TNF-α, a potent proinflammatory cytokine, is synthesized as a membrane-anchored precursor and proteolytically released from cells. Soluble TNF is the primary mediator of pathologies such as rheumatoid arthritis, Crohn’s disease, and endotoxin shock. The TNF-α converting enzyme (TACE), a disintegrin and metalloprotease 17 (ADAM17), has emerged as the best candidate TNF sheddase, but other proteinases can also release TNF. Because TACE-deficient mice die shortly after birth, we generated conditional TACE-deficient mice to address whether TACE is the relevant sheddase for TNF in adult mice. In this study, we report that TACE inactivation in myeloid cells or temporal inactivation at 6 wk offers strong protection from endotoxin shock lethality in mice by preventing increased TNF serum levels. These findings corroborate that TACE is the major endotoxin-stimulated TNF sheddase in mouse myeloid cells in vivo, thereby further validating TACE as a principal target for the treatment of TNF-dependent pathologies.

The proinflammatory cytokine TNF-α has potent beneficial and detrimental functions (1). Its dysregulation triggers autoimmune diseases such as rheumatoid arthritis and Crohn’s disease, but TNF is also critical for host defense against pathogens such as listeria. TNF is synthesized as a membrane-anchored precursor that is released by the TNF-α-converting enzyme (TACE)3 a disintegrin and metalloprotease (ADAM) 17 (ADAM17) (2, 3). However, other enzymes, including ADAM10 (4), ADAM19 (5), matrix metalloproteinase 7 (6), and proteinase 3 (7) have also been implicated in releasing TNF, raising questions about the identity of the relevant TNF convertase in primary cells in vivo. LPS-triggered endotoxin shock in mice, a model for septic shock, provides an excellent means to evaluate the mechanism and consequences of TNF processing in vivo (8, 9, 10). LPS stimulates TNF release via TLRs and the resulting endotoxin shock requires soluble, but not membrane-anchored, TNF (8) released from myeloid cells (9) and can be prevented with metalloprotease inhibitors (10). This has led to the hypothesis that TACE is the critical enzyme for endotoxin-triggered release of TNF from myeloid cells in vivo in mice. However, to date this hypothesis could not be tested because TACE-deficient mice die shortly after birth (11, 12).

To address the role of TACE in endotoxin shock in vivo, we generated conditional TACE-deficient mice. We found that the temporal systemic deletion of Tace by the inducible Mx1-Cre transgene (13) or its deletion in myeloid cells by the M lysozyme promoter (LysM-Cre) (14) protected the mice from endotoxin shock with serum TNF levels that were significantly lower than those in control animals. These observations corroborate the hypothesis that endotoxin shock in mice is caused by TACE-dependent shedding of TNF from myeloid cells.

Genomic 129/SVJ DNA was used to generate a 1.6-kb PCR product with a noncoding sequence 1.1 kb upstream of exon 2 and a 5.2-kb PCR product with exon 2 and the surrounding noncoding region, which were subcloned into pCR2.1 TOPO vectors (Invitrogen Life Technologies). A loxP and an EcoRI site were inserted 1.0 kb downstream of exon 2, and the long and short arms were subcloned into a pKOII vector (provided by Dr. R. DePinho, Dana-Farber Cancer Center, Boston, MA). 129P2/OlaHsd cell clones harboring floxed Tace were identified by Southern blot and PCR analysis (Fig. 1, A and B). Chimeric mice were generated by C57BL/6 blastocyst injection and bred with C57BL/6 mice to generate floxed Tace heterozygous mice (referred to as Taceflox/+; formal allele nomenclature: Adam17tm1Bbl). Homozygous Taceflox/flox mice were viable and fertile and did not display any evident pathological phenotype or histopathological defects (data not shown). Taceflox/+ mice were mated with Tg(EIIa-cre)C5379Lmgd transgenic mice (15) (referred to as EIIa-Cre) to generate Tace-null heterozygous mice (referred to as Tace+/−; formal allele nomenclature: Adam17tm1.1Bbl) or with Lyzstm1(cre)Ifo (referred to as LysM-Cre) or Tg(Mx1-cre)1Cgn (referred to as Mx1-Cre) transgenic mice (13, 14) to generate conditional TACE-deficient mice. For temporal deletion of floxed Tace, 6-wk-old Taceflox/flox/Mx1-Cre+ mice were injected i.p. with 250 μg of polyinosinic-polycytidylic acid (pIpC; Sigma-Aldrich) three times at 2-day intervals as described (13). In all experiments with Taceflox/flox/Cre+ mice, littermate Taceflox/flox/Cre mice served as controls.

