Characterization of TNF receptor type 2 isoform in the mouse

Molecular Immunology 44 (2007) 3563–3570

Characterization of TNF receptor type 2 isoform in the mouse Andrea Hauser, Thomas Hehlgans, Daniela N. M¨annel ∗ Department of Immunology, University of Regensburg, F.-J.-Strauss-Allee, D-93042 Regensburg, Germany Received 16 January 2007; received in revised form 14 March 2007

Abstract In this report a TNF receptor type 2 (TNFR2) isoform in the mouse, termed micp75TNFR, was compared to its human orthologue hicp75TNFR and to the known mouse TNFR2. The micp75TNFR is generated by the use of an alternative transcriptional start site within the mouse TNFR2 gene. This receptor isoform was found to be expressed in different macrophage-like and endothelial tumor cell lines and mouse spleen cells after stimulation. Lacking the leader sequence and part of the amino-terminal end of the mature TNFR2, the micp75TNFR was characterized by mainly intracellular expression. In contrast to its human paralogue, the micp75TNFR lacked the capacity to bind TNF. Therefore, micp75TNFR expression did not mediate protection from TNF-induced cytotoxicity but may interfere with TNFR2 signalling. © 2007 Elsevier Ltd. All rights reserved. Keywords: TNF receptor; Isoform; Cytotoxicity

1. Introduction TNF is a pleiotropic cytokine involved in a broad spectrum of inflammatory and immune responses including proliferation and cytotoxicity in a variety of different cell types. Two distinct receptor molecules with an apparent molecular weight of 55 kDa (TNFR type 1, TNFR1) and 75 kDa (TNFR type 2, TNFR2), respectively, have been identified and their corresponding cDNAs have been cloned (Loetscher et al., 1990; Schall et al., 1990; Smith et al., 1990; Lewis et al., 1991). The TNFR1 is expressed rather constitutively on a broad spectrum of different cell types and has been shown to mediate most of the commonly known biological effects of TNF (Vercammen et al., 1995; Wajant et al., 2003). In contrast, expression of the TNFR2 seems to be regulated and there are only a few cellular responses which can be attributed exclusively to signalling via the TNFR2, e.g. proliferation thymocytes and mature T cells, of NK and B cells, and GM-CSF secretion of T lymphocytes (Vandenabeele et al., 1992; Grell et al., 1998). Recently a function for TNFR2 has also been shown in expansion and activation of regulatory T cells (Scumpia et al., 2006; Chen et al., in press). Moreover, the TNFR2 has been shown to be preferentially activated by membrane-bound TNF (Seitz et al., ∗

Corresponding author. Tel.: +49 941 944 5460; fax: +49 941 944 5462. E-mail address: [email protected] (D.N. M¨annel).

0161-5890/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2007.03.017

1998). Although the intracellular receptor domains show only little similarity, TNFR1 and TNFR2 share activities such as NF␬B activation (Vercammen et al., 1995; Wajant et al., 2003). Here we characterize the mouse TNFR2 isoform termed micp75TNFR in analogy to the previously described human orthologue hicp75TNFR (Seitz et al., 1998). The elucidated open reading frame of micp75TNFR revealed that the leader sequence encoded by exon 1 of mouse TNFR2 is replaced by an alternative un-translated exon. Subsequently translation of micp75TNFR is initiated by the use of a translational initiation site (ATG) within the second exon of the mouse TNFR2 gene leading to a truncated form of the receptor molecule incapable of binding to TNF. 2. Materials and methods 2.1. Cell lines and reagents Mouse L929, RAW 264.7, P815, PU5-1.8 and bEnd3 cells (ATCC, American Type Culture Collection, Manassas, VA), were maintained in RPMI 1640 medium (Sigma–Aldrich Chemie GmbH, Deisenhofen, Germany) and NIH 3T3 and HEK 293 cells (ATCC) in Dulbecco’s MEM (Invitrogen, Karlsruhe, Germany) supplemented with 10% heat-inactivated fetal calf serum (FCS; PAN Biotech GmbH, Aidenbach, Germany) and 0.05-mg/ml gentamycine (PAA Laboratories GmbH, Linz, Austria). Purified recombinant human TNF (rhTNF) was kindly

