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Soluble tumor necrosis factor (TNF) receptor-1 induces apoptosis via reverse TNF signaling and autocrine transforming growth factor-ß1

Georg H. Waetzig^*,,1, Philip Rosenstiel^*,1, Alexander Arlt, Andreas Till^*, Karen Bräutigam^*, Heiner Schäfer, Stefan Rose-John, Dirk Seegert^,2 and Stefan Schreiber^*,2,3

^* Institute of Clinical Molecular Biology and
Laboratory of Molecular Gastroenterology and Hepatology, 1. Department of Medicine, Schleswig-Holstein University Medical Center, Kiel, Germany;
Institute of Biochemistry, Christian-Albrechts-University, Kiel, Germany; and
Conaris Research Institute AG, Kiel, Germany

³ Correspondence: Institute of Clinical Molecular Biology, Schleswig-Holstein University Medical Center, Schittenhelmstrasse 12, Kiel 24105, Germany. E-mail: s.schreiber{at}mucosa.de

SPECIFIC AIMS

Transmembrane forms of the pivotal proinflammatory cytokine tumor necrosis factor- (TNF-) and its two receptors are cleaved by the cell membrane-anchored proteinase TNF- converting enzyme (TACE), resulting in appreciable serum levels of soluble TNF- and TNF- receptors (sTNFR1 and -2). The aims of the present study were to 1) investigate whether sTNFR1 has any signaling functions beyond its known role of neutralizing and buffering TNF- in the circulation and 2) characterize these novel signaling pathways in primary monocytes and a monocytic model system.

PRINCIPAL FINDINGS

1. sTNFR1 induces apoptosis in primary human monocytes and THP-1 cells
Primary human peripheral monocytes obtained from seven healthy volunteers were stimulated with various concentrations of recombinant sTNFR1. A monoclonal anti-TNF- antibody (anti-TNF; infliximab) was used as a positive and specificity control. Luminometric caspase-3/7 activity assays showed significant and dose-dependent caspase activation after a 12 h stimulation with sTNFR1 (1.5-fold with 10 µg/ml, 2-fold with 50 µg/ml, and 3.3-fold with 100 µg/ml sTNFR1). A similar pattern was observed when phosphatidylserine externalization in THP-1 myelomonocytic cells was analyzed by annexin V/propidium iodide (PI) FACS after a 24 h stimulation with sTNFR1 or anti-TNF. Apoptotic cell death was further verified by Western blot analysis of cleaved poly(ADP-ribose) polymerase-1 (PARP-1), a substrate of the effector caspase-3, and by measuring caspase-9 activation and cytochrome c release from mitochondria. sTNFR1 (and anti-TNF) activated the MAP kinases p38 and extracellular signal-regulated kinase-1 and -2 (ERK1/2).

2. Apoptosis triggered by sTNFR1 depends on caspase-8 and the mitochondrial pathway, but not on the death receptor adaptor protein FADD
The p38 and ERK1/2 MAP kinase pathways can regulate apoptotic processes in various contexts and have been shown to be activated after binding of transmembrane TNF- (mTNF-) by anti-TNF- antibodies. Therefore, the effects of several specific MAP kinase inhibitors on sTNFR1-induced apoptosis were investigated. Inhibition of p38 by SB203580 (1 µM) protected THP-1 cells against apoptosis at various concentrations of sTNFR1 (e.g., 6% cell death vs. 36% with 10 µg/mL sTNFR1 alone; P<0.01). In contrast, inhibition of ERK1/2 by PD98059 (20 µM) or U0126 (2.5 µM) resulted in significantly increased apoptosis (e.g., 61% cell death instead of 36% with 10 µg/mL sTNFR1 alone; P<0.01). To dissect apoptotic signaling, dominant-negative Fas-associated death domain protein (dnFADD) or FADD-like IL-1ß-converting enzyme (dnFLICE/caspase-8) were overexpressed in THP-1 cells, which were then stimulated with sTNFR1 or anti-TNF. We found that sTNFR1-induced apoptosis is independent of death receptor signaling, as dnFADD could not rescue the cells. However, functional caspase-8 signaling is essential for mTNF--mediated apoptosis because overexpression of dnFLICE/caspase-8 abrogated apoptosis after stimulation with sTNFR1 or anti-TNF.

