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Originally published online as doi:10.1189/jlb.1007713 on April 24, 2008

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(Journal of Leukocyte Biology. 2008;84:162-169.)
© 2008 by Society for Leukocyte Biology

Enhanced susceptibility to Con A-induced liver injury in mice transgenic for the intracellular isoform of human TNF receptor type 2

Monika Bäumel, Anja Lechner, Thomas Hehlgans and Daniela N. Männel1

Department of Immunology, University of Regensburg, Regensburg, Germany

1Correspondence: Department of Immunology, University of Regensburg, F.-J.-Strauss-Allee, D-93042 Regensburg, Germany. E-mail: daniela.maennel{at}klinik.uni-regensburg.de


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ABSTRACT
 
TNF is a pleiotropic cytokine involved in a variety of inflammatory processes and immune responses. TNF effects are mediated via two distinct membrane receptors: TNFR1 and TNFR2. Investigations concerning regulation and function of TNFR2 revealed a novel TNFR2 isoform in human and mouse cells, termed icp75TNFR, with mainly intracellular localization. As human icp75TNFR is capable of functional interaction with mouse TNF, mouse lines transgenic for the human icp75TNFR were generated and characterized. Transgenic expression was identified in several organs, and soluble human (sh)TNFR2 was detected in serum. shTNFR2 released from transfected cells or peritoneal macrophages of transgenic mice protected from TNF-induced cytotoxicity. Although in vivo, no change in inflammatory reactions was observed in models of septic peritonitis, of colitis, or after stimulation with bacterial LPS, liver injury was strongly enhanced in transgenic mice after Con A challenge. Thus, the functional properties of human icp75TNFR seem to be similar to that of TNFR2, resulting in exacerbation of inflammatory tissue damage, thus revealing the functional importance of TNFR2 in pathophysiological processes.

Key Words: p75TNF receptor • hepatitis • cytotoxicity


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INTRODUCTION
 
TNF is a pleiotropic cytokine with strong inflammatory and immune-modulating properties. It belongs to a family of structurally related ligands with a high degree of homology in their extracellular domains [1 ]. The extracellular domain of the transmembrane precursor form of TNF is cleaved by membrane-bound metalloproteinases leading to release of soluble (s)TNF [2 ]. Membrane-bound as well as the sTNF form noncovalently linked homotrimers [3 ] and activate the two TNFRs, TNFR1 and TNFR2 [4 ].

Most inflammatory effects of TNF have been attributed to sTNF by activating TNFR1 [5 ], and it has been demonstrated that the endotoxin-induced detrimental effects in septic shock are caused by shedding TNF from myeloid cells [6 ]. The membrane-bound form of TNF preferentially signals through TNFR2 [7 ], and a crucial role of TNFR2 activation in chronic inflammatory disease is increasingly becoming apparent [8 ]. Interestingly, human (h)TNF does not activate the mouse (m)TNFR2, and mTNF is able to activate the hTNFR2.

Both TNFRs belong to the TNFR superfamily [1 ] and are coexpressed on most tissues. Although TNFR1 is constitutively expressed, TNFR2 expression is under transcriptional and post-transcriptional control [5 ]. The extracellular domains of both TNFRs are similar and possess multiple cysteine-rich motifs, and the intracellular domains differ in structure and induce different signaling pathways. In inflammatory diseases, shedding of the extracellular domains of both TNFRs occurs [9 ], possibly representing a regulatory mechanism to bind and inactivate the sTNF ligand. As the preferred ligand for TNFR2 is membrane-bound TNF [7 ], and TNFR2 activation can act in an autocrine manner to enhance TNF effects [10 ], a local immune regulatory role for TNFR2 is suggested.

Mice deficient for functional TNFR2 develop normally and have no structural or morphological abnormal phenotype. Overexpression of the hTNFR2, including the regulatory human promoter elements in a transgenic mouse model, resulted in development of a severe inflammatory syndrome with constitutively activated NF-{kappa}B in peripheral mononuclear cells of the animals [11 , 12 ]. Also, in the experimental hepatitis model induced by Con A, in which liver damage is dependent on the presence of TNF and both TNFRs, mice overexpressing the regulated hTNFR2 displayed increased pathology [13 ].