3564

A. Hauser et al. / Molecular Immunology 44 (2007) 3563–3570

provided by BASF (BASF Bioresearch, Ludwigshafen, Germany) and purified recombinant mouse TNF (rmTNF) by P. Scheurich, Stuttgart. Bacterial lipopolysaccharide (LPS E. coli 0127:38) and d-galactosamine were purchased from Sigma, mouse monoclonal anti-c-myc antibodies (9E10) from Invitrogen, polyclonal rabbit anti-mouse TNFR1 antibodies from HyCult (Caltag, Hamburg, Germany), biotinylated anti-mouse TNFR2 antibodies (TR-32.4) from BioLegend (San Diego, USA) and polyclonal rabbit anti-human TNFR2 antibodies (H202) from Santa-Cruz (Biotechnology, Santa Cruz, CA, USA). 2.2. micp75TNFR RT-PCR and Southern blot Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Hilden, Germany) and transcribed into cDNA with the Reverse Transcription System from Promega (Madison, USA). PCR amplification was performed using sense and antisense primers specific for micp75TNFR (5 -GAGAAGATGCAAACATTCATGTGGAG-3 and 5 -GATCTGGCACTCGTACCCAGGTTC-3 ). Agarose gels were blotted and the resulting nylon membranes were UV-crosslinked, prehybridized with ULTRAhyb Hybridization Buffer (Ambion, Austin, USA) and incubated with a radioactively labeled specific probe, that was generated by PCR, using an expression plasmid for micp75TNFR and a pair of specific sense and antisense primers (5 -CCTGAGCGGTGCCAACTTTG-3 and 5 -CACTCCTTCCTCTTCGAGAC-3 ), targeting the 5 untranslated region of micp75TNFR. After three washing steps (0.1% SDS, 0.1 N SSC) membranes were developed by exposure to X-ray films (BioMax MR films; Kodak, Cedex, France) for different periods of time. The quality of all cDNAs was tested by performing RT-PCR, amplifying ␤-actin. 2.3. Mouse TNFR2 and micp75TNFR expression plasmids Mouse TNFR2 and micp75TNFR expression plasmids were constructed by inserting the corresponding myc-tagged cDNAs into the modified proviral expression plasmid pQCXIP (BD Biosciences Clontech, CA, USA) containing the multiple cloning site and EYFP coding sequence of pEYFP-N1 (BD Biosciences Clontech, CA, USA). Constructs are therefore expressed as myc-tagged receptor proteins fused to EYFP at the intracellular portion of the receptor. All constructs have been verified by sequencing. 2.4. Transduction In order to produce retrovirus containing supernatants HEK 293 cells were transiently cotransfected with the packaging construct pCl-10 A1 (Naviaux et al., 1996) and the retroviral vector pQCXIP (BD Biosciences Clontech) containing the gene of interest using the HEPES-buffered saline Ca3 (PO4 )2 precipitation method (Sambrook and Russel, 2001). Retrovirus containing supernatants were collected 2 and 3 days after transfection of HEK 293 cells and filtered through a low protein binding 0.45 ␮m pore-size filter (Acrodisc Syringe filter; PALL Corporation, MI, USA) in presence of 8 ␮g/ml polybrene

(Sigma–Aldrich). Retroviral infection of target cells took place over 2 days by changing the virus containing medium every 12 h. Selection for positive clones was done via puromycin resistance of the pQCXIP vector using 13 ␮g/ml puromycin (Sigma–Aldrich) for L929 cells and 2 ␮g/ml for NIH 3T3 cells. 2.5. Flow cytometry Expression of the transduced mouse TNFR2 isoforms was detected by flow cytometry using a FACStar Plus (Becton Dickinson, San Jose, CA, USA). For FACS analysis cells (1 × 106 /tube) were blocked in PBS supplemented with 10% heat inactivated fetal calf serum for 30 min on ice and either examined directly, analyzing their EYFP-autofluorescence in channel FL-1H, or stained with a biotinylated anti-mouse TNFR2 antibody (TR-32.4; BioLegend) and Streptavidin-APC (Pharmingen, Becton Dickinson) as secondary antibody. Mouse TNFR2-Ig and micp75TNFR-Ig constructs were incubated with memTNF-expressing or control cells for 1 h at 4 ◦ C, washed, and stained with a polyclonal, PE-coupled donkey anti-human IgG F(ab )2 fragment (Jackson ImmunoResearch, Soham, UK). 2.6. Immunoprecipitation and Western blot Stably transduced L929 cells (1 × 107 ) expressing one of the two mouse TNFR2 isoforms or EYFP alone as negative control were lysed in 1 ml buffer (150 mM NaCl, 50 mM Tris·HCl pH 7.4, 1 mM EDTA, 1% Triton X-100, 1% NP-40, 0.25% Na-deoxycholate) containing a mixture of protease inhibitors (CompleteTM EDTA-free tablets, Roche, Mannheim, Germany). After centrifugation, lysates were precleared for 4 h with 20 ␮l protein G-Sepharose (Amersham Biosciences, Uppsala, Sweden) and incubated with a mouse anti-c-myc antibody (clone 9E10, Invitrogen) for 16 h at 4 ◦ C and immunoprecipitated with 20 ␮l protein G-Sepharose for 1 h at 4 ◦ C. Precipitates were washed three times in PBS and protein G-Sepharose-free samples were subjected to SDS-PAGE under reducing conditions and blotted onto a PVDF membrane. Immunoblot analysis was performed after blocking in 1% non-fat milk powder with a polyclonal rabbit antibody, directed against the intracellular portion of the human TNFR2 (H-202; Santa-Cruz Biotechnology) to detect the full length protein. A goat anti-rabbit IgG-HRP (Sigma–Aldrich) was used as secondary antibody. Blots were then incubated with HRP substrate (enhanced chemiluminescence substrate NOWA solution A and B, MoBiTec GmbH) and developed by exposure to film (Hyperfilm; Amersham Biosciences) using the Enhanced Chemiluminescence (ECL) Western Blot Detection System (Energene, Regensburg, Germany). 2.7. Expression and analysis of Ig fusion proteins Mouse TNFR2-Ig and micp75-Ig constructs were expressed in HEK 293 cells by transiently transfecting the pQCXIP vector containing the extracellular domain of the respective TNFR2