3. Autocrine TGF-ß1 is involved in sTNFR1-induced apoptotic signaling
The results so far closely resembled the signaling pattern described for apoptotic responses induced by the cytokine transforming growth factor-ß1 (TGF-ß1). Therefore, we measured TGF-ß1 levels in the culture supernatant of primary human monocytes 24 h after stimulation with sTNFR1. While the supernatant of unstimulated monocytes contained 35 pg/mL TGF-ß1, the concentration of this cytokine was significantly increased in a dose-dependent manner after stimulation with sTNFR1 (Fig. 1 A). Primary monocytes and THP-1 cells expressed both type I and II TGF-ß receptors, a prerequisite for functional TGF-ß signaling (Fig. 2B ). Introduction of an anti-TGF-ß antibody (1–1.5 µg/mL) or a blocking anti-TGF-ß receptor-2 antibody (10 µg/mL) into the culture medium specifically and significantly reduced sTNFR1-induced apoptosis, as determined by PARP-1 cleavage (Fig. 1C ), colorimetric cell viability assays (Fig. 1D ), and annexin V/PI FACS (Fig. 1F ). Correspondingly, anti-TGF-ß blocked the activation of p38 and ERK1/2 in response to sTNFR1 (Fig. 1E ). The same observations were made when a monoclonal anti-TNF- antibody was used instead of sTNFR1. Proapoptotic p38 activation and antiapoptotic ERK1/2 activation by sTNFR1 both depend on the presence of autocrine TGF-ß1 (Fig. 2 ).

View larger version (42K): [in this window] [in a new window]   Figure 1. Autocrine stimulation of monocytes by TGF-ß1 induces apoptosis and MAPK activation. A) Levels of TGF-ß1 in the supernatant of peripheral monocytes increased dose-dependently after a 24 h stimulation with sTNFR1. Baseline secretion yielded 35 pg/mL TGF-ß1. Data represent monocytes from 4 individuals with at least 3 measurements/sTNFR1 dose. *Statistical significance vs. control cells: **P < 0.01. B) RT-PCR detection of type I and type II TGF-ß receptors in peripheral monocytes and THP-1 cells. C) Western blot analysis of uncleaved (116 kDa) and cleaved (89 kDa) PARP-1 in THP-1 cells 24 h after apoptosis induction by sTNFR1 in the presence or absence of 1 µg/mL anti-TGF-ß antibody. Results of a representative experiment are shown (n=3). D) THP-1 cell viability as determined by colorimetric cell viability assays 48 h after stimulation with a low (5 µg/mL) and a high (100 µg/mL) dose of sTNFR1 in the presence of nonspecific control serum or anti-TGF-ß (1.5 µg/mL each); n = 3. **P < 0.01. E) Coincubation with anti-TGF-ß blocked p38 and ERK1/2 activation in THP-1 cells 24 h after stimulation with low (10 µg/mL) or high (100 µg/mL) doses of sTNFR1. Results of a representative experiment are shown (n=3). F) Levels of apoptotic THP-1 cells after a 24 h incubation with sTNFR1 alone or with anti-TGF-ß (1 µg/mL) or anti-TGF-ßR2 (10 µg/mL) as determined by annexin V/PI FACS analysis. Results of a representative experiment are shown (n=3).

View larger version (21K): [in this window] [in a new window]   Figure 2. Schematic representation of the proposed model of sTNFR1-induced monocyte apoptosis. Reverse signaling from sTNFR1 via mTNF- leads to enhanced TGF-ß1 release. Autocrine TGF-ß1 activates the proapoptotic effector p38 and the antiapoptotic protector ERK1/2. The balance of p38 and ERK1/2 signaling tightly regulates cell survival or death.

CONCLUSIONS AND SIGNIFICANCE

Our findings suggest that the role of sTNFR1 in the immune system far exceeds its basic functions of buffering and neutralizing circulating TNF-. The specific induction of apoptosis by sTNFR1 in cells bearing mTNF- on their cell membrane indicates that sTNFR1 functions as a ligand and mTNF- as a receptor. This so-called reverse signaling has been described for many receptor/ligand pairs of the TNF superfamily. Previous studies have shown that bivalent anti-TNF- antibodies and monovalent antigen binding fragments of these antibodies (just as the monovalent sTNFR1 in our experiments) can bind to mTNF- and induce apoptosis.

The mechanisms underlying these phenomena have remained elusive. All data available so far suggest that anti-TNF- antibodies selectively kill activated immune cells at sites of inflammation, but not resting or circulating cells. This selectivity is probably due mainly to the presence of much larger amounts of mTNF- on activated cells. The monocytic cell line THP-1 we used is an exception to this rule, because resting THP-1 cells constitutively express mTNF-. This allowed us to rule out activation-induced stress signaling as a cause for the MAP kinase activation and apoptosis seen after stimulation with sTNFR1.