We have identified and characterized a TNFR2 isoform in human and mouse generated by the use of an additional transcriptional start site and alternative splicing [14 , 15 ]. The alternatively spliced hTNFR2 isoform (termed hicp75TNFR) and the mouse ortholog of this isoform (termed micp75TNFR) are mainly localized intracellularly [16 ] but differ in their TNF-binding capacity. Upon emerging on the cell surface, the hicp75TNFR is functionally no longer distinguishable from hTNFR2 and can become activated by human as well as mTNF, while the micp75TNFR is incapable of binding mTNF.

In this report, we provide the phenotypic characterization of mice transgenic for the hicp75TNFR and show that transgenic expression of this new TNFR2 isoform induces a similar but more subtle phenotype as the known TNFR2, as demonstrated by exacerbation of inflammatory liver damage in Con A-induced hepatitis.


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MATERIALS AND METHODS
 
Mice
Confirmed hicp75TNFR-transgenic (tg) founder mice were crossed to C57BL/6 mice purchased from Charles River (Sulzfeld, Germany). The hicp75TNFR-tg mouse lines were generated as described in detail below. Nontransgenic littermates were used as controls. Mice were housed in the animal facility of the University of Regensburg (Germany) and handled in accordance with institutional guidelines. All experiments were performed in compliance with the federal guidelines for animal experimentation.

Cells and reagents
Human embryo kidney (HEK) 293 cells and mouse L929 cells (American Type Culture Collection, Manassas, VA, USA) were maintained in RPMI-1640 medium (Sigma-Aldrich Chemie GmbH, Deisenhofen, Germany) or DMEM (Invitrogen, Karlsruhe, Germany) supplemented with 10% heat-inactivated FCS (PAN Biotech GmbH, Aidenbach, Germany). Purified recombinant hTNF (rhTNF) was kindly provided by BASF Bioresearch (Ludwigshafen, Germany) and purified rmTNF by Peter Scheurich (University of Stuttgart, Institute of Cell Biology and Immunology, Stuttgart, Germany). LPS from Salmonella enterica serovar Abortus-Equi was a gift from Marina Freudenberg (Max Planck Institute of Immunobiology, Freiburg, Germany) [17 ].

Plasmid construction and generation of hicp75TNFR-tg mice
To generate hicp75TNFR-tg mice, full-length hicp75TNFR-cDNA was cloned into HindIII and XhoI sites of an expression vector driven by the human ubiquitin C promoter [18 ]. The sequence of the cloned hicp75TNFR construct was confirmed by automated sequencing. The transgenic hicp75TNFR fragment was released by using NdeI/KpnI digestion of the vector and microinjected into pronuclei of fertilized oocytes prepared from FvB mice using standard protocols. The offspring were screened for transgene integration by DNA extraction from tail biopsies. The purified DNA was analyzed by Southern blot analysis using a {alpha}-P32-dCTP-labeled hicp75TNFR-cDNA fragment as probe. As positive control, a part of the transgenic hicp75TNFR fragment was removed by EcoRI digestion of the vector. Confirmed founders were backcrossed into C57BL/6 background, and transgene transmission in the offspring was tested by PCR as detailed below.

PCR genotyping
For PCR analysis, DNA was obtained from the tail by phenol/chloroform extraction. Total RNA was isolated from the tissue using QIAshredder (Qiagen, Hilden, Germany) and RNeasy Mini kit (Qiagen). PCR was performed as follows: cDNA was generated from total RNA (1 µg) from each sample by RT using the RT system (Promega, Mannheim, Germany), according to the manufacturer’s recommendations. The cDNAs (5 µl) were amplified using following primers: hicp75 3' in (5'-GTC CAA CGT TCC GTT CGC GCG G-3'), hicp75 3' out (5'-GCT CCT CCC AAG CCA GCT CCA C-3'); icp-tg forward (5'-AAA CCC CAT CTC TAT AAA GAA ATC-3'), icp-tg reverse (5'-CGG GCG GCA CTT GCG CAG-3'); β-actin (5'-TGA CGG GGT CAC CCA CAC TGT-3'), β-actin (5'-CTA GAA GCA TTT GCG GTG GAC-3'). Annealing temperatures for each primer: hicp75 3' in, hicp75 3' out: 60°C; icp-tg forward, icp-tg reverse: 70°C; β-actin: 58.5°C. Samples were kept for 5 min at 94°C, and to amplify genomic DNA from the tail, the following 35 cycles were conducted: 94°C for 60 s, annealing temperature for 60 s, and 72°C for 60 s. To amplify cDNA, the following 35 cycles were conducted: 94°C for 30 s, annealing temperature for 30 s, and 72°C for 45 s. To confirm equal amounts of RNA, β-actin cDNA was amplified in each assay. Aliquots of the samples were analyzed by electrophoresis on 1.5% agarose gel and visualized by ethidium bromide staining.