A. Hauser et al. / Molecular Immunology 44 (2007) 3563–3570

isoforms fused to the Fc part of human IgG1 and tagged with a human c-myc epitope, by using the Ca3 (PO4 )2 -precipitation method (Sambrook and Russel, 2001). One milliliter of the resulting supernatant was incubated with an anti-mouse TNFR2 antibody (TR-32.4; BioLegend) for 16 h at 4 ◦ C and the resulting immune complexes were precipitated with 20 ␮l protein G-Sepharose (Amersham Biosciences) for 1 h at 4 ◦ C. The precipitated proteins were subjected to SDS polyacrylamide electrophoresis and transferred to a membrane. Immunoblot analysis was performed with a horse radish peroxidasecoupled anti-c-myc-antibody (clone 9E10, Invitrogen) using the ECL Western Blot Detection System (Energene). Blots were then incubated with HRP substrate (enhanced chemiluminescence substrate NOWA solution A and B; MoBiTec GmbH) and developed by exposure to film (Hyperfilm; Amersham Biosciences). 2.8. Fluorescence microscopy Stably transduced mouse TNFR2 and icp75TNFRexpressing NIH 3T3 cells were seeded in Lab-Tek II Chamber Slide systems (Nunc GmbH & Co. KG, Wiesbaden, Germany). Twenty-four hours later cells were fixed with 4% paraformaldehyde in PBS for 15 min, permeabilized with 0.1% Triton X-100 for 5 min and incubated in blocking solution (1% BSA in PBS) for 1 h at room temperature. Nuclei of cells were stained with DAPI (Sigma–Aldrich) at room temperature for 15 min. Both mouse TNFR2 isoforms were analyzed directly without additional staining, using their EYFP-autofluorescence. Coverslides were mounted with MobiGLOW Mounting Medium (MoBiTec GmbH, G¨ottingen, Germany) and analyzed with a Zeiss Axiovert microscope. Images were taken with a charge-coupled device (CCD) camera (Princeton Instruments, Trenton, USA). 2.9. TNF-induced cytotoxicity assay and ligand binding assay L929 cells (2.5 × 104 ) either wild-type or stably transduced with one of the mouse TNFR2 isoforms or with an empty vector, respectively, were seeded into 96-well microtiter plates. After 12 h cells were treated with serial dilutions of recombinant mouse TNF (rmTNF, specific activity 2 × 107 U/mg) or of agonistic polyclonal rabbit anti-mouse TNFR1 antibodies (HyCult) in presence of 2 ␮g/ml actinomycin D. In case of the ligand binding assay with membrane TNF (memTNF) L929 cells were treated with serial dilutions of stably transduced L929 cells, expressing uncleavable mouse TNF (Lewis et al., 1991) and fixed with Cytofix/CytoPerm (BD, Heidelberg, Germany). For the TNF binding assays both, actinomycin D and rmTNF or fixed memTNF expressing cells, respectively, were diluted in Ig-construct-containing supernatants. Cell viability was determined by adding 0.5 mg/ml 3-(4,5dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide dye (MTT, Sigma–Aldrich) for 4 h at 37 ◦ C. Cells were lysed at 37 ◦ C overnight by adding 70 ␮l 20% SDS per well. Optical density was measured at 540 nm. Each experiment was repeated at least three times.