FIGURE 1.

Generation of Taceflox/flox and Tace−/− mice. A, Schema for conditional targeting of Tace. E indicates EcoRI site and arrowheads indicate loxP sites placed surrounding exon 2 and PGK neo. Arrows indicate primers used for genotyping. B, Southern blotting and PCR analysis of genomic DNA isolated from wild-type (+/+), floxed Tace (f/f), and Tace null (−/−) mice. C, TACE Western blot of lysates of mouse embryonic fibroblasts from embryonic day 13.5 wild-type and Tace−/− embryos.

FIGURE 1.

Generation of Taceflox/flox and Tace−/− mice. A, Schema for conditional targeting of Tace. E indicates EcoRI site and arrowheads indicate loxP sites placed surrounding exon 2 and PGK neo. Arrows indicate primers used for genotyping. B, Southern blotting and PCR analysis of genomic DNA isolated from wild-type (+/+), floxed Tace (f/f), and Tace null (−/−) mice. C, TACE Western blot of lysates of mouse embryonic fibroblasts from embryonic day 13.5 wild-type and Tace−/− embryos.

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Tissues were fixed in 4% paraformaldehyde and PBS, sectioned, stained with H&E, and photographed with a DXM1200 camera (Nikon) on a BX50 microscope (Olympus). Images were processed with Adobe Photoshop CS2.

Embryonic fibroblasts isolated from E13.5 Tace−/− embryos as described (16) were immortalized with the SV40 large T Ag. Absence of TACE protein in Tace−/− fibroblasts was confirmed by Western blotting (see Fig. 1 C).

Immortalized fibroblasts were transfected with alkaline phosphatase-tagged TNF or TGFα (see Ref. 16 for details). Fresh Opti-MEM (Invitrogen Life Technologies) with or without 25 ng/ml PMA was added for 1 h after 1 day, and alkaline phosphatase activity was measured by colorimetry or visualized by in-gel staining of substrates as described (16).

Bone marrow was collected from the tibiae and femurs of 6-wk-old Taceflox/flox/LysM-Cre or littermate Taceflox/flox/LysM-Cre+ mice. RBCs were removed with RBC lysis buffer (Roche), and the remaining cells were plated on tissue culture plates. Adherent cells were grown in DMEM with 10% FCS, antibiotics, and 50 ng/ml recombinant mouse macrophage CSF (WAKO) for 4 days and then used as bone marrow macrophages and incubated with or without 1 μg/ml LPS (Sigma-Aldrich) for 3 h. Soluble TNF was measured by ELISA (R&D Systems).

Endotoxin shock was induced by i.p. injection of 5 μg of LPS (Sigma-Aldrich) and 20 mg of d-galactosamine (WAKO). All injected mice were closely monitored every hour for the first 16 h and every 3–6 h thereafter. To enhance serum TNF levels in a separate experiment (see Fig. 3 C), Taceflox/flox/LysM-Cre or littermate Taceflox/flox/LysM-Cre+ mice were treated with 100 μg of LPS and 20 mg of d-galactosamine and sacrificed after 3 h. Serum TNF was measured by ELISA. All animal experiments were approved by the Institutional Animal Care and Use Committee of the Keio University School of Medicine (Tokyo, Japan).

FIGURE 3.