Expression of shTNFR2 and hicp75TNFR
hTNFR2 and hicp75TNFR were expressed in HEK 293 cells by transiently transfecting the retroviral expression vector pQCXIP (BD Biosciences Clontech, Palo Alto, CA, USA) containing the respective hTNFR2 isoform tagged with a human c-myc epitope by using the Ca3(PO4)2 precipitation method as described recently [15 ]. Conditioned medium after 24 h was harvested and tested for inhibitory activity of TNF cytotoxic activity on L929 cells.

Preparation of peritoneal macrophages
Mice received 1 ml/mouse 3% thioglycollate i.p. After 3 days, peritoneal exudate cells were isolated from the peritoneal cavity by washing with ice-cold medium (RPMI 1640, 10% FCS). Cells were cultured for 1 h and washed with medium to remove nonadherent cells. Adherent cells were used as peritoneal macrophages and were cultured in DMEM supplemented with 5% FCS.

TNF-induced cytotoxic assays
Peritoneal macrophages (1x105 cells/culture) were seeded, and 1 h later, serial dilutions of rhTNF or rmTNF were added. After 24 h, cell viability was assessed by adding MTT (Sigma-Aldrich Chemie GmbH) for 4 h. Cells were lysed with SDS, and OD was determined at 540 nm.

To test TNF neutralizing capacity of shTNFR2 and hicp75TNFR, L929 cells (2x104 cells/culture) were seeded, and 16 h later, serial dilutions of rhTNF or rmTNF diluted in conditioned media from transfected HEK cells were added in the presence of actinomycin D (2 µg/ml, Sigma-Aldrich Chemie GmbH). Cell viability was assessed after 24 h as above.

Measurements of cytokine serum levels
Serum samples were taken 90 min after i.p. challenge with LPS from S. enterica serovar Abortus-Equi [17 ] at 1 µg/200 µl per mouse. Quantification of soluble hp75TNFR serum titers was performed by ELISA (R&D Systems, Wiesbaden, Germany) according to the manufacturer’s recommendations.

D-galactosamine (DGalN)/LPS treatment
Mice received 5 µg/kg LPS from S. enterica serovar Abortus-Equi and 700 mg/kg DGalN (Roth, Karlsruhe, Germany) in a volume of 200 µl PBS i.p. [18 ]. Five hours later, animals were killed by cervical dislocation, and liver damage was assessed by measuring serum liver enzymes and by histological examinations (see below).

Con A and hTNFR2-Ig treatment
Con A (30 mg/kg, Sigma-Aldrich Chemie GmbH) was injected i.v. The commercially available TNF inhibitor hTNFR2-Ig (Etanercept, Enbrel®, Wyeth Pharma GmbH, Münster, Germany; 5 mg/kg) was administered i.p. 1 h earlier as indicated. As control treatment, mice received the carrier solution (0.9% NaCl) only. After 8 h, mice were killed by cervical dislocation, the liver was removed, and blood was sampled.

Determination of alanine aminotransferase (ALT) and aspartate aminotransferase (AST)
Hepatocyte damage was assessed by the activity of ALT and AST in the serum. The activities were determined by an enzyme assay as described previously [13 ].