3565

3. Results While studying the transcriptional regulation of the mouse TNFR2 gene it was realized that the promoter sequence lacks a consensus TATA element within the first few hundred base pairs proximal to the translational start site ATG (+1) in the first exon, while TATA-like elements are located further upstream of the translational start site. Primer extension analysis identified two transcriptional start sites within the 5 flanking region of the mouse TNFR2 gene. Transcriptional initiation was located at positions −39/−35 (TS I) relative to the translational start site ATG (+1) of mouse TNFR2 while a second transcriptional start site can be localized at position −588 (TS II) relative to the translational start site ATG (Seitz et al., 1998). These findings resemble the situation of the human TNFR2 gene organization (Seitz et al., 1998). In order to identify the corresponding cDNA complementary to the mRNA originating from TSII we performed RT-PCR using a 5 primer located at position −529 to −550 and a 3 primer located at the 3 end of the mouse TNFR2 cDNA (Fig. 1B). Three independent cDNA clones were isolated and revealed sequence identity. Primary sequence analysis indicated that transcriptional initiation at TSII within the 5 flanking region of the mouse TNFR2 gene results in a novel mouse TNFR2 cDNA. Comparison of the two mouse TNFR2 cDNAs indicated sequence identity in the extracellular (ED), transmembrane (TM), and intracellular domain (ID). In contrast, the new mouse TNFR2 cDNA isoform lacks the 5 UTR and the first exon of mouse TNFR2 cDNA (Fig. 1A). Alignment with the genomic sequence further showed that the mRNA transcript initiated at TSII generates a splice donor site at position −198 which fuses the transcript to the splice acceptor site at the 5 end of exon 2 of the mouse TNFR2 transcript. The fusion of this new exon 1a generates an open reading frame with an ATG within exon 2 of the mouse TNFR2 transcript as a potential translational initiation codon (Fig. 1A and B). Thus, the ORF of the new mouse TNFR2 transcript generates a cDNA that encodes for a new mouse TNFR2 which is truncated by 52 amino acids at the N-terminal domain of the mouse TNFR2 protein. Since the first 26 amino acids of the mouse TNFR2 protein were identified as a signal peptide directing the nascient protein to the cell surface the truncation in the new mouse TNFR2 cDNA results in an effective deletion of 26 amino acids of the mature protein compared to mouse TNFR2 (Fig. 1B). In analogy to the human TNFR2 and its isoform (hicp75TNFR) (Seitz et al., 1998), the new mouse TNFR2 isoform cDNA was termed micp75TNFR cDNA. In order to determine the expression pattern of the new isoform of mouse TNFR2 mRNA we performed RT-PCR analysis using a primer specific for the 5 end of mouse TNFR2 cDNA and a 3 primer annealing in the second exon of the mouse TNFR2 cDNA resulting in a 360 bp PCR product. The cDNAs prepared from various cell lines and primary mouse tissue were subjected to RT-PCR and products were verified by hybridization with a specific probe generated with nested primers specific for the 5 untranslated region of the new micp75TNFR cDNA. Our results revealed expression of the new mouse TNFR2 iso-

3566

A. Hauser et al. / Molecular Immunology 44 (2007) 3563–3570

Fig. 1. Structure and sequence of the TNFR2 gene and the mRNAs of both mouse TNFR2 isoforms. (A) The mRNAs for both isoforms originate from different transcriptional start sites (TS I and TS II, respectively). In case of mouse TNFR2 exon 1 (black box) codes for a signal peptide whereas micp75TNFR uses a 5 UTR instead. Protein biosynthesis is initiated at an ATG within exon 2. Exons are indicated as boxes. SP, signal peptide; ED, extracellular domain; TM, transmembrane domain; ID, intracellular domain; 5 UTR, 5 untranslated region; 3 UTR, 3 untranslated region. Primer sites for micp75TNFR-specific RT-PCR are indicated. (B) Partial nucleotide sequence of the mouse TNFR2 gene showing the proximal promotor region, exon 1 of mouse TNFR2, the 5 UTR of micp75TNFR (underlined) and the N-terminus of exon 2. Translated nucleotide sequences are shown in capital letters. The first ATG (boxed) initiating exon 1 marks the startpoint of protein biosynthesis for mouse TNFR2 whereas micp75TNFR uses an ATG (boxed) within exon 2.

form in the macrophage-like cell lines RAW 264.7 and PU5-1.8, in the mastocytoma cell line P815, and in the mouse endothelial cell line bEnd3 after stimulation with LPS, a known inducer of TNFR2 transcription (Vercammen et al., 1995; Wajant et al., 2003). RAW 264.7 was the only cell line tested positive for the expression of the new mouse TNFR2 (icp75TNFR) mRNA without prior LPS stimulation, supporting the notion

that the micp75TNFR mRNA expression is up-regulated by proinflammatory stimuli (Fig. 2). The same cell lines which expressed the micp75TNFR mRNA were also found positive for the regular mouse TNFR2 mRNA expression in parallel experiments. Interestingly, the micp75TNFR transcript was also detectable in spleen cells but only from mice treated with LPS and d-galactosamine (Fig. 2). This combined treatment of d-

