelta1-12 transfectants as compared with TNFDelta1-9,K11E transfectants was not due to a lower protein expression level, since membrane-associated expression of TNFDelta1-12 and TNFDelta1-9,K11E showed similar levels (Fig. 3). The lower bioactivity of TNFDelta1-12 might be caused by removal of Pro, which could lead to a local structural disturbance, resulting indirectly in lower binding capacity/affinity for TNF-R. Carlino et al. (1987) also demonstrated a direct correlation between the bioactivity of hTNF mutants with N-terminal deletions and the physical binding to TNF-R. Analysis of the expression levels of uncleavable TNF mutants did not reveal increased numbers of TNF molecules on the cell membrane, despite the fact that these molecules are no longer cleaved off. This is analogous to findings that after inhibition of TNF secretion by metalloproteinase inhibitors, no accumulation of membrane-bound TNF was observed (Gearing et al., 1994). This lack of TNF accumulation might be due to the presence of a homeostatic feedback mechanism, which ensures that the proform of TNF is rapidly recycled and degraded inside the cells (Pradines-Figueres and Raetz, 1992).

Our data indicate that cleavage of the 26-kDa mTNF precursor is not aimed at generating an active TNF form from an inactive one, but rather at switching between two active TNF forms, one membrane-bound and the other diffusible. The proteolytic cleavage of TNF might therefore be an important regulatory step to switch from TNF effects mediated at the local level through cell-cell contact to effects mediated at the paracrine and systemic level. Also the mechanism of TNF signaling by membrane-bound TNF is different, since it does not involve ligand passing and therefore raises the threshold concentration for TNF-R55-signaling responses. Thus, the relative contribution of both forms of TNF to bioactivity is crucial for understanding the physiological impact of TNF. Elevated doses of soluble TNF in animals and humans are severely toxic (Beutler et al., 1985; Tracey et al., 1986). Recently, metalloproteinase inhibitors impairing TNF processing have been identified and found to protect animals against a lethal dose of endotoxin-induced TNF (Mohler et al., 1994). These data suggest that secreted TNF, and not membrane-bound TNF, is most harmful to the host. Our findings suggest that membrane-bound TNF in the presence of proteinase inhibitors would still be biologically active. Injection of TNF-expressing tumor cells in mice showed a reduced tumorigenicity (Vanhaesebroeck et al., 1991). In order to investigate the role therein of membrane-bound versus secreted TNF, the in vivo behavior of L929 cells expressing uncleavable TNF mutants will be studied. In this respect, biologically active, uncleavable TNF mutants will allow examination of the physiological relevance of membrane-bound TNF in vivo.


FOOTNOTES

*
This work was supported in part by the Interuniversitaire Attractiepolen, the Fonds voor Geneeskundig Wetenschappelijk Onderzoek, Levenslijn, and a European Community Biotech Program ``In Vitro Immunotoxicology.'' The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a fellowship from the Instituut tot Aanmoediging van het Wetenschappelijk Onderzoek in Nijverheid en Landbouw.

Present address: Ludwig Institute for Cancer Research, 91 Riding House Street, London W1P 8BT, UK.

**
Postdoctoral Research Assistant with the Nationaal Fonds voor Wetenschappelijk Onderzoek.

§§
To whom correspondence should be addressed. Tel.: 32-9-264-51-31; Fax: 32-9-264-53-48.

^1
The abbreviations used are: TNF, tumor necrosis factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; h, human; m, murine; neo^r, neomycin-resistant; TNF-R, TNF receptor; TNF-R55, 55-kDa TNF-R; TNF-R75, 75-kDa TNF-R; wt, wild type.

^2
W. Declercq, P. Vandenabeele, and W. Fiers, (1995) Cytolkine7, in press.


ACKNOWLEDGEMENTS

We thank A. Raeymaekers for providing TNF preparations; J. Van der Heyden (Roche Research, Gent, Belgium), Dr. R. Lucas (Brussels University, Brussels, Belgium), and Dr. M. Brockhaus (Hoffmann-La Roche, Basel, Switzerland) for antisera; and Dr. J. DeLamarter (Glaxo IMB, Geneva, Switzerland) for mGM-CSF. A. Meeus, M. Van den Hemel, W. Burm, D. Ginneberge, and F. Molemans are acknowledged for technical assistance.


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