Histological examinations
To investigate the degree of histopathological changes, livers from treated mice were removed and fixed in 10% formalin in PBS overnight, and sections (5 µm) of the paraffin-embedded material were made. Liver sections were stained with H&E.

Statistical analysis
For statistical analysis, the Mann-Whitney U-test was used. Error bars represent the SEM. It was considered statistically significant when P < 0.05.


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RESULTS
 
Generation of hicp75TNFR-tg mice
By injection of the ubi-hicp75TNFR fragment into oocytes of FvB mice, transgenic embryos were created and transplanted into pseudo-pregnant foster mice. Four out of five mice born were tested positive by Southern blot analysis. Mouse number 46 and mouse number 62 showed the most intensive bands characteristic for integration of the hicp75TNFR transgene (Fig. 1A ). These two founder mice were each backcrossed into C57BL/6 background, and their transgenic offspring and littermates, respectively, were used for experiments. The transgene was inherited in a Mendelian manner. Attempts to generate mice homozygous for the hicp75TNFR transgene failed with both lines. No homozygous transgenic mice could be generated from parents both transgenic for the hicp75TNFR gene.


Figure 1
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  		Figure 1. Expression of hicp75TNFR in transgenic mice. (A) Genomic DNA isolated from founder mice (numbers 20, 24, 25, 46, and 62) and wild-type mice (wt) was analyzed by Southern blot for expression of the hicp75TNFR transgene. A mixture of wild-type DNA and a fragment of the vector (wt/control) was used as positive control. (B) Total RNA extracted from organs of hicp75TNFR-tg mice (lines 46 and 62) was analyzed by RT-PCR. As negative control, total RNA isolated from wild-type mice was used. The quality of all cDNAs was tested by RT-PCR, amplifying β-actin. (C) LPS-induced, soluble hp75TNFR was measured by ELISA in mouse sera from hicp75TNFR-tg (n=12) and wild-type mice (n=3). Data are expressed as the mean ± SEM.

Expression of hicp75TNFR in transgenic mice
RT-PCR of the transgenic lines 46 and 62 demonstrated that the hicp75TNFR transgene was expressed in lymphoid and nonlymphoid organs. The characteristic hicp75TNFR RT-PCR product at 430 bp was detected in thymus, mesenteric lymph node, spleen, liver, and colon in both mouse lines (Fig. 1B) . β-Actin has been amplified with each sample as an internal control. After LPS treatment, only sera from transgenic mice showed high levels of soluble hicp75TNFR, which is identical with shTNFR2 and known to be cleaved off the membrane after LPS stimulation (Fig. 1C) . The presence of shTNFR2 proves biosynthesis of hicp75TNFR in transgenic mice. No histological differences in the structure of organs (spleen, lymph nodes, thymus, Peyer’s patches, colon, intestinum, and liver) from hicp75TNFR-transgenic or nontransgenic mice were found (data not shown).

Soluble hicp75TNFR neutralizes mTNF cytotoxicity
Supernatants from transiently transfected cells expressing the extracellular domains of hTNFR2 or hicp75TNFR, respectively, were compared in TNF-induced cytotoxicity assays on sensitive L929 for their TNF neutralizing capacity. Supernatants from hTNFR2 and hicp75TNFR transfectants acted equally well as inhibitors by competitive binding of rhTNF as well as rmTNF and neutralizing the cytotoxic activity (Fig. 2A and 2B ). Therefore, it was examined whether cells from transgenic mice would be protected from TNF-induced cytotoxicity by shedding soluble hicp75TNFR/hTNFR2. Peritoneal exudate macrophages from wild-type and hicp75TNFR-tg mice, respectively, were stimulated with different concentrations of rmTNF. As shown in Figure 2C , the survival rate of macrophages from transgenic mice was slightly increased at low concentrations of rmTNF. Although in the presence of low concentrations of rmTNF, the survival rate of macrophages from wild-type mice was ~80%, and nearly 100% of macrophages isolated from hicp75TNFR-tg mice survived. Only at rmTNF concentrations higher than 1.25 ng/ml did the cytotoxic effect of rmTNF increase in wild-type and transgenic macrophages in a dose-dependent manner.