A. Hauser et al. / Molecular Immunology 44 (2007) 3563–3570

Fig. 2. Expression pattern of micp75TNFR and mouse TNFR2 mRNA. RTPCR products were hybridized with a radioactively labeled probe specific for the 5 end of the micp75TNFR cDNA. Cells of the lines PU5-1.8, P815, bEnd3, and RAW 264.7 were either not treated or stimulated with LPS (10 ␮g/ml) for 12 h and spleen cells from mice treated with LPS alone (1 ␮g/20 g mouse) or with a combination of LPS (100 ng/20 g mouse) and d-galactosamine (d-GalN; 14 mg/20 g mouse) 5 h earlier were used to prepare RNA. The quality of all cDNAs was tested by RT-PCR, amplifying ␤-actin.

galactosamine together with LPS sensitizes mice for the LPS toxicity and leads to endotoxic shock (Galanos et al., 1979). Compared to the mouse TNFR2 mRNA expression level, the relative abundance of the micp75TNFR mRNA seemed much

3567

lower in all tested cell lines and also in the primary cells of the LPS/d-galactosamine-treated mice. To further characterize the new mouse TNFR2 isoform, cell lines were generated that stably express one of either receptor proteins with an N-terminal c-myc-tag and fused to enhanced yellow fluorescent protein (EYFP). Transduced cells expressing EYFP served as negative controls in the experiments. To verify expression of both receptor isoforms, transduced L929 cells (Fig. 3A) and NIH 3T3 cells (data not shown) were analyzed by flow cytometry using the EYFP-fluorescence of both receptor constructs. All analyzed cell lines were positive for EYFP fluorescence. Both receptor constructs together with the negative control were immune precipitated to determine their molecular weight (Fig. 3B). The precipitating antibody was directed against the c-myc-tag fused to the receptor and detection was performed using polyclonal antibodies against the intracellular portion of mouse TNFR2 to exclusively detect full length proteins and exclude shedded forms of the receptors. The apparent molecular weight of mouse TNFR2 was determined with approximately 116 kDa resulting in approximately 80 kDa when the molecular weight of the EYFP fusion protein is subtracted. This result is consistent with earlier publications and inspired to the name “p80TNFR” for TNFR2 used by several groups. The

Fig. 3. Expression and characterization of micp75TNFR. (A) Flow cytometric analysis of transduced cells. L929 cells were transduced with expression plasmids either encoding mouse TNFR2 or micp75TNFR, both expressed as fusion proteins with eYFP and selected for positive clones with puromycin. The resulting cell populations were then analyzed in a flow cytometer for expression of the corresponding receptor using their eYFP fluorescence. Untransduced L929 cells were used for determination of autofluorescence (shaded peak). (B) Biochemical characterization of micp75TNFR. L929 cells stably transduced with either mouse TNFR2, micp75TNFR, or the control vector, expressing EYFP only, were lysed and immunoprecipitated with monoclonal antibodies against the c-myc epitope (9E10). After blotting, the membrane was stained using polyclonal antibodies against the intracellular portion of mouse TNFR2 (H-202) to exclusively detect full length protein. (C) Localization of the micp75TNFR. Expression patterns of both mouse TNFR2 isoforms were analyzed using fluorescent microscopy. Stably transduced NIH 3T3 cells were fixed, permeabilized and nuclei were stained with DAPI. Both receptors were analyzed directly without further staining using their intracellular EYFP-tag. While mouse TNFR2 is mainly expressed on the cell membrane micp75TNFR shows an intracellular, perinuclear staining (1000-fold magnification). (D) Cell surface expression of micp75TNFR. Expression of both mouse TNFR2 isoforms was analyzed by flow cytometry using L929 cells stably transduced with expression plasmids encoding either mouse TNFR2 (gray line) or micp75TNFR (black line) or empty vector (shaded peak). Receptor expressing cells were gated for equal expression of EYFP to eliminate differences in transduction efficiency. All three cell lines were stained with monoclonal antibodies against the extracellular domain of mouse TNFR2 (TR-32.4). The histogram shows a weaker but detectable expression of micp75TNFR on the cell surface.