Temporal ablation of Tace or ablation in myeloid cells prevents lethality from LPS-induced shock. A, The efficiency of Cre-induced Tace excision in different organs in Taceflox/flox/Mx1-Cre+(Mx1+) mice was evaluated by PCR using comparable amounts of genomic DNA from Mx1-Cre+ and Mx1-Cre (Mx) Taceflox/flox mice as template. B, Seven- to 9-wk-old Taceflox/flox/Mx1-Cre or Taceflox/flox/Mx1-Cre+ mice treated with pIpC were injected i.p. with 5 μg LPS and 20 mg d-galactosamine and monitored for survival up to 36 h (n = 8). C, Systemic TNF production induced by LPS. LPS (100 μg) was injected i.p. into wild-type, Taceflox/flox/Cre (Cre), Taceflox/flox/Mx1-Cre+ pIpC-treated (Mx1+) or Taceflox/flox/LysM-Cre+ (LysM+) mice, sera were collected 3 h after injection, and TNF was measured by ELISA. *, p < 0.005 between Mx1+ or LysM+ and wild-type or Cre−. D, PCR analysis and Western blotting of cell lysates from Taceflox/flox/LysM-Cre and Taceflox/flox/LysM-Cre+ bone marrow macrophages. E, LPS-stimulated TNF secretion from Taceflox/flox/LysM-Cre and Taceflox/flox/LysM-Cre+ bone marrow-derived macrophages. LPS (1 μg/ml) was added to bone marrow macrophages isolated from 6-wk-old wild-type Taceflox/flox/LysM-Cre or Taceflox/flox/LysM-Cre+ mice and incubated for 3 h and TNF was measured by ELISA. n.d., Not detectable. F, Seven- to 9-wk-old mice were injected i.p. with 5 μg of LPS and 20 mg of d-galactosamine and monitored for survival up to 36 h (n = 13).

FIGURE 3.

Temporal ablation of Tace or ablation in myeloid cells prevents lethality from LPS-induced shock. A, The efficiency of Cre-induced Tace excision in different organs in Taceflox/flox/Mx1-Cre+(Mx1+) mice was evaluated by PCR using comparable amounts of genomic DNA from Mx1-Cre+ and Mx1-Cre (Mx) Taceflox/flox mice as template. B, Seven- to 9-wk-old Taceflox/flox/Mx1-Cre or Taceflox/flox/Mx1-Cre+ mice treated with pIpC were injected i.p. with 5 μg LPS and 20 mg d-galactosamine and monitored for survival up to 36 h (n = 8). C, Systemic TNF production induced by LPS. LPS (100 μg) was injected i.p. into wild-type, Taceflox/flox/Cre (Cre), Taceflox/flox/Mx1-Cre+ pIpC-treated (Mx1+) or Taceflox/flox/LysM-Cre+ (LysM+) mice, sera were collected 3 h after injection, and TNF was measured by ELISA. *, p < 0.005 between Mx1+ or LysM+ and wild-type or Cre−. D, PCR analysis and Western blotting of cell lysates from Taceflox/flox/LysM-Cre and Taceflox/flox/LysM-Cre+ bone marrow macrophages. E, LPS-stimulated TNF secretion from Taceflox/flox/LysM-Cre and Taceflox/flox/LysM-Cre+ bone marrow-derived macrophages. LPS (1 μg/ml) was added to bone marrow macrophages isolated from 6-wk-old wild-type Taceflox/flox/LysM-Cre or Taceflox/flox/LysM-Cre+ mice and incubated for 3 h and TNF was measured by ELISA. n.d., Not detectable. F, Seven- to 9-wk-old mice were injected i.p. with 5 μg of LPS and 20 mg of d-galactosamine and monitored for survival up to 36 h (n = 13).

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A Student’s t test for two samples assuming equal variances was used to calculate the p values. p < 0.05 was considered statistically significant.

Mice containing a floxed Tace allele were crossed with a germline “Cre-deleter” strain (EIIa-Cre) (15) to generate Tace+/− animals that were then bred to produce Tace−/− mice (Fig. 1,A; see Materials and Methods for details). Removal of floxed Tace was confirmed by PCR and Southern blotting (Fig. 1,B) and the lack of TACE protein was corroborated by Western blotting (Fig. 1,C). The Tace−/− animals generated in this study were indistinguishable from the previously described TaceΔZnZn mice that lack the exon carrying the catalytic site of TACE (2, 11); they died shortly after birth (Table I), with open eyes and defects in the aortic, pulmonic, and tricuspid heart valves (Fig. 2, A and B; pulmonic valves not shown). In addition, constitutive and phorbol ester-stimulated shedding of transfected TGF-α and TNF was strongly reduced in Tace−/− mouse embryonic fibroblasts (mEF) compared with wild-type controls (Fig. 2 C) as previously described for TaceΔZnZn mEF (5, 11, 16). Thus, Cre-mediated excision of floxed Tace results in the inactivation of TACE.