Figure 2
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  		Figure 2. TNF cytotoxicity. Survival of L929 cells was tested after exposure to increasing concentrations of rhTNF (A) or rmTNF (B) in the absence or presence of supernatant conditioned by HEK cells control-transfected (•), transfected with hicp75TNFR ({circ}), or hTNFR2 ({blacktriangledown}). (C) Survival of macrophages of hicp75TNFR-tg ({blacksquare}) and wild-type ({square}) mice stimulated with increasing concentrations of rmTNF was determined.

DGalN/LPS-induced hepatotoxicity
TNF is an important inflammatory cytokine in acute inflammation. To determine whether overexpression of hicp75TNFR is able to modify acute liver toxicity, mice were treated with DGalN and LPS [19 ]. As indicated in Figure 3A 3B 3C , liver histology and liver enzyme levels in sera of wild-type and transgenic mice demonstrated similar inflammatory reactions: Liver sections isolated from DGalN/LPS-treated wild-type and transgenic mice displayed comparable necrosis and areas with hemorrhage (Fig. 3A) . Also, a similar DGalN/LPS-induced increase in serum levels of AST and ALT was observed in wild-type and transgenic mice (Fig. 3B and 3C) . Therefore, icp75TNFR does not seem to be involved in DGalN/LPS-induced liver inflammation.


Figure 3
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  		Figure 3. LPS-induced liver injury in sensitized hicp75TNFR-tg mice. (A) Histological analysis of liver sections 5 h after DGalN/LPS treatment of hicp75TNFR-transgenic and wild-type mice. Liver sections from untreated mice served as negative controls. Paraffin sections were stained with H&E (original magnification, x100). Liver enzyme activity of AST (B) and ALT (C) was quantified in sera from hicp75TNFR-transgenic (n=3) and wild-type (n=3) mice 5 h after DGalN/LPS treatment. Data are expressed as the mean ± SEM.

Con A-induced hepatotoxicity in hicp75TNFR-tg mice
i.v. injection of Con A in mice induces hepatotoxicity within 8 h [20 ]. In this model, liver biopsies from Con A-treated, transgenic mice showed coherent, bridging areas of necrosis with strong hemorrhage. Inside necrotic tissue-condensed nuclei were present, which are characteristic for cell death (data not shown). In contrast, liver sections from wild-type mice solely indicated insular regions of necrosis (Fig. 4A ), and IL-2 and IFN-{gamma} serum levels 3 h after Con A treatment were not significantly different in transgenic versus nontransgenic littermates (data not shown). Measurements of ALT and AST in sera resulted in significantly higher levels in transgenic compared with wild-type mice (Fig. 4B and 4C) . Therefore, administration of Con A resulted in considerably augmented hepatotoxicity in hicp75TNFR-tg compared with wild-type mice.


Figure 4
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  		Figure 4. Con A-induced liver injury in hicp75TNFR-tg mice. (A) Histological analysis of liver sections of hicp75TNFR-transgenic and wild-type mice. Paraffin sections were stained with H&E (original magnification, x100). (B) Liver enzyme activity of AST (B) and ALT (C) was quantified in sera from hicp75TNFR-transgenic (n=8) and wild-type (n=6) mice 8 h after Con A treatment. Data are expressed as the mean ± SEM.

TNF blockade protects from Con A-induced hepatotoxicity
Endogenous TNF was neutralized in mice before Con A administration to discriminate whether soluble hicp75TNFR/hTNFR2 in transgenic mice acting as a TNF inhibitor or overexpression of hicp75TNFR allowing augmented binding and signaling of endogenous TNF is causing the observed, increased hepatotoxicity. For that purpose, the dimeric fusion protein of the extracellular domain of hTNFR2 and the Fc part of hIgG1 (hTNFR2-Ig) was used as a TNF inhibitor in vivo in wild-type and hicp75TNF-tg mice. Histological analysis of liver sections revealed that pretreatment with the TNF inhibitor ameliorated hepatic damage in both mouse lines (Fig. 5A ). The massive damage of liver tissue following Con A administration with partially disrupted cell structures, whereby liver sections of transgenic mice again exhibited more vigorous necrotic areas and apoptotic nuclei than sections from wild-type mice, was completely, equally prevented in both mouse lines. Also, the increased serum levels of ALT after Con A injection were equally normalized by blocking TNF in transgenic and wild-type mice (Fig. 5B) .