Interestingly, only high-affinity anti-TNF- antibodies such as infliximab, but not the low-affinity TNFR2 fusion protein etanercept, have been shown to induce apoptosis in mTNF--bearing cells. However, etanercept neutralizes soluble TNF- as effectively as infliximab. Therefore, the induction of apoptosis by high-affinity TNF- binding agents such as sTNFR1 or anti-TNF- antibodies is due to ligation of mTNF- and not to the neutralization of secreted TNF-, which can be a survival factor for monocytic cells. Previous findings and our experiments with dominant-negative proapoptotic mediators show that mTNF--mediated apoptosis is independent of death receptors but involves the mitochondrial apoptotic pathway.

The apoptotic effector mechanisms found for sTNFR1 and anti-TNF- antibodies correspond to known signaling pathways triggered by TGF-ß. TGF-ß functions as a molecular switch that antagonizes and modifies the action of other cytokines and has a critical role for directing and resolving inflammatory processes. The severe and uncontrolled inflammatory reactions observed in a TGF-ß^–/– knockout model and in dominant-negative TGF-ß receptor-2 transgenic mice underline the importance of TGF-ß for the containment of inflammation. It has been shown that TGF-ß can act in an autocrine fashion and that TGF-ß exerts its inhibitory function in immunological homeostasis and inflammatory responses by inducing a form of apoptosis that is dependent on the activation of p38 and, subsequently, caspase-8, caspase-9, and caspase-3, but independent of the death receptor signaling adaptor FADD.

The striking similarities between the characteristics of TGF-ß1- and sTNFR1/anti-TNF--induced apoptosis led us to the hypothesis that reverse signaling via mTNF- might increase the constitutive release of TGF-ß1 from monocytes and that TGF-ß1 could activate p38 and ERK1/2 in an autocrine manner. The present results support this hypothesis, since we found that monocytes secreted significantly more TGF-ß1 in response to stimulation with sTNFR1 or anti-TNF-. Blocking antibodies directed against TGF-ß1 and TGF-ß receptor-2, but not nonspecific control antiserum, strongly reduced p38 and ERK1/2 activation and apoptosis in response to sTNFR1. Our findings suggest that p38 is a pivotal TGF-ß1-dependent effector of apoptosis triggered by sTNFR1. In addition, ligation of mTNF- by anti-TNF- or sTNFR1 probably sensitizes the cells to TGF-ß1-induced apoptosis, as TGF-ß1 alone is not cytotoxic to THP-1 cells at the concentrations we used but induces differentiation of these cells to a macrophage-like phenotype. A plausible explanation is that reverse signaling via mTNF- has been shown to cause a strong increase in intracellular Ca²⁺. This may lead to a rapid Ca²⁺ influx into mitochondria, which can cause cytochrome c release and apoptosis. The protective role of ERK1/2 in TGF-ß1-induced apoptosis is in accordance to previous studies. For example, it has been demonstrated that the ERK1/2 pathway directly inhibits caspase-9 activity and subsequent caspase-3-dependent apoptosis. Future studies are needed to determine the mechanisms of TGF-ß1 release after mTNF- ligation and the cross-talk between the plethora of pathways activated by mTNF- reverse signaling.

Besides possible physiological functions of this sTNFR1/mTNF-/TGF-ß1 signaling pathway in the resolution of inflammation, reduced autocrine TGF-ß1-mediated apoptosis after mTNF- ligation may also play an important role in the pathophysiology of chronic autoimmune diseases, where apoptosis resistance of activated immune cells is a common feature. In Crohn’s disease, the therapeutic efficacy of anti-TNF- agents has been linked to their ability to induce apoptosis in mTNF--expressing immune cells. Given the findings of the present study, it is plausible that the efficacy of apoptosis induction by anti-TNF- drugs depends on triggering the autocrine TGF-ß1/p38 axis and sensitizing mTNF--bearing cells to TGF-ß1-induced apoptosis, whereas endogenous levels of TGF-ß1 in the inflamed tissue may not be sufficient to overcome the apoptosis resistance of the disease-perpetuating immune cells.

The activation of both p38 as a proapoptotic effector and ERK1/2 as an antiapoptotic protector via an sTNFR1-induced autocrine TGF-ß1 signaling loop suggests a careful cellular balance between selective killing of immune cell subsets and the protection of necessary immune functions. We postulate that cross-talk from other pathways will be necessary to decide whether the proapoptotic or antiapoptotic signaling induced by sTNFR1 and TGF-ß1 will prevail. A strict localization and targeted secretion of apoptosis-inducing amounts of sTNFR1 could be pivotal for a selective removal of certain immune cell subpopulations in relevant anatomical compartments. In the context of inflammatory microenvironments and immunological synapses, local sTNFR1 concentrations may well reach the range in which this regulation was observed. The mechanisms of sTNFR1-induced apoptosis could be crucial for the pathophysiology and TNF--blocking therapy of chronic autoimmune disorders.

FOOTNOTES

¹ These authors contributed equally to this work.

² These authors share senior authorship.

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2073fje;

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