3568

A. Hauser et al. / Molecular Immunology 44 (2007) 3563–3570

new mouse TNFR2 isoform showed a reduction in the apparent molecular weight of approximately 15 kDa resulting from the truncation of exon 2 (Fig. 1A). Stably transduced NIH 3T3 cells expressing one or the other of the mouse TNFR2 isoforms N-terminally fused to an EYFP-tag were analyzed in fluorescent microscopy to determine the subcellular location of the new mouse TNFR2 isoform protein (Fig. 3C). Both receptor isoforms could be detected directly without additional staining by use of their EYFP fluorescence. Microscopic analysis revealed a clear cell surface staining for mouse TNFR2 whereas the new mouse TNFR2 isoform seemed primarily localized intracellularly in a perinuclear region. No specific staining was detected in untransfected cells (data not shown). Due to its intracellular expression and in analogy to the human TNFR2 isoform (Seitz et al., 1998; Scher¨ubl et al., 2005) the newly identified receptor isoform was termed micp75TNFR for intracellularly expressed mouse TNFR2. Although cell surface expression could not be detected by fluorescent microscopy, potential membrane expression of micp75TNFR was additionally examined by flow cytometry (Fig. 3D). L929 cells stably expressing either of the two mouse TNFR2 isoforms N-terminally fused to EYFP were gated on equal expression of EYFP first to eliminate possible differences in transduction efficiency. Staining with monoclonal antibodies directed against the extracellular domain of mouse TNFR2 (TR-32.4) confirmed cell surface expression of the TNFR2 (Fig. 3D, gray line) but surprisingly also showed membrane staining for micp75TNFR however to a lesser extent (Fig. 3D, black line). To investigate functional characteristics of micp75TNFR, L929 cells stably expressing either mouse TNFR2 isoform or empty vector were analyzed in a TNF-induced cytotoxicity assay (Fig. 4A). Whereas cells overexpressing mouse TNFR2 mediate protection from cytotoxicity induced by mouse TNF, cells expressing mcip75TNFR behaved like negative control cells transfected with EYFP alone. As actinomycin D, a potent RNApolymerase inhibitor, is present in the cytotoxicity assays no

NF␬B can be activated to mediate protection by expression of anti-apoptotic genes. Hence additionally, a TNF-induced cytotoxicity assay was performed with delayed (after 1 h) addition of actinomycin D. Again protection from mTNF-induced cytotoxicity could only be detected for mouse TNFR2-transduced cells (data not shown). Fig. 4B demonstrates that the protection is restricted to mouse TNF as cytotoxicity induced by human TNF was not inhibited by either cell line indicating that competitive binding of toxic TNF is the reason for the observed protection by mouse TNFR2 in Fig. 4A. Human TNF exclusively binds to the mouse TNFR1, thereby inducing cell death but does not bind to mouse TNFR2 (Lewis et al., 1991). Therefore, competition for TNF can only take place in case of mTNF but not when hTNF is used. While in the case of hicp75TNFR, the human TNFR2 isoform, binding of TNF was demonstrated (Seitz et al., 1998; Scher¨ubl et al., 2005), these data led to the question, whether the new mouse TNFR2 isoform, lacking 26 amino acids of exon 2, is still capable of binding TNF. To address this question extracellular domains of either mouse TNFR2 isoform originating at the translational start site within exon 2 in case of micp75TNFR and right at the beginning of exon 2 in case of mouse TNFR2, respectively, were fused to the Fc part of human IgG1 carrying a C-terminal c-myc-tag. Both constructs together with a control protein (Fc part of human IgG1 alone) were expressed individually in HEK 293 cells followed by immune precipitation and Western blot analysis using precipitating antibodies against the extracellular domain of mouse TNFR2 and anti-cmyc antibodies for detection. Analysis of both Ig-constructs resulted in detection of a single protein band for each construct with an apparent molecular mass of about 85 kDa for mouse TNFR2-Ig and a slightly reduced molecular weight for micp75TNFR-Ig, respectively, while no protein expression was detected in control protein expressing HEK 293 cells (data not shown). To further test whether the new mouse TNFR2 isoform as a fusion protein is capable of binding TNF, the supernatants

Fig. 4. Role of micp75TNFR in TNF-induced cytotoxicity assay. (A) TNF-induced cytotoxicity assay using recombinant mTNF. A TNF-induced cytotoxicity assay was performed using recombinant mTNF, diluted to a concentration of 10 ng/ml and added in serial dilution in the presence of 2 ␮g/ml actinomycin D. Mouse TNFR2-expressing cells in contrast to micp75TNFR- or EYFP-expressing cells mediate protection from TNF-induced cytotoxicity. (filled circle: L929-EYFP, open circle: L929-micp75TNFR, triangle: L929-mouse TNFR2). (B) TNF-induced cytotoxicity assay using recombinant hTNF. Recombinant hTNF was used at a stock concentration of 50 ng/ml (filled circle: L929-EYFP, open circle: L929-micp75TNFR, triangle: L929-mouse TNFR2). (C) TNF-induced cytotoxicity assay using agonistic antibodies to mouse TNFR1. Polyclonal rabbit anti-mouse TNFR1 antibodies were added in serial dilutions in presence of 2 ␮g/ml actinomycin D (filled circle: L929-EYFP, open circle: L929-micp75TNFR, triangle: L929-mouse TNFR2).