Table I.

Mendelian distribution of offspring from Tace+/− × Tace+/− mating

AgeaTotal+/++/−−/−
E17.5–18.5 87 23 45 19 
P1–2 87 28 49 10 
P13–15 78 26 52 
AgeaTotal+/++/−−/−
E17.5–18.5 87 23 45 19 
P1–2 87 28 49 10 
P13–15 78 26 52 
a

E, Embryonic; P, postnatal.

FIGURE 2.

Characterization of Tace−/− mice. A, Newborn Tace−/− (−/−) and wild-type (+/+) mice (upper panel); Tace−/− embryos have open eyes (arrowhead, lower panel). B, Histological analysis of the eyes and the aortic and tricuspid heart valves of newborn wild-type and Tace−/− mice (arrowheads). Bar, 500 μm. C, Evaluation of TNF and TGF-α shedding from immortalized wild-type and Tace−/− mEFs in the presence or absence of the phorbol ester PMA. An in-gel assay of substrate expression in lysates and shedding into the supernatant is shown in the left panel and a colorimetric assay of shedding into the supernatant is shown in the right panel (see Materials and Methods for details).

FIGURE 2.

Characterization of Tace−/− mice. A, Newborn Tace−/− (−/−) and wild-type (+/+) mice (upper panel); Tace−/− embryos have open eyes (arrowhead, lower panel). B, Histological analysis of the eyes and the aortic and tricuspid heart valves of newborn wild-type and Tace−/− mice (arrowheads). Bar, 500 μm. C, Evaluation of TNF and TGF-α shedding from immortalized wild-type and Tace−/− mEFs in the presence or absence of the phorbol ester PMA. An in-gel assay of substrate expression in lysates and shedding into the supernatant is shown in the left panel and a colorimetric assay of shedding into the supernatant is shown in the right panel (see Materials and Methods for details).

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To evaluate the role of TACE in adult mice, we generated Taceflox/flox/Mx1-Cre animals to allow excision of the floxed exon by i.p. injection of pIpC (13) in 6-wk-old mice. The pIpC-induced recombination in Mx1-Cre mice occurs in various organs with different efficiency, with almost complete recombination in bone marrow, liver, and spleen (13). Accordingly, we found strongly decreased amounts of floxed TACE in these tissues in Taceflox/flox/Mx1-Cre+ mice 1 wk after the pIpC injection (Fig. 3,A). After treatment with pIpC, Taceflox/flox/Mx1-Cr+ and Taceflox/flox/Mx1-Cre mice displayed no evident pathological phenotypes or histopathological defects for at least 2 wk (data not shown). We next tested the survival of Taceflox/flox/Mx1-Cre+ animals in a murine model for endotoxin shock, which depends on soluble TNF in vivo (8). When Taceflox/flox/Mx1-Cre+ mice or Taceflox/flox/Mx1-Cre controls were injected i.p. with LPS and d-galactosamine, six of eight Mx1-Cre+ mice were protected from endotoxin shock whereas seven of eight Mx1-Cre controls died within the first 16 h after injection (Fig. 3,B). Serum TNF levels after LPS injection were also significantly lower in Taceflox/flox/Mx-Cre+ mice (230 pg/ml, ± 177 SD; n = 7) compared with wild-type controls (852 pg/ml, ± 349 SD; n = 8) or Taceflox/flox/Cre animals (779 pg/ml, +/− 310 SD; n = 10) (Fig. 3 C). These results support the hypothesis that the shedding of TNF by TACE is crucial for LPS-induced endotoxin shock in mice, although protection from endotoxin shock could also be due, at least in part, to the reduction in cleavage of other TACE substrates.