Figure 5
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  		Figure 5. Con A-induced liver injury in hicp75TNFR-tg mice after neutralization of TNF. (A) Wild-type and hicp75TNFR-transgenic mice were pretreated with TNF inhibitor or saline (control) 1 h before Con A treatment, and histological analysis of liver sections was performed. Paraffin sections were stained with H&E (original magnification, x100). Liver enzyme activity of ALT (B) was quantified in sera from hicp75TNFR-transgenic (n=5) and wild-type (n=4) mice, pretreated with TNF inhibitor (solid bars) or saline (control, open bars) 8 h after Con A treatment. Data are expressed as the mean ± SEM.


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DISCUSSION
 
The functional role of TNFR2 is still not entirely clear. Unchallenged, TNFR2-deficient mice are normal with respect to size and composition of their lymphocyte compartments. They are protected from TNF-induced death and tissue necrosis [21 ], experimental cerebral malaria [22 ], mortality in septic peritonitis models [23 ], and tissue damage in glomerulonephritis [24 ]. Therefore, the hypothesis has evolved that TNFR2 serves as an accessory role in enhancing TNFR1-mediated inflammatory reactions by amplifying apoptotic signal transduction initiated by TNFR1 [25 , 26 ].

On the other hand, TNFR2 acted in an anti-inflammatory way by preventing TNF binding to TNFR1 through shedding of sTNFR2 and competitive binding of TNF [25 , 27 , 28 ]. In addition, TNFR2-specific activation reduced neurodegeneration after retinal ischemic injury by [29 ] and susceptibility to edema formation [30 ]. TNFR2 activation was required for development of LPS-induced protection from lethal inflammation [31 ] as well as sepsis-induced immune suppression [32 ], also indicating a role for this receptor in dampening inflammation. Recently, a function for TNFR2 has been shown as a T cell costimulatory molecule in controlling cell fate duringTCR/CD28 stimulation [33 ] and in expansion and activation of regulatory T cells [34 ]. Activation of the transcription factor NF-{kappa}B is the main cellular response upon TNFR2 activation [35 36 37 38 ]. Members of the TNFR-associated factor (TRAF) family interact with TNFR2, with TRAF1 acting as a negative regulator of TNF signaling [39 ] and TRAF2 leading to up-regulation of NF-{kappa}B [40 ] and inhibition of TNF-induced apoptosis [41 , 42 ].

We recently described new isoforms of the TNFR2 in the mouse and in the human system, termed micp75TNFR and hicp75TNFR, respectively [14 , 15 ]. The discovery of these new TNFR2 isoforms, which had developed independently in mice and men during evolution, opened up the possibility for further TNFR2 functions [43 ]. In mice, the alternatively spliced form of TNFR2 consisted of a TNFR2 protein, not only lacking the leader sequence but also the first 26 amino-terminal amino acids, resulting in a TNFR incapable of binding TNF [15 ]. In contrast, in humans, introduction of a transposable alu element and subsequent, alternative splicing contributed to the creation of a new, alternative first exon and production of a functional TNFR2 isoform also lacking a leader sequence [14 , 43 ]. As shown for the mouse ortholog [15 ], this new hicp75TNFR was also found to be retained in the trans-Golgi network, where it colocalized with endogenous TNF [16 ]. Upon emerging on the cell surface, this mainly intracellularly localized hicp75TNFR was functionally no longer distinguishable from hTNFR2.

Two independent mouse lines transgenic for the hicp75TNFR described in this report demonstrated no histological change in structure of lymphatic or nonlymphatic organs (data not shown). Numbers of CD4+ or CD8+ splenic T cells were comparable in hicp75TNFR-transgenic and nontransgenic littermates (data not shown). No difference was detectable in the production of IL-2 or IFN-{gamma} or in proliferation of transgenic CD4+ T cells when compared with wild-type T cells when stimulated with anti-CD3 and anti-CD28 antibodies under conditions where TNFR2-deficient cells clearly exhibit reduced effects (data not shown). However, macrophages from transgenic mice were protected from TNF toxicity at low concentrations of TNF, which can be attributed to shed shTNFR2, which acts as inhibitor by competitive binding of TNF.