A. Hauser et al. / Molecular Immunology 44 (2007) 3563–3570

3569

Fig. 5. Ligand-binding capacity of micp75TNFR. Mouse TNFR2 and micpTNFR-ECD were fused to the Fc-part of a human IgG1 as depicted schematically in (A). Both Ig-constructs, together with a control protein (Fc-part of human IgG1 alone) were separately expressed in HEK 293 cells. Ig-constructs were used as soluble proteins in TNF-induced cytotoxicity assays. (B) Binding capacity of micp75TNFR to soluble mouse TNF (mTNF). Soluble recombinant mTNF and actinomycin D were diluted in Ig-construct- or control protein-containing supernatants to a concentration of 10 ng/ml and 4 ␮g/ml, respectively. Soluble mTNF in conditioned medium was added in serial dilutions and an equal amount of actinomycin D was added resulting in an effective concentration of 2 ␮g/ml actinomycin D (filled circle: control protein, open circle: micp75TNFR-Ig, triangle: mouse TNFR2-Ig). (C) Binding capacity of micp75TNFR to soluble human TNF (hTNF). A TNF-induced cytotoxicity assay as described in (B) was performed, using a concentration of 50 ng/ml recombinant human TNF (hTNF) (filled circle: control protein, open circle: micp75TNFR-Ig, triangle: mouse TNFR2-Ig). (D) Binding capacity of micp75TNFR to membrane expressed mTNF (memTNF). A TNF-induced cytotoxicity assay was performed as described in (B) using L929 cells, stably transduced with uncleavable mTNF (memTNF) instead of soluble mTNF as toxic agent. Transduced and fixed memTNF-expressing L929 cells were diluted in Ig-construct- or control protein-containing medium to a concentration of 1 × 107 cells/ml and added in serial dilutions in presence of 2 ␮g/ml actinomycin D (filled circle: control protein, open circle: micp75TNFR-Ig, triangle: mouse TNFR2-Ig).

of HEK 293 cells transfected with either Ig construct or control protein were tested in TNF-induced cytotoxicity assays. The addition of supernatant derived from HEK 293 cells transfected with the mouse TNFR2-Ig expression construct inhibited cytotoxicity induced by soluble mouse TNF whereas supernatant of micp75TNFR-Ig expressing cells or control protein expressing cells, respectively, showed no protective effect (Fig. 5B). To prove that the observed neutralization of TNF is a specific effect resulting from competitive binding of TNF by its receptor construct, the experiment was repeated with human TNF (Fig. 5C). As expected none of the two receptor fusion proteins protected from hTNF-induced cytotoxicity as human TNF does not bind to mouse TNFR2 (Lewis et al., 1991). For TNFR2 it has been shown that – in contrast to TNFR1 – this receptor type is primarily activated by membrane expressed TNF (memTNF) (Grell et al., 1995). Thus, the ligand-binding capacity of micp75TNFR to memTNF was examined (Fig. 5D) using memTNF-expressing L929 cells as inducers of cytotoxicity. Again mouse TNFR2-Ig fusion protein was able to neutralize memTNF-induced cytotoxicity whereas the micp75TNFR-Ig

fusion protein showed only a small inhibitory effect compared to control protein. To more precisely investigate binding of micp75TNFR-Ig to memTNF, TNF-expressing cells were incubated with either Ig-construct or control protein and the Fc-part of all Ig-constructs was stained with PE-conjugated polyclonal antibodies directed against human IgG1. Flow cytometric analysis demonstrated that the micp75TNFR-Ig construct exhibited nonspecific binding to a broad variety of cell lines not expressing any TNF at all (data not shown). This indicates that the new micp75TNFR protein isoform is no longer capable of binding mouse TNF. 4. Discussion A human TNFR2 isoform generated by the use of an additional transcriptional start site has previously been described by us and was termed hicp75TNFR (Seitz et al., 1998). In the case of hicp75TNFR, exon 1 contributing to the signal peptide in human TNFR2 was replaced by exon1a consisting of an Alu element which was exonized during evolution (Singer et al., 2004). The resulting alternative splice variant of human TNFR2 was found

3570

A. Hauser et al. / Molecular Immunology 44 (2007) 3563–3570

to be retained in the trans-Golgi network where it co-localized with endogenous TNF (Scher¨ubl et al., 2005). Upon emerging on the cell surface the hicp75TNFR was functionally no longer distinguishable from human TNFR2. This study shows that the human and the mouse TNFR2 gene organization are very similar (Seitz et al., 1998). However, while in the human alternatively spliced hicp75TNFR protein the extracellular domain of the mature protein is complete and only the signal peptide is lacking, in the mouse not only the leader sequence but also the first 26 amino-terminal amino acids of the protein are missing. The N-terminal region of the TNFR2 molecule is part of the so-called pre-ligand assembly domain (PLAD) of the TNF receptor which is required for non-covalent homotypic receptor interactions that allow efficient ligand binding (Chan et al., 2000). Therefore, the human alternatively spliced TNFR2 isoform and the mouse orthologue of this isoform differ in their TNF binding capacity while both are mainly localized intracellularly. The fact that the micp75TNFR is incapable of binding TNF excludes a function for micp75TNFR as TNF inhibitor competing for TNF. The structural features and functions of this mouse TNFR2 isoform resemble those described for other forms of TNFR. Analysis of TNFR1 polymorphisms and of mutant TNFR1 molecules associated with TNFR-associated periodic syndrome (TRAPS) with mutations or in-frame deletions of amino acids in the extracellular domain showed that these mutant receptors did not bind TNF, had defective surface expression, and failed to function as dominant negative inhibitors of TNFR1-induced apoptosis (Lobito et al., 2006). Instead, the TRAPS mutant TNFR1 exhibiting a high-degree of PLADindependent oligomerization failed to interact with wild-type TNFR1 molecules through the PLAD that normally governs receptor self-association. Retention in the endoplasmatic reticulum of mutant receptors, reduced ability to form soluble receptors, and altered signalling are described as the key common features of the misfolding and abnormal oligomerization. It was suggested that the inflammatory phenotype of TRAPS may be due to consequences of the mutant TNFR1 protein misfolding. It remains to be tested whether micp75TNFR can interact with wild-type TNFR2 to act as a dominant negative inhibitor or in a homotypic way such as the TRAPS mutant TNFR1. In the latter case interference with TNFR2 signal transduction could be envisaged as a possible function.