To further narrow down the cell type in which TACE is required for endotoxin-induced release of TNF and the resulting lethality, we generated Taceflox/flox/LysM-Cre+ mice to remove TACE in myeloid cells (14), which are critical for LPS-induced shock (9). Taceflox/flox/LysM-Cre+ mice were viable and fertile and did not display any evident abnormalities (data not shown). Macrophages isolated from Taceflox/flox/LysM-Cre+ mice had very low amounts of the floxed Tace allele, as confirmed by PCR, and of the TACE protein, as detected by Western blot analysis, compared with the Taceflox/flox/LysM-Cre controls (Fig. 3,D). LPS-stimulated release of soluble TNF was strongly reduced in macrophages isolated from Taceflox/flox/LysM-Cre+ animals compared with Taceflox/flox/LysM-Cre controls (Fig. 3,E), which is consistent with the recently reported ablation of TNF shedding from TaceΔZnZn leukocytes isolated from a radiation chimera (17). When Taceflox/flox/LysM-Cre+ animals and Taceflox/flox/LysM-Crecontrols were subjected to LPS and d-galactosamine to trigger endotoxin shock, 11 of 13 control animals perished within 8 h of injection whereas only three of 13 Taceflox/flox/LysM-Cre+ animals succumbed to this challenge (Fig. 3,F). Following endotoxin challenge, the average TNFserum levels in Taceflox/flox/LysM-Cre+ (212 pg/ml, ± 158 SD; n = 7) were very similar to the levels in Taceflox/flox/Mx1-Cre+ mice (230 pg/ml, ± 177 SD; n = 7) and, thus, significantly lower than in Taceflox/flox/Cre animals or wild-type controls (see above and Fig. 3,C). The remaining endotoxin-stimulated shedding of TNF in Taceflox/flox/LysM-Cre+ and Taceflox/flox/Mx-Cre+ is most likely due to cells in which one or both floxed Tace alleles escaped Cre excision (see Fig. 3, A and D), although it cannot be ruled out that other candidate TNF sheddases also contribute to TNF shedding following Cre excision of TACE.

Taken together, these results provide strong evidence that TACE is indeed a principal enzyme responsible for the release of soluble TNF from the relevant primary cells (myeloid cells) during endotoxin shock in an intact organism in vivo (a discussion of criteria to link enzymes and relevant substrates can be found in Ref. 18). These results are consistent with studies of mice lacking an endogenous inhibitor of TACE, the tissue inhibitor of matrix metalloproteinases 3 (TIMP3) (19), that have an exacerbated response to an endotoxin challenge (20). In addition to releasing TNF, TACE is known to have a role in the shedding of numerous other membrane proteins from mEFs (11, 16, 21), and TACE activation in leukocytes also triggers shedding of the TNF receptors I and II (17). Shedding of these and other membrane anchored cytokines and growth factors could thus potentially contribute to the anergic state of the immune system at later stages of septic shock. It would therefore be interesting to determine whether or not TACE inhibitors might offer advantages in the treatment of septic shock in humans over the blocking of TNF, at least in those cases where an involvement of TNF is likely (reviewed in (22). Moreover, our results further support the notion that TACE is likely also a good target for treatment of other pathologies caused by soluble TNF, most notably rheumatoid arthritis, Crohn’s disease, and psoriasis. The Taceflox/flox animals described here will help address these questions, and permit an evaluation of other TACE functions during mouse development and in disease models, including those related to the role of TACE in epidermal growth factor receptor-ligand activation in cancer (23).

We thank Dr. Ronald A. DePinho, Dana-Farber Cancer Institute (Boston, MA) for the pKOII vector, Dr. Irmgard Förster, University of Düsseldorf (Düsseldorf, Germany) for the LysM-Cre transgenic mouse line, Dr. Ursula Lichtenberg, University of Cologne (Cologne, Germany) for the Mx1-Cre transgenic mouse line, Dr. Chingwen Yang from the Rockefeller University transgenic facility (New York, NY) for ES-cell targeting, Dr. Willie Mark, Sloan-Kettering Institute (New York, NY) for blastocyst injection and production of chimeric mice, Ms. Yuko Hashimoto and Ms. Shizue Tomita for technical assistance, and Dr. Roy Black for critically reading this manuscript.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by National Institutes of Health Grant R01GM64750 (to C.P.B.) and the Nagao Memorial Fund, the Nakatomi Foundation, and Grants-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (19591765) (to K.H.).

3

Abbreviations used in this paper: TACE, TNF-α-converting enzyme; ADAM, a disintegrin and metalloprotease; mEF, mouse embryonic fibroblast; pIpC, polyinosinic-polycytidylic acid.

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