It has been shown that hepatocyte damage upon LPS injection in sensitized mice entirely depends on TNFR1 [44 ]. Accordingly, no enhanced hepatotoxicity to LPS was observed in hicp75TNFR-transgenic mice, even after sensitization by DGalN. This finding is in contrast to mouse lines transgenic for regulated hTNFR2 in which increased production of hTNFR2 potently sensitized the transgenic mice to the toxicity of LPS or TNF and sustained overproduction-triggered, multiorgan, inflammatory pathology [11 ]. This difference between the mouse lines transgenic for the two different hTNFR2 isoforms is also seen in models of intestinal inflammation. In a Th1 T cell-mediated mouse model of experimental colitis induced by reconstitution of SCID mice with CD62 ligand+ CD4+ T cells from wild-type or regulated, hTNFR2-transgenic mice, respectively, overexpression of the regulated hTNFR2 augmented development of chronic, intestinal inflammation with a decreased apoptosis rate of the inflammatory infiltrate [12 ]. In contrast, no difference was observed with respect to weight loss or histology of colonic tissue between hicp75TNFR-transgenic mice and their nontransgenic littermates using models of acute or chronic colitis induced by dextran sulfate sodium, in which TNFR2-deficient mice were protected during the acute phase but exhibited stronger intestinal inflammation in the chronic phase (data not shown). Also, survival of septic peritonitis induced by cecal ligation and puncture was not changed in hicp75TNFR-transgenic mice (data not shown).

The mouse model of Con A-induced inflammatory liver injury resembles active autoimmune hepatitis in humans characterized by CD4+T cell infiltration, apoptosis of hepatocytes, release of transaminases into circulation, and liver damage shown to be mediated by TNF and to be dependent on the presence of TNFR1 and TNFR2 [13 , 20 ]. Inactivation of metalloproteinases such as TNF-converting enzyme efficiently reduced shedding of TNF [6 ] and exacerbated hepatic injury in this hepatitis model [45 ]. Mice transgenic for noncleavable TNF and lacking sTNF developed hepatitis to the same degree as wild-type mice, indicating a pathogenic role of membrane-bound TNF locally activating TNFR2 on intrinsic liver cells [13 ]. In this model of hepatic injury, the hicp75TNFR-transgenic mice resembled the mice described previously, transgenic for regulated hTNFR2 [11 ] in developing exacerbated liver damage upon Con A treatment.

The findings that ligand-induced clustering of TNFR is essential for signaling [46 ] and forced production, potentially leading to spontaneous signaling and cell death, even in the absence of ligand [11 , 35 , 47 , 48 ], might explain why it was not possible for us to generate a mouse line with homozygous expression of hicp75TNFR. Although hicp75TNFR-transgenic mice appear to expose little hTNFR2 on the cell membrane, selective pressure for viable offspring obviously prevented generation of stable homozygous expression of the hicp75TNFR transgene. This is in line with the results from mice transgenic for regulated hTNFR2, where severity of pathology was dependent on transgene copy number and expression of the membrane form of hTNFR2 [11 ]. In addition, homozygous expression of this regulated hTNFR2 gene led to a constitutive hTNFR2 appearance on the membrane and development of a severe, multiorgan, inflammatory syndrome with death early after birth.

These findings, indicating a more subtle effect of the transgenic expression of hicp75TNFR than expression of the regulated hTNFR2 but still inducing enhanced susceptibility for TNF-dependent tissue damage, support the functional importance of TNFR2 in pathophysiological processes and make the hicp75TNFR2-transgenic mice a suitable model for studies of the functional role of TNFR2 in vivo.


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ACKNOWLEDGEMENTS
 
This work was supported by a grant of the Deutsche Forschungsgemeinschaft (DFG), Ma760, to D. N. M.

Received October 26, 2007; revised February 28, 2008; accepted March 18, 2008.


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