Acknowledgments This work was supported by a grant from the Deutsche Forschungsgemeinschaft SFB 585 to D.N.M. and T.H. References Chan, F.K., Chun, H.J., Zheng, L., Siegel, R.M., Bui, K.L., Lenardo, M.J., 2000. Science 288, 2351–2354. Chen, X., B¨aumel, M., M¨annel, D.N., Howard, O.M.Z., Oppenheim, J.J., in press. Interaction of TNF with TNF receptor type 2 promotes expansion and function of mouse. J. Immunol. Galanos, C., Freudenberg, M.A., Reutter, W., 1979. Proc. Natl. Acad. Sci. U.S.A. 76, 5939–5943. Grell, M., Becke, F.M., Wajant, H., M¨annel, D.N., Scheurich, P., 1998. Eur. J. Immunol. 28, 257–263. Grell, M., Douni, E., Wajant, H., Lohden, M., Clauss, M., Maxeiner, B., Georgopoulos, S., Lesslauer, W., Kollias, G., Pfizenmaier, K., Scheurich, P., 1995. Cell 83, 793–802. Lewis, M., Tartaglia, L.A., Lee, A., Bennet, G.L., Rice, G.C., Wong, G.H., Chen, E.Y., Goeddel, D.V., 1991. Proc. Natl. Acad. Sci. U.S.A. 88, 2830–2834. Lobito, A.A., Kimberley, F.C., Muppidi, J.R., Komarow, H., Jackson, A.J., Hull, K.M., Kastner, D.L., Screaton, G.R., Siegel, R.M., 2006. Blood 108, 1320–1327. Loetscher, H., Pan, Y.C., Lahm, H.W., Gentz, R., Brockhaus, M., Tabuchi, H., Lesslauer, W., 1990. Cell 61, 351–359. Naviaux, R.K., Costanzi, E., Haas, M., Verma, I.M., 1996. J. Virol. 70, 5701–5705. Sambrook, J., Russel, D.W., 2001. Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Schall, T.J., Lewis, M., Koller, K.J., Lee, A., Rice, G.C., Wong, G.H., Gatanaga, T., Granger, G.A., Lentz, R., Raab, H., Kohr, W.J., Goeddel, D.V., 1990. Cell 61, 361–370. Scher¨ubl, C., Schneider-Brachert, W., Sch¨utze, S., Hehlgans, T., M¨annel, D.N., 2005. J. Inflamm. 2, 7. Scumpia, P.O., Delano, M.J., Kelly, K.M., O’Malley, K.A., Efron, P.A., McAuliffe, P.F., Brusko, T., Ungaro, R., Barker, T., Wynn, J.L., Atkinson, M.A., Reeves, W.H., Salzler, M.J., Moldawer, L.L., 2006. J. Immunol. 177, 7943–7949. Seitz, C., M¨annel, D.N., Hehlgans, T., 1998. Genomics 48, 111–116. Singer, S.S., M¨annel, D.N., Hehlgans, T., Brosius, J., Schmitz, J., 2004. J. Mol. Biol. 341, 883–886. Smith, C.A., Davis, T., Anderson, D., Solam, L., Beckmann, M.P., Jerzy, R., Dower, S.K., Cosman, D., Goodwin, R.G., 1990. Science 248, 1019–1023. Vandenabeele, P., Declercq, W., Vercammen, D., Van de Craen, M., Grooten, J., Loetscher, H., Brockhaus, M., Lesslauer, W., Fiers, W., 1992. J. Exp. Med. 176, 1015–1024. Vercammen, D., Vandenabeele, P., Declercq, W., Van de Craen, M., Grooten, J., Fiers, W., 1995. Cytokine 7, 463–470. Wajant, H., Pfizenmaier, K., Scheurich, P., 2003. Cell Death Differ. 10, 45–65.